WO2017123592A1

WO2017123592A1 - Bacteria engineered to treat disorders associated with bile salts

Info

Publication number: WO2017123592A1
Application number: PCT/US2017/012946
Authority: WO
Inventors: Dean Falb; Jonathan W. KOTULA; Vincent M. ISABELLA; Paul F. Miller; Alex TUCKER
Original assignee: Synlogic, Inc.
Priority date: 2016-01-11
Filing date: 2017-01-11
Publication date: 2017-07-20
Also published as: WO2017123592A8

Abstract

The present disclosure provides recombinant bacterial cells comprising a heterologous gene encoding a bile salt hydrolase enzyme and/or a 7α-dehydroxylating enzyme. In another aspect, the recombinant bacterial cells further comprise a bacterial kill switch. The disclosure further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease (such as cirrhosis or nonalcoholic steatohepatitis (NASH), C. difficile infection, and cancer, using the pharmaceutical compositions of the disclosure.

Description

BACTERIA ENGINEERED TO TREAT DISORDERS

ASSOCIATED WITH BILE SALTS

Related Applications

[01] This application claims priority to U.S. Provisional Application No.

62/277,346, filed on January 11, 2016; U.S. Provisional Application No. 62/336,012, filed on May 13, 2016; U.S. Provisional Application No. 62/362,863, filed on July 15, 2016; U.S. Provisional Application No. 62/347,576, filed on June 8, 2016; U.S. Provisional Application No. 62/348,620, filed on June 10, 2016; PCT Application No. PCT/US2016/039444, filed on June 24, 2016; PCT Application No. PCT/US2016/069052, filed on December 28, 2016; and PCT Application No. PCT/US2016/032565, filed on May 13, 2016, the entire contents of each of which are expressly incorporated herein by reference.

Background

[02] Bile salts (also called conjugated bile acids) are cholesterol derivatives synthesized in the liver which comprise a steroid ring component conjugated with either taurine (taurocholic acid; TCA) or glycine (glycochenodeoxycholic acid; GCDCA). Bile salts act as signaling molecules to regulate systemic endocrine functions, including triglyceride, cholesterol, and glucose homeostasis (Houten et al., EMBO J., 25: 1419-1425 (2006) and Watanabe et al, Nature, 439:484-489 (2006)) and may trigger cellular farnesoid X receptor (FXR)- and G-protein coupled receptor (TGR4)-mediated host responses.

Additionally, bile salts have been shown to facilitate lipid absorption and repress bacterial cell growth in the small intestine, thereby influencing both host metabolic pathways and the microflora present in the gut (Jones et al, PNAS, 105(36): 13580- 13585 (2008) and Ridlon et al., J. Lipid Research, 47(2):241-259 (2006)).

[03] Bile salts are stored in the gallbladder and then subsequently released into the duodenum via the common bile duct. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules, a process referred to as

deconjugation, to produce the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non- reabsorbed bile acids enter the large intestine. Once in the large intestine, bile acids are amenable to further modification by microbial 7a-dehydroxylating enzymes to yield secondary bile acids, such as deoxycholic acid (DC A) and lithocholic acid (LCA) (Joyce et al, Gut Microbes, 5(5):669-674 (2014); Bhowmik et al, Accepted Article,

doi: 10.1002/prot.24971 (2015); see also Figure 1).

[04] Bile acids (also called unconjugated bile salts) are also ligands for the FXR nuclear hormone receptor.T he bile acid-FXR interaction regulates bile acid synthesis, transport and cholesterol metabolism. Furthermore, bile acid-FXR regulation has been shown to affect hepatic and intestinal inflammation, atherosclerosis, and inflammation and autoimmune disease in the CNS. For example, CDC A and obeticholic acid, also known as 6a-ethyl-chenodeoxycholic acid (6-ECDCA), a synthetic bile acid analogue that is a 6a-ethyl derivative of CDCA, have each been shown to ameliorate experimental autoimmune encephalomyelitis (EAE) in mice (Ho and Steinman, Proc. Natl. Acad. Sci. U.S.A.,

113(6): 1600-1605 (2016)). In various other studies using animal models for fatty liver disease, 6-ECDCA has also been shown to reduce liver fat and fibrosis. More specifically, 6- ECDCA has been found to improve glucose and insulin tolerance and decrease steatohepatitis (Vignozzi et al., Journal of Sexual Medicine, 8:57-77 (2011); Cipriani et al., J. Lipid Res., 51:771-784 (2010)), decrease hepatic expression of genes involved in fatty acid synthesis and reduce TNF-a and elevated peroxisome-proliferator activated receptor alpha expression, thereby improving NASH phenotype (Carr et al., Pharm. Res., 17(16): 1-16 (2015); Cipriani et al., J. Lipid Res., 51:771-784 (2010)), prevent hepatic stellate cell activation by inhibiting osteopontin production (Fiorucci et al., Gastroenterology, 127: 1497-1512 (2004)), and prevent fibrosis progression, reverse fibrosis and cirrhosis development and reduce portal hypertension (Carr et al., Pharm. Res., 17(16): 1-16 (2015); see also Khalid et al., Liver Res. Open J., 1:32-40 (2015)). Furthermore, a small number of clinical trial studies have shown that the FXR agonist, 6-ECDCA, can improve insulin sensitivity and decrease the levels of markers for inflammation and fibrosis in patients with type II diabetes and NAFLD (Mudaliar et al., Gastroenterology, 127: 1497-1512 (2013)), and improve liver histology in patients with non-alcoholic steatohepatitis (Neuschwander-Tetri et al., (Lancet, 385:956-1065 (2014)).

[05] It has been shown that bile salt metabolism is involved in host physiology (Ridlon et al., Current Opinion Gastroenterol., 30(3):332 (2014) and Jones et al., 2008). For example, it is known that the expression of bile salt hydrolase enzymes functionally regulate host lipid metabolism and play a role in cholesterol metabolism and transport, circadian rhythm, gut homeostasis/barrier function, weight gain, adiposity, and possibly gastrointestinal cancers in the host (Joyce et al., PNAS, 111(20):7421-7426 (2014); Zhou and Hylemon, Steroids, 86:62-68, (2014); Mitchell et al., Expert Opinion Biolog. Therapy, 13(5):631-642 (2013); and W014/198857, the entire contents of each of which are expressly incorporated herein by reference). Specifically, potential effects of bile salt hydrolase-expressing bacteria on cholesterol metabolic pathways have been shown to upregulate the ATP binding cassette Al (ABCA1), the ATP binding cassette Gl (ABCG1), the ATP binding cassette G5/G8 (ABCG5/G8), cholesterol 7 alpha-hydroxylase (CYP7A1), and liver X receptor (LXR), and to downregulate farnesoid X receptor (FXR), Niemann-Pick Cl-like 1 (NPC1L1), and small heterodimer partner (SHP), which impacts cholesterol efflux, plasma HDL-C levels, biliary excretion, cholesterol catabolism, bile acid synthesis, cholesterol levels, and decreased intestinal cholesterol absorption, among other effects (Mitchel et al. (2014) and Zhou and Hy lemon (2014)). Additionally, bile salt hydrolase activity has been shown to impact bile detoxification, gastrointestinal persistence, nutrition, membrane alterations, altered digestive functions (lipid malabsorption, weight loss), cholesterol lowering, cancer, and formation of gallstones (see Begley et al, Applied and Environmental Microbiology, 72(3): 1729-1738 (2006)).

[06] Moreover, a Clostridium scindens bacterium expressing a 7a-dehydroxylating enzyme has been shown to produce resistance to C. difficile infection in hosts (Buffie et al., Nature, 517:205-208 (2015), and bile salt metabolism has been shown to play a role in both regulating the microbiome as well as in cirrhosis (Ridlon et al., Gut Microbes, 4(5):382-387 (2013) and Kakiyama et al, J. Hepatol, 58(5):949-955 (2013)). Thus, a need exists for treatments which address the metabolism of bile salts in subjects in order to treat and prevent diseases and disorders in which bile salts play a role, such as cardiovascular disease, metabolic disease, cirrhosis, gastrointestinal cancer, and C. difficile infection.

Summary

[07] The present disclosure provides recombinant bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with bile salts. Specifically, the recombinant bacteria disclosed herein have been engineered to comprise genetic circuits encoding, for example, a bile salt hydrolase enzyme and/or a 7a- dehydrolase enzyme to treat disease, disorders, and/or conditions associated with bile salts and bile salt metabolism. In some embodiments, the recombinant bacteria comprise genetic circuits encoding a bile salt hydrolase enzyme and/or a 7a-dehydrolase enzyme, eas well as other circuitry in order to guarantee the safety and non-colonization of a subject, such as auxotrophies, kill switches, etc. These recombinant bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect.

[08] In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s). In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise a heterologous gene encoding a 7a-dehydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and a 7a-dehydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and/or a 7a-dehydrolase enzyme and is capable of processing and reducing levels of bile salts, e.g., deconjugation of bile salts. In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and/or a 7a-dehydrolase enzyme and is capable of processing and reducing levels of bile salts, e.g., deconjugation of bile salts. In some embodiments, the disclosure provides a bacterial cell genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and/or a 7a-dehydrolase enzyme and is capable of processing and reducing levels of bile salts, e.g., deconjugation of bile salts in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacterial cells and

pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess bile salts into non-toxic molecules (e.g., bile salt metabolites) in order to treat and/or prevent disorders associated with bile salts, such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection.

[09] In some embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme. In some embodiments, the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s). In some

embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the glycine or taurine molecules from a bile salt to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDC A). In some embodiments, the recombinant bacterial cell engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme is capable of removing the glycine or taurine molecules from a bile salt to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDC A). In some embodiments, the recombinant bacterial cell is engineered to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDCA). In some embodiments, the

recombinant bacterial cell is genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the glycine from

glycochenodeoxycholic acid (GCDCA) to produce chenodeoxycholic acid (CDCA). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the taurine from taurocholic acid (TCA) to produce cholic acid (CA).

[010] In some embodiments, a recombinant bacterial cell is engineered to comprise a heterologous gene sequence encoding a bile salt hydrolase (BSH) enzyme that removes the glycine or taurine molecules from a bile salt to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDCA) in low-oxygen environments, e.g., the gut. In some embodiments, a recombinant bacterial cell is engineered to comprise a heterologous gene sequence encoding a bile salt hydrolase (BSH) enzyme to produce the primary bile acids cholic acid (CA) and/or chenodeoxycholic acid (CDCA), wherein the primary bile acid stimulates FXR.

[Oi l] In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is operably linked to an inducible promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is operably linked to an inducible promoter that is induced directly or indirectly induced by exogenous environmental conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is operably linked to an inducible promoter that is directly or indirectly induced by exogenous environmental conditions found in a mammalian gut. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is operably linked to an inducible promoter that is directly or indirectly induced by low oxygen or anaerobic conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is operably linked to a constitutive promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is present on a plasmid. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt hydrolase (BSH) enzyme(s), the gene sequence encoding one or more bile salt hydrolase (BSH) enzymes is present on a chromosome in the bacterial cell.

[012] In some embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding one or more 7a- dehydroxylating enzyme(s). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s) to produce secondary bile salts, e.g., deoxycholic acid (DCA) and/or lithocholic acid (LCA). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise gene sequence encoding a 7a-dehydroxylating enzyme to produce deoxycholic acid (DCA). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise gene sequence encoding a 7a-dehydroxylating enzyme to produce lithocholic acid (LCA). In some embodiments, the 7a-dehydroxylating enzyme is a bacterial enzyme. In some embodiments, the 7a-dehydroxylating enzyme is a bai gene. In some embodiments, the 7a-dehydroxylating enzyme is produced by a bacterial bai operon. In some

embodiments, the bai operon is from C. scindens. In some embodiments, the bai operon of C. scindens encodes baiB, baiCD, baiE, baiAl, baiA2, baiA3, baiF, baiG, baiH, and/or bail. In some embodiments, the bai operon is from C. hiranonis.

[013] In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is operably linked to an inducible promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is operably linked to an inducible promoter that is induced directly or indirectly induced by exogenous environmental conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is operably linked to an inducible promoter that is directly or indirectly induced by exogenous environmental conditions found in a mammalian gut. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a- dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is operably linked to an inducible promoter that is directly or indirectly induced by low oxygen or anaerobic conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a- dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is operably linked to a constitutive promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a- dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is present on a plasmid. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s), the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) is present on a chromosome in the bacterial cell.

[014] In some embodiments, the recombinant bacterial cell is genetically engineered to comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) and to further comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s). In some embodiments, the recombinant bacterial cell is capable of producing secondary bile acids, e.g., DCA and/or LCA. In some embodiments, the recombinant bacterial cell is engineered to comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) to produce the primary bile acids cholic acid (CA) and/or chenodeoxycholic acid (CDCA) and to further comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s) to produce deoxycholic acid (DCA) and/or lithocholic acid (LCA). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) and to further comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s) to produce primary bile acids and/or secondary bile acids in low-oxygen environments, e.g., the gut.

[015] In some embodiments, the recombinant bacterial cell further comprises gene sequence encoding one or more bile salt and/or bile acid transporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more bile salt hydrolase enzymes and gene sequence encoding one or more bile salt and/or bile acid transporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more 7a-dehydroxylating enzymes and gene sequence encoding one or more bile salt and/or bile acid transporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more bile salt hydrolase enzymes, gene sequence encoding one or more 7a-dehydroxylating enzymes and gene sequence encoding one or more bile salt and/or bile acid transporters.

[016] In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is operably linked to an inducible promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is operably linked to an inducible promoter that is induced directly or indirectly induced by exogenous environmental conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more 7a-dehydroxylating enzyme(s) bile salt and/or bile acid transporters is operably linked to an inducible promoter that is directly or indirectly induced by exogenous environmental conditions found in a mammalian gut. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is operably linked to an inducible promoter that is directly or indirectly induced by low oxygen or anaerobic conditions. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is operably linked to a constitutive promoter. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is present on a plasmid. In some embodiments in which the recombinant bacterial cell comprises a heterologous gene sequence encoding one or more bile salt and/or bile acid transporters, the gene sequence encoding one or more bile salt and/or bile acid transporters is present on a chromosome in the bacterial cell. [017] In some embodiments, the recombinant bacterial cell further comprises gene sequence encoding one or more bile acid exporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more bile salt hydrolase enzymes and gene sequence encoding one or more bile acid exporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more 7 a- dehydroxylating enzymes and gene sequence encoding one or more bile acid exporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more bile salt and/or bile acid transporters and gene sequence encoding one or more bile acid exporters. In some embodiments, the recombinant bacterial cell comprises gene sequence encoding one or more bile salt hydrolase enzymes, and/or gene sequence encoding one or more 7a-dehydroxylating enzymes and/or gene sequence encoding one or more bile salt and/or bile acid transporters and/or gene sequence encoding one or more bile acid exporters.

Brief Description of the Drawings

[018] Fig. 1 depicts bile salt metabolism. Bile salts are synthesized from cholesterol in the liver and stored in the gallbladder. After release into the duodenum, microbial bile salt hydrolase activity in the small intestine deconjugates the glycine or taurine molecules to produce primary bile acids (also known as unconjugated bile acids). Most bile acids are reabsorbed into the enterohepatic portal system, but some enter the large intestine where they are further metabolized by microbial 7a-dehydroxylase to produce secondary bile acids. Excess bile acids are also lost in the stool (200 mg - 600 mg per day).

[019] Fig. 2 depicts the structure of bile salts and the location at which bile salt hydrolase enzymes deconjugate the bile salts.

[020] Fig. 3 depicts the state of one non-limiting embodiment of the bile salt hydrolase enzyme construct under inducing conditions. Expression of the bile salt hydrolase enzyme and a bile salt transporter are both induced by the FNR promoter in the absence of oxygen. The thyA gene has been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth. The recombinant bacterial cell may further comprise an auxotrophic mutation, a type III secretion system, and/or a kill switch, as further described herein.

[021] Fig. 4 depicts exemplary circuit designs for the recombinant bacteria of the disclosure. In some embodiments, two bile salt hydrolase (BSH) genes from Lactobacillus salivarius (BSH1 and BSH2) are under the control of an aTc-inducible promoter in a single operon. In some embodiments, two bile salt hydrolase (BSH) genes (BSHl and BSH2) are each under the control of an aTc-inducible promote for individual expression. In some embodiments, the BSHl and BSH2 genes encode the same bile salt hydrolase enzyme. In other embodiments, the BSHl and BSH2 genes encode different bile salt hydrolase enzymes.

[022] Figs. 5A-B depict exemplary schematics of the E. coli 1917 Nissle

chromosome comprising multiple MoAs. Fig. 5A depicts an embodiment wherein a bile salt hydrolase (BSH) gene and a bile salt transporter, e.g., an importer,are inserted at two or more different chromosomal insertion sites. Fig. 5B depicts an embodiment wherein a bile salt hydrolase (BSH) gene, a bile salt transporter, e.g., an importer, and other expression circuits, for example, a Glp-1 expression circuit, and a butyrate production circuit are inserted at four or more different chromosomal insertion sites.

[023] Fig. 6 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown Table 3 (Pfnrl-5). Different FNR-responsive promoters were used to create a library of anaerobic/low oxygen conditions inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+0₂) or anaerobic conditions (-0₂). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.

[024] Fig. 7A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P_fnrs)- LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. Fig. 7B depicts FNR promoter activity as a function of β- galactosidase activity in SYN-PKU904. SYN-PKU904, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr. under anaerobic and/or low oxygen conditions . Fig. 7C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.

[025] Fig. 8 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.

[026] Fig 9A depicts a "Oxygen bypass switch" useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more propionate catabolism enzyme(s) (POI1) and /or one or more transporter(s)/importer(s) and/or exporter(s) (POI2) under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active propionate catabolism enzyme(s) prior to

administration. This can be done by pre-inducing the expression of these enzymes as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites.

[027] FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ, The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar

24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar

24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.

[028] In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y. [029] Fig. 9B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of Fnr expression can be fine- tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bioinformatics tools for optimization of RBS are known in the art.

[030] Fig. 9C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bioinformatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of P_fnrs-POI constructs are maintained to allow for strong in vivo induction.

[031] Fig. 10 depicts the gene organization of an exemplary construct, comprising a cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.

[032] Fig. 11 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI). In some embodiments, this construct is useful for pre-induction and preloading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a bile salt transporter construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. Bile salt transporter expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is used in combination with a 7a-dehydroxylating enzyme expression construct. [033] In some embodiments, the constructs bile salt hydrolase sequences are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ ID NO: 29. In some embodiments, the bile salt hydrolase sequences are codon optimized for expression in E coli. In some embodiments, the bile salt hydrolase sequences are codon optimized for expression in Lactococcus. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is employed in a biosafety system, such as the system shown in Fig. 26AA, Fig. 26B, Fig. 26C, and Fig. 26D. In some embodiments, the construct is integrated into the genome at one or more locations described herein.

[034] Figs. 12A-C depict schematics of non-limiting examples of constructs expressing a protein of interest (POI). Fig 12A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, the construct comprises SEQ ID NO: 65. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower). [035] Fig. 12B depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only

the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some

embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

[036] Fig. 12C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter. The arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POI(s) prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low- oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.

[037] Fig. 13A depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta-galactosidase activity was measured. Fig. 13B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1 : 100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).

[038] Fig. 14 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites.

[039] Fig. 15 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent. [040] Fig. 16 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

[041] Fig. 17 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating.

[042] Fig. 18 depicts an exemplary schematic of the E. coli 1917 Nissle

chromosome comprising multiple mechanisms of action (MoAs).

[043] Figs. 19A-19C depict other non-limiting embodiments of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. Fig. 19A depicts an embodiment of heterologous gene expression in which, in the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (ParaBAo), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. Fig. 19A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. [044] Fig. 19B depicts a non-limiting embodiment of the disclosure, where an antitoxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araB AD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit.

[045] Fig. 19C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araB AD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter {e.g. , AraC promoter) in this circuit.

[046] Fig. 20 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated

conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[047] Fig. 21 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.

[048] Fig. 22 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the

recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested

recombinases can be used to further control the timing of cell death.

[049] Fig. 23 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.

[050] Fig. 24 depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived antitoxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies. [051] Fig. 25 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al, 2015.

[052] Figs. 26A-26D depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 26A and Fig. 26B), which also contains a chromosomal component (shown in Fig. 26C and Fig. 26D). The Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 26A) and thyA (Fig. 26B) and promoter elements driving expression of these components. In a non-limiting example, the plasmid comprises SEQ ID NO: 81. In a non- limiting example, the plasmid comprises SEQ ID NO: 82. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, in which a bile salt hydrolase and/or a bile salt transporter is expressed from an inducible or constitutive promoter. Fig. 26C and Fig. 26D depict schematics of the gene organization of the chromosomal component of a biosafety system. Fig. 26C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter. In some embodiments, the construct comprises SEQ ID NO: 89. Fig. 26D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. In some embodiments, the construct comprises SEQ ID NO: 90. If the plasmid containing the functional DapA is used (as shown in Fig. 26A), then the chromosomal constructs shown in Fig. 26C and Fig. 26D are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in Fig. 26B), then the chromosomal constructs shown in Fig. 26C and Fig. 26D are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.

[053] Fig. 27 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. [054] Fig. 28 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N- terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta- domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.

[055] Fig. 29 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP- binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.

[056] Fig. 30 depicts a schematic of the outer and inner membranes of a gram- negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g. , therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g. , lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g. , degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.

[057] Fig. 31 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons). [058] Fig. 32A, Fig. 32B, and Fig. 32C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of proteins of interest, e.g., therapeutic proteins of interest, which are secreted using components of the flagellar type III secretion system. A protein of interest, such as a bile salt hydrolase and/or a 7a-dehydrolase enzyme , is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (Fig. 32A and Fig. 32B) or a Tet-inducible promoter (Fig. 32C). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR- inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in Fig. 32B and Fig. 32C.

[059] Fig. 33A and Fig. 33B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a Tet promoter (Fig. 33A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, Fig. 33B), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose.

[060] Fig. 34A depicts a schematic diagram of a wild-type clbA construct.

[061] Fig. 34B depicts a schematic diagram of a clbA knockout construct.

[062] Fig. 35 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animal disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.

[063] Fig. 36 depicts a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. Step A depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm. Step B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SCI, duration 1.5 hours, temperature 37° C, shaking at 250 rpm. Step C depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. Step D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. Step E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.

Description of the Embodiments

[064] The disclosure provides genetically engineered microorganisms,

pharmaceutical compositions thereof, and methods of treating disorders associated with bile salts and/or bile acids. Specifically, the recombinant bacteria disclosed herein have been engineered to comprise genetic circuits encoding, for example, a bile salt hydrolase enzyme and/or a 7a-dehydrolase enzyme to treat the disease, as well as, in some embodiments, other circuitry that guarantees the safety and non-colonization of the subject that is administered the recombinant bacteria, such as auxotrophies, kill switches, etc. These recombinant bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect.

[065] In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme. In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and/or a 7a- dehydrolase enzyme. In some embodiments, the engineered bacteria are capable of processing and reducing levels of bile salts and/or bile acids. In some embodiments, the engineered bacteria are capable of processing and reducing levels of bile salts and/or bile acids in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess bile salts into non-toxic molecules in order to treat and/or prevent disorders associated with bile salts, such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection. In some embodiments, the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess bile acids into non-toxic molecules in order to treat and/or prevent disorders associated with bile salts and bile salt metabolites (e.g., bile acids), such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection.

[066] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[067] As used herein, the terms "degrade", "degrading", and their cognates is meant to refer to the changing, converting, or processing of a macromolecule into one or more smaller, simpler, or less complex molecule(s), compound(s), component(s), or unit(s), e.g., peptides, amino acids, monosaccharides or other carbohydrates, nucleic acids, lipids, fatty acids, and the like.

[068] As used herein, "payload" refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria, yeast, or a virus. In some embodiments, the payload is a therapeutic payload. In one embodiment, the term "payload" is one or more bile salt hydrolase enzymes, e.g., one or more enzymes that catabolize bile salts into unconjugated bile acids, such as CA and CDCA. In another embodiment, "payload" refers to one or more 7a-dehydroxylating enzymes, e.g., enzymes that metabolize primary bile acids into secondary bile acids. In one embodiment, the term "payload" refers to one or more bile salt hydrolase enzymes that catabolize bile salts into unconjugated bile acids (such as CA and CDCA) and one or more 7a-dehydroxylating enzymes that metabolize primary bile acids into secondary bile acids. In another

embodiment, the term "payload" refers to a bile salt transporter, e.g. which facilitates import of bile salt into the bacterial cell. In some embodiments, the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments, the payload is encoded by a gene or multiple genes or an operon. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.

[069] As used herein, the term"gene" or "gene sequence" is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene or gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene or gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other nonprotein coding sequence.

[070] "Microorganism" refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa. In some aspects, the microorganism is engineered ("engineered microorganism") to produce one or more therapeutic molecules or proteins of interest. In certain aspects, the

microorganism is engineered to take up and catabolize certain metabolites or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.

[071] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria do not contain lipopolysaccharides (LPS). In some embodiments, non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to certain strains belonging to the genus Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum,

Clostridium butyricum, Enterococcus faecium, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri,

Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al, 2009; Dinleyici et al , 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

[072] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. In some embodiments, the probiotic bacteria are Gram-negative bacteria. In some embodiments, the probiotic bacteria are Gram- positive bacteria. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to certain strains belonging to the genus Bifidobacteria, Escherichia,

Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al, 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al, 2010; Olier et al, 2012; Nougayrede et al, 2006). Nonpathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered or

programmed to enhance or improve probiotic properties.

[073] As used herein, the term "recombinant bacterial cell" or "recombinant bacteria" refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably

incorporated into their chromosome.

[074] A "programmed or engineered recombinant bacterial cell" is a recombinant bacterial cell that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered recombinant bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered recombinant bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.

[075] As used herein, a "heterologous" gene or "heterologous sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. "Heterologous gene" includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non- native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, the term "transgene" refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

[076] As used herein, a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g. , an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al. , 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in a gene cassette. In some embodiments, "non-native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In addition, multiple copies of any regulatory region, promoter, gene, and/or gene cassette may be present in the bacterium, wherein one or more copies of the regulatory region, promoter, gene, and/or gene cassette may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same regulatory region, promoter, gene, and/or gene cassette in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions. In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a bile salt hydrolase enzyme that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g. , an FNR promoter operably linked to a gene encoding a bile salt hydrolase.

[077] As used herein, the term "coding region" refers to a nucleotide sequence that codes for a specific amino acid sequence. The term "regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.

[078] As used herein the term "codon-optimized" refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism.

[079] "Operably linked" refers a nucleic acid sequence, e.g., a gene encoding a bile salt hydrolase enzyme, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[080] A "promoter" as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5' of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue- specific manner, in response to different environmental or physiological conditions, or in response to specific compounds.

Prokaryotic promoters are typically classified into two classes: inducible and constitutive.

[081] "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli σ promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli σ³² promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110

(BBa_M13110)), a constitutive Bacillus subtilis σ^Α promoter (e.g., promoter veg

(BBa_K143013), promoter 43 (BBa_K143013), Pi_iaG (BBa_K823000), Pi_epA

(BBa_K823002), P_veg (BBa_K823003)), a constitutive Bacillus subtilis σ promoter (e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_Kl 13010; BBa_Kl 13011; BBa_Kl 13012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof.

[082] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region.

[083] A "directly inducible promoter" refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a phenylalanine-metabolizing enzyme, e.g., PAL; in the presence of an inducer of said regulatory region, the phenylalanine- metabolizing enzyme is expressed. An "indirectly inducible promoter" refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a gene encoding a first molecule, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a gene encoding a phenylalanine-metabolizing enzyme. In the presence of an inducer of the first regulatory region, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the phenylalanine-metabolizing enzyme. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by "inducible promoter."

[084] As used herein, "stably maintained" or "stable" bacterium or virus is used to refer to a bacterial or viral host cell carrying non-native genetic material, e.g., one or more a bile salt hydrolase enzymes, such that the non-native genetic material is retained, expressed, and propagated. For example, the non-native genetic material may be incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid. The stable bacterium or virus is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium or virus may be a genetically engineered bacterium or genetically engineered virus comprising non-native genetic material encoding a bile salt hydrolase enzyme, in which the plasmid or chromosome carrying the non-native genetic material is stably maintained in the bacterium or virus, such that the bile salt hydrolase enzyme can be expressed in the bacterium or virus, and the bacterium or virus is capable of survival and/or growth in vitro and/or in vivo.

[085] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide. [086] As used herein, the term "plasmid" or "vector" refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell' s genome.

Plasmids are usually circular and capable of autonomous replication. Plasmids may be low- copy, medium-copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding a bile salt hydrolase enzyme.

[087] As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically- stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

[088] The term "genetic modification," as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising a bile salt hydrolase enzyme operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the

chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.

[089] As used herein, the term "genetic mutation" refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example,

substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.

[090] An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.

[091] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR- responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1.

[092] In a non-limiting example, a promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global

transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS. Table 1. Examples of transcription factors and responsive genes and regulatory regions

[093] "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.

[094] In some embodiments, the genetically engineered bacteria are active {e.g., express one or more BSHs) in the gut. In some embodiments, the genetically engineered bacteria are active {e.g., express one or more BSHs) in the large intestine. In some

embodiments, the genetically engineered bacteria are active {e.g., express one or more BSHs) in the small intestine. In some embodiments, the genetically engineered bacteria are active in the small intestine and in the large intestine. In some embodiments, the genetically

engineered bacteria transit through the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the small intestine. In some

embodiments, the genetically engineered bacteria colonize the small intestine. In some embodiments, the genetically engineered bacteria do not colonize the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the gut. In some embodiments, the genetically engineered bacteria colonize the small gut. In some embodiments, the genetically engineered bacteria do not colonize the gut.

[095] As used herein, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0₂) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% 0_2; <160 torr 0₂>). Thus, the term "low oxygen condition or conditions" or "low oxygen environment" refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of 0₂ that is 0-60 mmHg 0₂ (0-60 torr 0₂₎ (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg 0₂), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg 0₂, 0.75 mmHg 0₂, 1.25 mmHg 0₂, 2.175 mmHg 0₂, 3.45 mmHg 0₂, 3.75 mmHg 0₂, 4.5 mmHg 0₂, 6.8 mmHg 0₂, 11.35 mmHg 02, 46.3 mmHg 0₂, 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments, "low oxygen" refers to about 60 mmHg 0₂ or less (e.g. , 0 to about 60 mmHg 0₂₎. The term "low oxygen" may also refer to a range of 0₂ levels, amounts, or concentrations between 0-60 mmHg 0₂ (inclusive), e.g., 0-5 mmHg 0₂, < 1.5 mmHg 0₂, 6-10 mmHg, < 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al, Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al, J Clin. Invest., 41(11): 1971- 1980 (1962); Crompton et al, J Exp. Biol., 43: 473-478 (1965); He et al, PNAS (USA), 96: 4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi:

10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorportated by reference herewith in their entireties. In some embodiments, the term "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments, "low oxygen" is meant to refer to the level, amount, or concentration of oxygen (0₂) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table 2 summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (0₂) is expressed as the amount of dissolved oxygen ("DO") which refers to the level of free, non-compound oxygen (0₂) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; lmg/L = 1 ppm), or in micromoles (umole) (1 umole 0₂ = 0.022391 mg/L 0₂). Fondriest Environmental, Inc., "Dissolved Oxygen", Fundamentals of Environmental Measurements, 19 Nov 2013, www.fondriest.com/environmental- measurements/parameters/water-quality/dissolved- oxygen/>. In some embodiments, the term "low oxygen" is meant to refer to a level, amount, or concentration of oxygen (0₂) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g. , 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (0₂) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term "low oxygen" is meant to refer to 40% air saturation or less, e.g. , 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g. , 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g. , 0- 5%, 0.05 - 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0- 10%, 5- 10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term "low oxygen" is meant to refer to 9% 0₂ saturation or less, e.g. , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, 0₂ saturation, including any and all incremental fraction(s) thereof (e.g. , 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of 0₂ saturation levels between 0-9%, inclusive (e.g. , 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0-8%, 5- 7%, 0.3-4.2% 0_2, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. Table 2.

[096] "Non-pathogenic bacteria" refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus,

Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii,

Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al, 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.

[097] "Exogenous environmental condition(s)" or "environmental conditions" refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject

environment. Thus, "exogenous" and "endogenous" may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some

embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease-state, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue- specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.

[098] As used herein, "exogenous environmental conditions" or "environmental conditions" also refers to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the

microorganism. "Exogenous environmental conditions" may also refer to the conditions during growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations. Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium. Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions.

[099] Thus, as used herein, the phrase "exogenous environmental condition" or "exogenous environment signal" refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. An exogenous environmental condition or signal is a condition or signal that is external to or outside of the recombinant bacterial cell of the disclosure. For example, in one embodiment, the exogenous environmental condition is specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS). In some embodiments, exogenous environmental conditions are biological molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.

[0100] In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous

environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). [0101] As used herein, the term "auxotroph" or "auxotrophic" refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient, to support its growth. An "auxotrophic modification" is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient. As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).

[0102] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.

[0103] As used herein, the term "plasmid" or "vector" refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell' s genome.

[0104] As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically- stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

[0105] The term "genetic modification," as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising a bile salt hydrolase enzyme operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.

[0106] As used herein, the term "genetic mutation" refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example,

substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity ( e.g., enzymatic activity) of the gene's polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.

[0107] "Probiotic" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei,

Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et ah, 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et ah, 2012; Cuevas-Ramos et ah, 2010; Olier et ah, 2012; Nougayrede et ah, 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g. , survivability. Nonpathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

[0108] As used herein, the term "auxotroph" or "auxotrophic" refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient, to support its growth. An "auxotrophic modification" is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient. As used herein, the term "essential gene" refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).

[0109] As used herein, the terms "modulate" and "treat" and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment, "modulate" and "treat" refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "modulate" and "treat" refer to inhibiting the progression of a disease, disorder, and/or condition, either physically {e.g. , stabilization of a discernible symptom),

physiologically {e.g. , stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition. As used herein, "prevent" and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.

[0110] Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease. Disorders associated with bile salts, e.g., cardiovascular disease, metabolic disease, liver disease, such as cirrhosis and nonalcoholic steatohepatitis, cancer, and/or C. difficile infection, may be caused by inborn genetic mutations for which there are no known cures. Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection. Treating diseases associated with bile salts may encompass reducing excess levels of bile salts, reducing normal levels of bile salts, or eliminating bile salts, and does not necessarily encompass the elimination of the underlying disease.

[0111] As used herein, the term "bile salt" or "conjugated bile acid" refers to a cholesterol derivative that is synthesized in the liver and consists of a steroid ring component that is conjugated with either glycine (glycochenodeoxycholic acid; GCDCA) or taurine (taurocholic acid; TCA). Examples of bile salts include, but are not limited to, taurocholic acid (TCA) and glycochenodeoxycholic acid (GCDCA). Bile salts are stored in the gallbladder and then subsequently released into the duodenum. Bile salts act as signaling molecules to regulate systemic endocrine functions including triglyceride, cholesterol, and glucose homeostasis, and also facilitate lipid absorption. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDC A).

[0112] As used herein, the term "bile acid" or "unconjugated bile salt" refers to a cholesterol moiety that that consists of a steroid ring that is synthesized in the liver via a classic bile acid biosynthetic pathway wherein cholesterol is converted to 7a- hydroxycholesterol by the cholesterol 7a-hydroxylase enzyme (CYP7A1), or via an alternative pathway carried out by the microsomal enzyme sterol 12-hydroxylase (CYP8B 1) (see Khalid et ah, Liver Res. Open J., 1:32-40 (2015)). The immediate products of each of these pathways are the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA). The term "primary bile acids" refers to, for example, CA and CDCA. CA and CDCA can subsequently be conjugated with taurine or glycine by the bile acid transferase and bile acid coenzyme A synthesas to form the bile acid salts taurocholic acid (TCA) and glycochenodeoxycholic acid (GCDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. In the large intestine, bile acids are amenable to further modification by microbial 7a-dehydroxylating enzymes to yield secondary bile acids, such as deoxycholic acid (DC A) and lithocholic acid (LCA). The term "secondary bile acids" refers to, for example, deoxycholic acid (DC A) and lithocholic acid (LCA).

[0113] As used herein, the term "farnesoid X receptor" or "FXR" refers to a nuclear bile acid receptor that is expressed in liver, intestine, kidneys, and adrenal glands mainly, and also at lower levels in adipose tissue and the heart (see Ho and Steinman, Proc. Natl. Acad. Sci. U.S.A., 113(6): 1600- 1605 (2016)). FXR is the primary receptor for bile acid. The CDCA, DCA and LCA bile acids are the natural ligands that bind to and activate FXR. The bile acid-FXR interaction has been shown to regulate, for example, hepatic inflammation and regeneration, liver injury, bacterial outgrowth and inflammatory responses in the intestinal tract, preservation of intestinal barriers, and inflammation in the central nervous system (see Khalid et al., Liver Res. Open J., 1:32-40 (2015); Ho and Steinman, Proc. Natl. Acad. Sci. U.S.A., 113(6): 1600-1605 (2016); Neuschwander-Tetri et al., (Lancet, 385:956-1065 (2014); and Joyce et al., PNAS, l l l(20):7421-7426 (2014)).

[0114] As used herein, the term "FXR agonist" or "FXR activator" refers to a molecule that activates the farnesoid X receptor (FXR), thereby stimulating FXR activity. As used herein, the FXR agonist can be a naturally occurring molecule, such as a natural FXR ligand, or the FXR agonist can be a molecule that is not naturally produced in vivo. FXR can be stimulated to varying degrees by many bile acids. CDC A is the highest affinity natural ligand for FXR, and stimulates FXR with the highest potency, with an EC50 of about 10 μΜ (see Khalid et al., Liver Res. Open J., 1:32-40 (2015); Ho and Steinman, Proc. Natl. Acad. Sci. U.S.A., 113(6): 1600- 1605 (2016)). CDCA stimulates FXR to a greater degree than LCA and DCA, and LCA and DCA each stimulate FXR to a greater degree than CA. In some embodiments, the FXR agonist can be a naturally occurring ligand such as CDCA, LCA, DCA, or CA. In some embodiments, the FXR agonist can be a molecule that is not naturally produced in a mammal, such as a bile acid analogue, including, e.g., obeticholic acid (OCA), a 6a-ethyl derivative of CDCA (see, e.g., Pellicciari et al., J. Med. Chem., 45:3569-3572 (2002)), GW4064 (see, e.g., Zhang et al., Proc. Natl. Acad. Sci., U.S.A., 103: 1006-1011 (2006)), or WAY-362450 (Zhang et al., J. Hepatol., 51:380-388 (2009)). In some embodiments, the recombinant bacterial cell comprises one FXR agonist. In some embodiments, the recombinant bacterial cell comprises two or more FXR agonists. In some embodiments, the recombinant bacterial cell FXR agonist is CDCA.

[0115] In some embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme. In some embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the glycine or taurine molecules from a bile salt to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDCA). In some embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDCA). In some

embodiments, a recombinant bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the glycine or taurine molecules from a bile salt to produce the primary bile acids cholic acid (CA) or chenodeoxycholic acid (CDCA) in low-oxygen environments, e.g., the gut, wherein the primary bile acid stimulates FXR. In some embodiments, the recombinant bacterial cell is genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the glycine from glycochenodeoxycholic acid (GCDCA) to produce chenodeoxycholic acid (CDCA). In some embodiments, the recombinant bacterial cell is genetically engineered to comprise a heterologous gene encoding a bile salt hydrolase (BSH) enzyme that removes the taurine from taurocholic acid (TCA) to produce cholic acid (CA). In some embodiments, the recombinant bacterial cell is genetically engineered to additionally comprise a 7a-dehydroxylating enzyme to produce deoxycholic acid (DC A) or lithocholic acid (LCA).

[0116] As used herein, the term "7a-dehydroxylating enzyme" or "7a-dehydroxylase" refers to an enzyme that is involved in the biosynthesis of secondary bile acids. In some embodiments, the 7a-dehydroxylating enzyme is necessary for the biosynthesis of the secondary bile acid(s), deoxycholic acid (DCA) and/or lithocholic acid (LCA). DCA and LCA are produced through the action of microbial enzymes in the human large intestines (Ridlon et al, J. Lipid Res., 47(2):241-259 (2006)). In some embodiments, the 7a- dehydroxylating enzyme is a bacterial enzyme. In some embodiments, the 7a- dehydroxylating enzyme is a bai gene. In some embodiments, the 7a-dehydroxylating enzyme is produced by a bacterial bai operon. In some embodiments, the bai operon is from C. scindens. In some embodiments, the bai operon of C. scindens encodes baiB, baiCD, baiE, baiAl, baiA2, baiA3, baiF, baiG, baiH, and/or bail. In some embodiments, the bai operon is from C. hiranonis.

[0117] As used herein, the term "catabolism" refers to the processing, breakdown and/or degradation of a complex molecule, such as a bile salt and/or bile acid, into compounds that are non-toxic or which can be utilized by the bacterial cell. In one embodiment, the term "bile salt catabolism" refers to the processing, breakdown, and/or degradation of bile salts into unconjugated bile acid(s).

[0118] In one embodiment, "abnormal catabolism" refers to any condition(s), disorder(s), disease(s), predisposition(s), and/or genetic mutations(s) that result in increased levels of bile salts. In one embodiment, "abnormal catabolism" refers to an inability and/or decreased capacity of a cell, organ, and/or system to process, degrade, and/or secrete bile salts. In healthy adult humans, 600 mg of bile salts are secreted daily. In one embodiment, said inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts is caused by the decreased endogenous deconjugation of bile salts, e.g., decreased endogenous deconjugation of bile salts into bile acids by the intestinal microbiota in the gut. In one embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts results from a decrease in the number of or activity of intestinal bile salt hydrolase (BSH)-producing microorganisms.

[0119] In one embodiment, a "disease associated with bile salts" or a "disorder associated with bile salts" is a disease or disorder involving the abnormal, e.g., increased, levels of bile salts in a subject. In one embodiment, a "disease associated with bile salts" may also refer to a disease or disorder involving the abnormal levels of bile salts and bile acids in a subject. Alternatively, a disease or disorder associated with bile salts is a disease or disorder wherein a subject exhibits normal levels of bile salts, but wherein the subject would benefit from decreased levels of bile salts. Bile salts function to solubilize dietary fat and enable its absorption into host circulation, and healthy adult humans secrete about 600 mg of bile salts daily through the stool. Thus, decreasing increased levels of bile salts, or normal levels of bile salts, in a subject would result in less uptake of dietary fat, causing the subject' s liver to pull cholesterol from systemic circulation as it attempts to synthesize more. Thus, in one embodiment, a subject having a disease or disorder associated with bile salts secretes about 600 mg of bile salts in their stool daily. In another embodiment, a subject having a disease or disorder associated with bile salts secretes more than 600 mg, 700 mg, 800 mg, 900 mg, or 1 g of bile salts in their stool daily.

[0120] In one embodiment, a disease or disorder associated with bile salts is a cardiovascular disease. In another embodiment, a disease or disorder associated with bile salts is a metabolic disease. In another embodiment, the disease or disorder associated with bile salts is an inflammatory and/or autoimmune disease. In another embodiment, a disease or disorder associated with bile salts is a liver disease, such as cirrhosis, nonalcoholic steatohepatitis (NASH), or progressive familialintrahepatic cholestasis type 2 (PFIC2). In another embodiment, the disease or disorder associated with bile salts is a disease of the central nervous system (CNS), such as an autoimmune disease, a multiple sclerosis, and/or experimental autoimmune encephalomyelitis (EAE). In another embodiment, a disease or disorder associated with bile salts is a cancer, such as a gastrointestinal cancer, hepatocellular carcinoma, or colon cancer. In another embodiment, a disease or disorder associated with bile salts is a C. difficile infection. In another embodiment, a disease or disorder associated with bile salts is inflammatory bowel disease (IBD) or colitis.

[0121] As used herein, the terms "cardiovascular disease" or "cardiovascular disorder" are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body, and encompasses diseases and conditions including, but not limited to hypercholesterolemia, diabetic dyslipidemia, hypertension, arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease. As used herein, a subject having "hypercholesterolemia" may have a total cholesterol of greater than 4 mmol/L, and a low-density lipoprotein cholesterol (LDL) of greater than 3 mmol/L.

[0122] As used herein, the terms "metabolic disease" or "metabolic disorder" refer to diseases caused by lipid and cholesterol metabolic pathways that are regulated by or affected by bile salts and bile acids. For example, cholesterol metabolic diseases and disorders include diabetes (including Type 1 diabetes, Type 2 diabetes, and maturity onset diabetes of the young (MODY)), obesity, weight gain, gallstones, hypertriglyceridemia,

hyperfattyacidemia, and hyperinsulinemia.

[0123] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered bacteria of the disclosure with other components such as a

physiologically suitable carrier and/or excipient.

[0124] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial or viral compound. An adjuvant is included under these phrases.

[0125] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0126] The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., a disorder associated with bile salts. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder associated with bile salts. Methods for diagnosing diseases or disorders associated with bile salts are known in the art (see, for example, U.S. 2007/0116671 and W014/198857, the entire contents of each of which are expressly incorporated herein by reference). A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[0127] As used herein, the term "polypeptide" includes "polypeptide" as well as "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e. , peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, "peptides," "dipeptides," "tripeptides, "oligopeptides," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "dipeptide" refers to a peptide of two linked amino acids. The term "tnpeptide" refers to a peptide of three linked amino acids. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occu ing amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the cureent invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are refened to as folded, and polypeptides, which do not possess a defined three- dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term "peptide" or "polypeptide" may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.

[0128] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms "fragment," "variant," "derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non- conservative amino acid substitutions, deletions or additions.

[0129] Polypeptides also include fusion proteins. As used herein, the term "variant" includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term "fusion protein" refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins. "Derivatives" include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; - Phe, Tyr, Trp, His; and -Asp, Glu.

[0130] As used herein, the term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention.

Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or

substitution(s). These may be naturally occurring variants as well as artificially designed ones.

[0131] As used herein, the term "polypeptide of interest" or "polypeptides of interest", "protein of interest", "proteins of interest", etc., includes any or a plurality of any of the bile salt hydrolase enzymes, 7a-dehydroxylating enzymes, and/or bile salt transporters described herein. As used herein, the term "gene of interest" or "gene sequence of interest" includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more bile salt hydrolase enzymes, bile salt transporters, and/or 7a-dehydroxylating enzymes described herein.

[0132] As used herein the term "linker", "linker peptide" or "peptide linkers" or "linker" refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g. , that link two polypeptide domains. As used herein the term "synthetic" refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.

[0133] As used herein the term "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. The term "codon-optimized" refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A "codon-optimized sequence" refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. In some embodiments, the improvement of transcription and/or translation involves increasing the level of transcription and/or translation. In some embodiments, the improvement of transcription and/or translation involves decreasing the level of transcription and/or translation. In some embodiments, codon optimization is used to fine-tune the levels of expression from a construct of interest, e.g. , bile salt hydrolase enzyme levels. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. [0134] Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, on the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0135] As used herein, the terms "secretion system" or "secretion protein" refers to a native or non-native secretion mechanism capable of secreting or exporting a protein(s) of interest from the microbial, e.g. , bacterial, cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex

e.g. ,HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g. , hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the protein(s) of interest includes a "secretion tag" of either RNA or peptide origin to direct the protein(s) of interest to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the protein(s) of interest from the engineered bacteria. For example, in Type V auto- secretion-mediated secretion the N- terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g. , OmpT cleavage thereby releasing the protein(s) of interest into the extracellular milieu. In some embodiments, the secretion system involves the generation of a "leaky" or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nip I, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g. , selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g. , selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[0136] As used herein, the term "transporter" is meant to refer to a mechanism, e.g. , protein or proteins, for importing a molecule, e.g. , amino acid, toxin, metabolite, substrate, etc. into the microorganism from the extracellular milieu. For example, a bile salt transporter imports a bile salt int the microorganism.

[0137] As used herein, the term "bacteriostatic" or "cytostatic" refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure.

[0138] As used herein, the term "bactericidal" refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.

[0139] As used herein, the term "toxin" refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure. The term "toxin" is intended to include bacteriostatic proteins and bactericidal proteins. The term "toxin" is intended to include, but not limited to, lytic proteins, bacteriocins (e.g. , microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term "anti-toxin" or "antitoxin," as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term antitoxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.

[0140] The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary. [0141] The phrase "and/or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase "and/or" may be used interchangeably with "at least one of or "one or more of the elements in a list.

[0142] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Bacteria

[0143] The genetically engineered microorganisms, or programmed microorganisms, such as genetically engineered bacteria of the disclosure are capable of producing one or more bile salt hydrolase enzymes.

[0144] In certain embodiments, the genetically engineered bacteria are obligate anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are facultative anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are aerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram- positive bacteria and lack LPS. In some embodiments, the genetically engineered bacteria are Gram-negative bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and obligate anaerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some

embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria,

Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55,

Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, Clostridium paraputrificum, Clostridium

pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and Vibrio cholera. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei,

Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus,

Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

[0145] In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.

[0146] In some embodiments, the genetically engineered bacteria are Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram- negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et ah, 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et ah, 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors {e.g., E. coli - hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et ah, 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo

(Rembacken et ah, 1999), to treat inflammatory bowel disease, Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et ah, 2004). It is commonly accepted that E. coli Nissle' s therapeutic efficacy and safety have convincingly been proven (Ukena et a/., 2007).

[0147] In one embodiment, the recombinant bacterial cell does not colonize the subject having the disorder associated with bile salts.

[0148] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria. Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et ah, 2009). In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention.

[0149] In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells.

[0150] In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells. In one aspect, the disclosure provides a recombinant bacterial culture which reduces levels of bile salts in the media of the culture. In one embodiment, the levels of bile salts are reduced by about 50%, about 75%, or about 100% in the media of the cell culture. In another embodiment, the levels of bile salts are reduced by about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold in the media of the cell culture. In one embodiment, the levels of bile salts are reduced below the limit of detection in the media of the cell culture.

[0151] In some embodiments of the above described genetically engineered bacteria, the bacteria comprise gene sequence encoding one or more bile salt hydrolase enzymes. In some embodiments of the above described genetically engineered bacteria, the bacteria comprise gene sequence encoding one or more 7a-dehydroxylase enzymes In some embodiments of the above described genetically engineered bacteria, the bacteria comprise gene sequence encoding one or more bile salt hydrolase enzymes and one or more 7a- dehydroxylase enzymes. In some embodiments, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present on a plasmid in the bacterium. In some embodiments, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present in the bacterial chromosome. In other embodiments, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

[0152] In some embodiments, the genetically engineered bacteria comprising a bile salt hydrolase and/or 7a-dehydroxylase is an auxotroph. In one embodiment, the genetically engineered bacteria comprising a bile salt hydrolase is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[0153] In some embodiments, the genetically engineered bacteria comprising a bile salt hydrolase and/or 7a-dehydroxylase further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more

recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of an promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD- In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.

[0154] In some embodiments, the genetically engineered bacteria is an auxotroph comprising a bile salt hydrolase and/or 7a-dehydroxylase and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.

[0155] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding a bile salt hydrolase and/or 7a-dehydroxylase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

Bile Salt Hydrolase (BSH) Enzymes

[0156] As used herein, the term "bile salt hydrolase" enzyme refers to an enzyme involved in the cleavage of the amino acid sidechain of glycol- or tauro-conjugated bile acids to generate unconjugated bile acids (Figure 2). Bile salt hydrolase (BSH) enzymes are well known to those of skill in the art. For example, bile salt hydrolase activity has been detected in Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Clostridum spp., Bacteroides spp., Methanobrevibacter spp., and Listeria spp. See, for example, Begley et al., Applied and Environmental Microbiology, 72(3): 1729- 1738 (2006); Jones et al., Proc. Natl. Acad. Sci., 105(36): 13580-13585 (2008); Ridlon et al, J. Lipid Res., 47(2):241-259 (2006); and

WO2014/198857, the entire contents of each of which are expressly incorporated herein by reference.

[0157] The bacterial cells described herein comprise a heterologous gene sequence encoding a bile salt hydrolase enzyme. In some embodiments, the bacterial cells described herein comprise gene sequence encoding a bile salt hydrolase enzyme and are capable of deconjugating bile salts into unconjugated bile acids (see Figures 1 and 2). In some embodiments, the bacterial cells described herein are capable of reducing the levels of bile salts in a subject or cell. In some embodiments, the bacterial cells described herein are capable of increasing the levels of bile acids in a subject or cell. In some embodiments, the bacterial cells described herein are capable of decreasing the level of TCA in a subject or cell. In some embodiments, the bacterial cells described herein are capable of decreasing the level of GCDCA. In some embodiments, the bacterial cells described herein are capable of increasing the levels of primary bile acids in a subject or cell. In some embodiments, the bacterial cells described herein are capable of increasing the level of CA in a subject or cell. In some embodiments, the bacterial cells described herein are capable of increasing the level of CDCA in a subject or cell. In some embodiments, the bacterial cells described herein are capable of increasing the levels of CA and CDCA in a subject or cell. In one embodiment, the bile salt hydrolase enzyme increases the rate of bile salt catabolism in the cell. In one embodiment, the bile salt hydrolase enzyme decreases the level of bile salts in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of taurocholic acid (TCA) in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of glycochenodeoxycholic acid (GCDCA) in the cell or in the subject. Methods for measuring the rate of bile salt catabolism and the level of bile salts and bile acids are well known to one of ordinary skill in the art. For example, bile salts and acids may be extracted from a sample, and standard LC/MS methods may be used to determine the rate of bile salt catabolism and/or level of bile salts and bile acids.

[0158] In another embodiment, the bile salt hydrolase enzyme increases the level of bile acids in the cell or in the subject as compared to the level of bile salts in the cell or in the subject. In another embodiment, the bile salt hydrolase enzyme increases the level of cholic acid (CA) in the cell. In another embodiment, the bile salt hydrolase enzyme increases the level of chenodeoxycholic acid (CDCA) in the cell.

[0159] Enzymes involved in the catabolism of bile salts may be expressed or modified in the bacteria of the disclosure in order to enhance catabolism of bile salts.

Specifically, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts into unconjugated bile acids when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts, such as TCA or GCDCA, into CA and CDCA when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme can catabolize bile salts to treat disorders associated with bile salts, including cardiovascular diseases, metabolic diseases, liver disease, such as cirrhosis or NASH, inflammatory and autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancers, and C. difficile infection.

[0160] The farnesoid X receptor (FXR) is a nuclear hormone receptor that acts as a bile acid sensor, regulates bile acid synthesis and secretion, and regulates cholesterol homeostasis (see Wang et al., Molecular Cell, 3:543-553 (1999); Lew et al., J. Biol. Chem., 279(10):8856-8861 (2004), the entire contents of which are expressly incorporated herein by reference). Bile acids are the natural ligands for FXR. Chenodeoxycholate (CDCA), cholate (CA, deoxycholate (DCA), and ursodeoxycholate (UDCA) act as FXR agonists, while lithocholate (LCA) acts as an FXR antagonist (Lew et al, J. Biol. Chem., 279(10):8856-8861 (2004). CDC A is a highly effective activator of FXR {see Wang et ah, Molecular Cell, 3:543-553 (1999)). FXR-bile acid interaction have been shown to affect numerous diseases associate with bile salts and bile acids. FXR-bile acid regulation affects hepatic and intestinal inflammation, atherosclerosis, and inflammation and autoimmune disease in the CNS. For example, CDCA and obeticholic acid (also known as 6-ECDCA, a synthetic 6a-ethyl derivative of CDCA) have each been shown to ameliorate experimental autoimmune encephalomyelitis (EAE) in mice (Ho and Steinman, Proc. Natl. Acad. Sci. U.S.A.,

113(6): 1600-1605 (2016), the entire contents of which are expressly incorporated herein by reference). In various other studies using animal models for fatty liver disease, FXR ligands such as 6-ECDCA have been shown to reduce liver fat and fibrosis. More specifically, FXR- ligand interactions, e.g., with 6-ECDCA, have been found to improve glucose and insulin tolerance and decrease steatohepatitis (Vignozzi et al., Journal of Sexual Medicine, 8:57-77 (2011); Cipriani et ah, J. Lipid Res., 51:771-784 (2010)), decrease hepatic expression of genes involved in fatty acid synthesis and reduce TNF-a and elevated peroxisome- proliferator activated receptor alpha expression, thereby improving NASH phenotype (Carr et al, Pharm. Res., 17(16): 1-16 (2015); Cipriani et al, J. Lipid Res., 51:771-784 (2010)), prevent hepatic stellate cell activation by inhibiting osteopontin production (Fiorucci et ah, Gastroenterology , 127: 1497-1512 (2004)), and prevent fibrosis progression, reverse fibrosis and cirrhosis development and reduce portal hypertension (Carr et ah, Pharm. Res., 17(16): 1- 16 (2015); see also Khalid et ah, Liver Res. Open J., 1:32-40 (2015)). Furthermore, a small number of clinical trial studies have shown that the FXR agonist, 6-ECDCA, can improve insulin sensitivity and decrease the levels of markers for inflammation and fibrosis in patients with type II diabetes and NAFLD, and improve liver histology in patients with non-alcoholic steatohepatitis (Mudaliar et ah, Gastroenterology , 127: 1497-1512 (2013); Neuschwander- Tetri et ah, Lancet, 385:956-1065 (2014), the entire contents of which are expressly incorporated herein by reference).

[0161] Thus, in some embodiments, the recombinant bacteria of the disclosure are capable of activating FXR. Thus, in some embodiments, the recombinant bacteria of the disclosure are capable of activating FXR activity. In some embodiments, the engineered bacteria comprises gene sequence encoding one or more bile salt hydrolase enzyme(s), which enzyme(s)convert bile salts into unconjugated bile acids, the natural ligands of the farnesoid X receptor (FXR), in the cell or subject. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria increase the levels of unconjugated bile acids that are natural ligand agonists of FXR in the cell or subject, thereby increasing FXR activity in the cell or subject. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) that increase the levels of CDCA in the cell or subject, thereby enhancing the CDCA-FXR interaction and increasing FXR activity. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) that increase the levels of CA in the cell or subject, thereby enhancing the CA- FXR interaction and increasing FXR activity. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) that increase the levels of DCA in the cell or subject, thereby enhancing the DCA-FXR interaction and increasing FXR activity. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s) that increase the levels of UDCA in the cell or subject, thereby enhancing the UDCA-FXR interaction and increasing FXR activity.

[0162] In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and reduce intestinal inflammation, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and reduce atherosclerosis, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and reduce inflammation and/or autoimmune disease in the CNS. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and reduce liver fat and fibrosis. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and increase glucose and insulin tolerance. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and decrease steatohepatitis. the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and decrease the hepatic expression of genes involved in fatty acid synthesis and/or reduce TNF-a and/or reduce elevated peroxisome-proliferator activated receptor alpha expression, thereby improving NASH phenotype. In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more bile salt hydrolase enzyme(s), wherein the bacteria produce CDCA and prevent fibrosis progression, and/or decrease fibrosis and/or decrease cirrhosis development and/or reduce portal hypertension.

[0163] In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a bile salt hydrolase enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a bile salt hydrolase enzyme, as well as at least one copy of a gene encoding a bile salt hydrolase enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a bile salt hydrolase enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a bile salt hydrolase enzyme.

[0164] Multiple distinct bile salt hydrolase enzymes are known in the art. In some embodiments, bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a non-bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the bile salt hydrolase enzyme is derived from an organism of the genus or species that includes, but is not limited to, Lactobacillus spp., such as Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus brevis, or Lactobacillus gasseri; Bifidobacterium spp., such as Bifidobacterium longum, Bifidobacterium bifidum, or Bifidobacterium adolescentis; Bacteroides spp., such as Bacteroides fragilis or Bacteroides vlugatus;

Clostridium spp., such as Clostridium perfringens; Listeria spp., such as Listeria

monocytogenes, Enterococcus spp., such as Enterococcus faecium or Enterococcus faecalis; Brucella spp., such as Brucella abortus; Methanobrevibacter spp., such as

Methanobrevibacter smithii, Staphylococcus spp., such as Staphylococcus aureus, Mycobacterium spp., such as Mycobacterium tuberculosis; Salmonella spp., such as

Salmonella enterica; Listeria spp., such as Listeria monocytogenes.

[0165] In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in Lactococcus. When the gene encoding the bile salt hydrolase enzyme is expressed in the recombinant bacterial cells, the bacterial cells catabolize more bile salt than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme may be used to catabolize excess bile salts to treat a disorder associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH, inflammatory and autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancer, and/or C. difficile infection. Alternatively, the genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme may be used to increase the levels of unconjugated bile salts, e.g., bile acids, up to normal levels or to greater than normal levels, to treat a disorder associated with bile salts, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancer, and/or C. difficile infection. The genetically engineered bacteria comprising a heterologous gene encoding a bile salt hydrolase enzyme may be used to increase the levels of the natural ligands or agonists of FXR, thereby stimulating FXR or increasing the activity of FXR to treat a disorder associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH, inflammatory and autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancer, and/or C. difficile infection.

[0166] The present disclosure further comprises genes encoding functional fragments of a bile salt hydrolase enzyme or functional variants of a bile salt hydrolase enzyme. As used herein, the term "functional fragment thereof or "functional variant thereof of a bile salt hydrolase enzyme relates to an element having qualitative biological activity in common with the wild-type bile salt hydrolase enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated bile salt hydrolase enzyme is one which retains essentially the same ability to catabolize bile salts as the bile salt hydrolase enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having bile salt hydrolase enzyme activity may be truncated at the N-terminus or C-terminus and the retention of bile salt hydrolase enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional variant. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional fragment.

[0167] Assays for testing the activity of a bile salt hydrolase enzyme, a bile salt hydrolase enzyme functional variant, or a bile salt hydrolase enzyme functional fragment are well known to one of ordinary skill in the art. For example, bile salt catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous bile salt hydrolase enzyme activity. Bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner, Applied Environ. Microbiol., 55: 11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992), the entire contents of each of which are expressly incorporated herein by reference. Briefly, bacterial cultures that are grown overnight can be spotted onto LB bile agar supplemented with either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile. BSH activity can be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al, PNAS, 105(36): 13580-13585 (2008), the entire contents of which are expressly incorporated herein by reference). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3): 186-202 (2012), the entire contents of which are expressly incorporated herein by reference. Alternatively, a mouse model can be used to assay bile salt and bile acid signatures in vivo (see, for example, Joyce et al, PNAS, l l l(20):7421-7426 (2014), the entire contents of which are expressly incorporated herein by reference).

[0168] As used herein, the term "percent (%) sequence identity" or "percent (%) identity," also including "homology," is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0169] The present disclosure encompasses nucleic acids comprising gene sequence encoding one or more bile salt hydrolase enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding one or more bile salt hydrolase enzyme(s) that comprise amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, He, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val).

[0170] In some embodiments, the gene encoding a bile salt hydrolase enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the bile salt hydrolase enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase enzyme is mutagenized. [0171] In some embodiments, the nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, may comprise gene sequence encoding bile salt hydrolase from various different species of bacteria. For example, in one embodiment, the gene encoding the bile salt hydrolase enzyme is from Lactobacillus spp. In one embodiment, the Lacotbacillus spp. is Lactobacillus plantarum WCFS 1, Lactobacillus plantarum 80, Lactobacillus johnsonii NCC533, Lactobacillus johnsonii 100-100, Lactobacillus acidophilus NCFM ATCC700396, Lactobacillus brevis ATCC 367, Lactobacillus gasseri ATCC 33323, or Lactobacillus acidophilus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from a Bifidobacterium spp. In one embodiment, the Bifidobacterium spp. is Bifidobacterium longum NCC2705, Bifidobacterium longum DJO10A,

Bifidobacterium longum BB536, Bifidobacterium longum SBT2928, Bifidobacterium bifidum ATCC 11863, or Bifidobacterium adolescentis. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Bacteroides spp. In one embodiment, the Bacteroides spp. is Bacteroides fragilis or Bacteroides vlugatus. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Clostridium spp. In one embodiment, the Clostridum spp. is Clostridum perfringens MCV 185 or Clostridum perfringens 13. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from Listeria spp. In one embodiment, the Listeria spp. is Listeria monocytogenes. In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Methanobrevibacter spp. In one embodiment, the Methanobrevibacter spp. is Methanobrevibacter smithii. Other genes encoding bile salt hydrolase enzymes are well-known to one of ordinary skill in the art and described in, for example, Jones et al, PNAS, 105(36): 13580-13585 (2008) and

WO2014/198857.

[0172] In some embodiments, the nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the gene sequence is selected from any of SEQ ID NOs: 1, 3, 15, 17, 19, 21, 23, 25, 27, and 29.

[0173] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: l. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: l. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: l. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: l. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: l. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 1.

[0174] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:3. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:3. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:3. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:3. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:3. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:3.

[0175] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 15. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 15. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 15. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 15. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 15. [0176] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 17. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 17. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 17. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 17. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 17. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 17.

[0177] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 19. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 19. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 19. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 19. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 19. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 19. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 19. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 19.

[0178] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:21. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:21. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:21. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:21. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:21. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:21. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:21. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:21.

[0179] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:23. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:23. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:23. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:23. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:23. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:23. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:23. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:23.

[0180] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:25. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:25. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:25. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:25. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:25. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:25. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:25. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:25. [0181] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:27. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:27. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:27. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:27. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:27. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:27. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:27. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:27.

[0182] In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO:29. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO:29. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO:29. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO:29. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:29. Accordingly, in one embodiment, the bile salt hydrolase gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:29. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO:29. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO:29.

[0183] In some embodiments, the gene encoding the bile salt hydrolase enzyme is directly operably linked to a first promoter. In other embodiments, the gene encoding the bile salt hydrolase enzyme is indirectly operably linked to a first promoter. In some

embodiments, the the gene encoding the bile salt hydrolase enzyme is operably linked to a promoter that it is not naturally linked to in nature. In some embodiments, the the gene encoding the bile salt hydrolase enzyme is indirectly or directly operably linked to an inducible promoter. Thus, in some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is directly or indirectly operably linked to an inducible promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is directly or indirectly operably linked to a promoter that it is not naturally linked to in nature.

[0184] In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the bile salt hydrolase enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is directly or indirectly operably linked to a constitutive promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is directly or indirectly operably linked to a promoter that is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is directly or indirectly operably linked to a promoter that is directly or indirectly induced by low oxygen or anaerobic conditions.

[0185] The gene encoding the bile salt hydrolase enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the bile salt hydrolase is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding a bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example, E. coli comprises a native bile salt hydrolase gene. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is located on a plasmid in the bacterial cell. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is located in the chromosome of the bacterial cell. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a low-copy plasmid. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile salt hydrolase enzyme, thereby increasing the catabolism of bile salts. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is expressed on a low-copy plasmid in the bacterial cell. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt hydrolase enzymes, wherein the bile salt hydrolase gene is expressed on a high-copy plasmid in the bacterial cell.

Bile Acid 7a-dehydroxylating Enzymes

[0186] C. difficile is an intestinal pathogen that is a significant cause of antibiotic- induced diarrhea and is known to increase morbidity and mortality in hospital patients (see Buffie et ah, Nature, 517(7533):205-208 (2015), the entire contents of which are expressly incorporated herein by reference). Bile acid 7a-dehydroxylating intestinal bacteria, such the cluster XlVa Clostridium, C. scindens, have been found to increase resitance to C. difficile infection and reduce weight loss and mortality when administered to mice (Buffie et ah, Nature, 517(7533):205-208 (2015)). It has been shown that mice that were administered bacteria encoding 7-dehydoxysteoid dehydrogenase enzyme exhibited enhanced resitance to C. difficile. Moreover, increased levels of the secondary bile acids deoxycholate (DC A) or lithocholate (LCA) inhibit C. difficile in a dose-dependent manner. [0187] The secondary bile acids DCA and LCA are produced solely by microbial metabolic reations in the large intestine. Enzymes involved in secondary bile acid synthesis are known in the art, but an extremely small fraction of intestinal bacteria carry a complete secondary bile acid synthesis pathway (see, e.g., Buffie et al., Nature, 517(7533):205-208 (2015); Ridlon et al, J. Lipid Res., 47(2):241-259 (2006)). Bile acid 7a-dehydroxylation is carried out by only a few specieis of intestinal Clostridia which harbor a multi-gene bile acid inducible (bai) operon. For example, a bai regulon encoding at least 10 open reading frames has been identified in C. scindens, and a bai operon has also been identified in C. hiramonis (see Ridlon et al., J. Lipid Res., 47(2):241-259 (2006); Kang et al., Biochem Biophys Acta., 1781(1-2): 16-25 (2008), the entire contents of which are expressly incorporated herein by reference). The C. scrindens (bai) operon includes baiB (bile acid CoA ligase), baiCD (3- dehydro-4-CDCA/CA oxidoreductase), baiE (7a-dehydatase), baiF (putative bile acid CoA transferase), baiG (H+ dependent bile acid transporter), baiH (3-dehydro-4-UDCA/7-epiCA oxidoreductase, bail (putative 7 β -dehydratase), and baiA (3a-HSDH) (Ridlon et al., J. Lipid Res., 47(2):241-259 (2006)). The baiCD gene encodes the 7a-hydroxysteroid dehydrogenase enzyme that has been found to be critical to secondary bile acid biosynthesis, and conferred resitance to C. difficile (Buffie et al., Nature, 517(7533):205-208 (2015)).

[0188] A bile acid inducible (bai) operon may be expressed or modified in the recombinant bacterial cells of the disclosure in order to increase the biosynthesis of secondary bile acids in a cell or a subject. Specifically, when the bile acid inducible (bai) operon is expressed by the recombinant bacterial cells, more secondary bile acids

(deoxycholic acid (DCA) and litocholic acid (LCA)) are metabolized compared to

unmodified bacterial cells of the same bacterial subtype under the same conditions. In some embodiments, the recombinant bacterial cells of the disclosure are engineered to contain an entire bai operon. In some embodiments, the bacterial cells comprising gene sequence encoding one or more 7a-dehydroxylating enzyme(s). In some embodiments, the

recombinant bacterial cells comprise gene sequence or a gene cassette encoding a bai operon. In some embodiments, the recombinant bacterial cells of the disclosure are engineered to contain part of a bai operon, e.g., one or more genes from a bai operon. In some

embodiments, the recombinant bacterial cells comprise gene sequence or a gene cassette encoding one or more genes from a bai operon. In some embodiments, the recombinant bacterial cells express an entire bai operon. In some embodiments, the recombinant bacterial cells express one or more genes from a bai operon. In some embodiments, the recombinant bacterial cells are engineered to contain one or more genes from a bai operon. In some embodiments, the recombinant bacterial cells are engineered to express baiCD. In some embodiments, the recombinant bacterial cells comprise gene sequence or a gene cassette encoding baiCD. In some embodiments, the bai operon or one or more bai operon genes are from C. scindens. In some embodiments, the bai operon or one or more bai operon genes are from C.hiramonis. In some embodiments, the recombinant bacteria comprise gene encoding one or more 7a-dehydroxylating enzyme(s), wherein the recombinant bacterial cells increase the biosynthesis of secondary bile acids in a cell or a subject. In some embodiments, the recombinant bacteria comprise gene encoding one or more 7a-dehydroxylating enzyme(s), wherein the recombinant bacterial cells increase the biosynthesis of DCA in a cell or a subject. In some embodiments, the recombinant bacteria comprise gene encoding one or more 7a-dehydroxylating enzyme(s), wherein the recombinant bacterial cells increase the biosynthesis of LCA in a cell or a subject. In some embodiments, the recombinant bacteria comprise gene encoding one or more 7a-dehydroxylating enzyme(s), wherein the

recombinant bacterial cells increase the biosynthesis of DCA and LCA in a cell or a subject.

[0189] In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more 7a-dehydroxylating enzyme(s). In some embodiments, the nucleic acid comprises gene sequence or a gene cassette encoding a bai operon. In some embodiments, the nucleic acid comprises gene sequence containing a portion of a bai operon, e.g., one or more genes from a bai operon. In some embodiments, the nucleic acid comprises gene sequence encoding one or more genes from a bai operon. In some embodiments, the nucleic acid comprises gene sequence encoding baiCD. In some embodiments, the nucleic acid comprises gene sequence encoding one or more bai operon genes from C. scindens. In some embodiments, the nucleic acid somprises gene sequence encoding one or more bai operon genes are from C.hiramonis.

[0190] In some embodiments, the recombinant bacteria comprise gene sequence encoding a bai operon that has at least about 80% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the bai operon has at least about 85% identity with the entire sequence of SEQ ID NO:79. In one embodiment, the bai operon has at least about 90% identity with the entire sequence of SEQ ID NO:79. In one embodiment, the bai operon has at least about 95% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the bai operon has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:79. Accordingly, in one embodiment, the bai operon has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the bai operon comprises the sequence of SEQ ID NO:79. In yet another embodiment the bai operon consists of the sequence of SEQ ID NO:79. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a bai operon that has at least about 80% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the nucleic acid comprises gene sequence encoding a bai operon that has at least about 85% identity with the entire sequence of SEQ ID NO:79. In one

embodiment, the nucleic acid comprises gene sequence encoding a bai operon that has at least about 90% identity with the entire sequence of SEQ ID NO:79. In one embodiment, the nucleic acid comprises gene sequence encoding a bai operon that has at least about 95% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the nucleic acid comprises gene sequence encoding a bai operon that has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:79. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a bai operon that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:79. In another embodiment, the nucleic acid comprises gene sequence encoding a bai operon comprising the sequence of SEQ ID NO:79. In yet another embodiment the nucleic acid comprises gene sequence encoding a bai operon that consists of the sequence of SEQ ID NO:79.

[0191] In some embodiments, the recombinant bacteria comprise gene sequence encoding a bai operon has at least about 80% identity with the entire sequence of SEQ ID NO: 111. In another embodiment, the bai operon has at least about 85% identity with the entire sequence of SEQ ID NO: 111. In one embodiment, the bai operon has at least about 90% identity with the entire sequence of SEQ ID NO: 111. In one embodiment, the bai operon has at least about 95% identity with the entire sequence of SEQ ID NO: 111. In another embodiment, the bai operon has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 111. Accordingly, in one embodiment, the bai operon has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 111. In another embodiment, the bai operon comprises the sequence of SEQ ID NO: 111. In yet another embodiment the bai operon consists of the sequence of SEQ ID NO: 111. [0192] In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a bai operon having at least about 80% identity with the entire sequence of SEQ ID NO: 111. In another embodiment, the nucleic acid comprises gene sequence encoding a bai operon having at least about 85% identity with the entire sequence of SEQ ID NO: 111. In one embodiment, the nucleic acid comprises gene sequence encoding a bai operon having at least about 90% identity with the entire sequence of SEQ ID NO: 111. In one embodiment, the nucleic acid comprises gene sequence encoding a bai operon having at least about 95% identity with the entire sequence of SEQ ID NO: 111. In another

embodiment, the nucleic acid comprises gene sequence encoding a bai operon having at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 111.

Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a bai operon having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 111. In another embodiment, the nucleic acid comprises gene sequence encoding a bai operon comprising the sequence of SEQ ID NO: 111. In yet another embodiment the nucleic acid comprises gene sequence encoding a bai operon consisting of the sequence of SEQ ID NO: 111. A 7a-dehydroxylating enzyme may be expressed or modified in the recombinant bacterial cells of the disclosure in order to increase the biosynthesis of secondary bile acids in a cell or a subject. Specifically, when the 7a- dehydroxylating enzyme is expressed by the recombinant bacterial cells, more primary bile acids are metabolized into deoxycholic acid (DCA) and litocholic acid (LCA) compared to unmodified bacterial cells of the same bacterial subtype under the same conditions. In one embodiment, when a 7a-dehydroxylating enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells metabolize more TCA into DCA, and/or more CDCA into LCA than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a gene encoding a 7a- dehydroxylating enzyme may be used to to increase the levels of DCA and/or LCA to prevent or treat C. difficile infection. In some embodiments, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme. In some embodiments, the recombinant bacteria are capable of increasing the biosynthesis of secondary bile acids in a cell or a subject. In some embodiments, the recombinant bacteria are capable of increasing the biosynthesis of DCA in a cell or a subject. In some embodiments, the recombinant bacteria are capable of increasing the biosynthesis of LCA in a cell or a subject. In some embodiments, the recombinant bacteria are capable of increasing the biosynthesis of DCA and LCA in a cell or a subject. In some embodiments, the recombinant bacteria are capable of metabolizing primary bile acids in a cell or a subject. In some embodiments, the recombinant bacteria are capable of metabolizing CA in a cell or a subject. In some embodiments, the recombinant bacteria are capable of metabolizing CDCA in a cell or a subject. In some embodiments, the recombinant bacteria are capable of metabolizing CA and CDCA in a cell or a subject.

[0193] In one embodiment, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme and increase the rate of DCA and/or LCA production in a cell. In one embodiment, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme and increases the rate of DCA production in the cell. In one embodiment, the recombinant bacteria comprise gene sequence encoding a 7 a- dehydroxylating enzyme and increases the rate of LCA production in the cell. In another embodiment, the recombinant bacteria comprise gene sequence encoding a 7 a- dehydroxylating enzyme and increases the levels of the secondary bile acids DCA and LCA in the cell or in the subject. In another embodiment, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme and increases the levels of the secondary bile acids DCA and LCA in the cell or in the subject as compared to the levels of the primary bile acids CA and CDA in the cell or in the subject. In another embodiment, the 7a- dehydroxylating enzyme increases the level of DCA in the cell. In another embodiment, the 7a-dehydroxylating enzyme increases the level of LCA in the cell.

[0194] In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a 7a-dehydroxylating enzyme. In some embodiments, the disclosure provides a recombinant bacterial cell that comprises a heterologous gene encoding a 7a- dehydroxylating enzyme operably linked to a promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the bacterial cell comprises a gene sequence encoding a 7a-dehydroxylating enzyme from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a 7 a- dehydroxylating enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a 7a-dehydroxylating enzyme, as well as at least one copy of a gene encoding a 7a-dehydroxylating enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a 7a-dehydroxylating enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a 7a- dehydroxylating enzyme.

[0195] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme. In some embodiments, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme operably linked to a promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme from a different organism, e.g., a different species of bacteria. In another embodiment, the nucleic acid comprises more than one copy of a native gene encoding a 7a-dehydroxylating enzyme. In yet another embodiment, the nucleic acid comprises at least one native gene encoding a 7a-dehydroxylating enzyme, as well as at least one copy of a gene encoding a 7a-dehydroxylating enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the nucleic acid comprises at least one, two, three, four, five, or six copies of a gene encoding a 7a-dehydroxylating enzyme. In one embodiment, the nucleic acid comprises multiple copies of a gene or genes encoding a 7a-dehydroxylating enzyme.

[0196] In some embodiments, the 7a-dehydroxylating enzyme is encoded by a gene encoding a 7a-dehydroxylating enzyme derived from a bacterial species. In one

embodiment, the gene encoding the7a-dehydroxylating enzyme is derived from a bacterium of the genus or species that includes, but is not limited to Clostridium spp., such as

Clostridium scindens or Clostridium hiranonis. In one embodiment, the gene encoding the7a-dehydroxylating enzyme is derived from a bacterium of the Clostridium cluster XlVa. In one embodiment, the gene encoding the bile salt hydrolase enzyme is from Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium scindens. In one embodiment, the Clostridium spp. is Clostridium scindens (ATCC35704). In one embodiment, the Clostridium spp. is Clostridium hiranonis.

[0197] In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme derived from a bacterial species. In one embodiment, the gene sequence encoding the7a-dehydroxylating enzyme is derived from a bacterium of the genus or species that includes, but is not limited to Clostridium spp., such as Clostridium scindens or Clostridium hiranonis. In one embodiment, the gene sequence encoding the7a-dehydroxylating enzyme is derived from a bacterium of the Clostridium cluster XlVa. In one embodiment, the gene sequence encoding the bile salt hydrolase enzyme is from Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium scindens. In one embodiment, the Clostridium spp. is Clostridium scindens (ATCC35704). In one embodiment, the Clostridium spp. is Clostridium hiranonis.

[0198] In one embodiment, the bai operon or the gene encoding the 7a- dehydroxylating enzyme has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the bai operon or the gene encoding the 7a-dehydroxylating enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the bai operon or the gene encoding the 7a-dehydroxylating enzyme has been codon-optimized for use in Lactococcus. When the bai operon or the gene encoding the 7a-dehydroxylating enzyme is expressed in the recombinant bacterial cells, the bacterial cells metabolize more primary bile acids {e.g., cholic acid (CA) and chenodeoxycholic acid (CDCA)) into secondary bile acids {e.g., deoxycholic acid (DCA) and litocholic acid (LCA)) than unmodified bacteria of the same bacterial subtype under the same conditions {e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a bai operon or heterologous gene encoding a 7a-dehydroxylating enzyme may be used to produce increased levels of secondary bile acids to prevent or treat C. difficile infection.

[0199] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a bai operon, wherein the gene sequence encoding the bai operon has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the gene sequence encoding the bai operon or the gene sequence encoding the 7a-dehydroxylating enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the gene sequence encoding the bai operon or the gene sequence encoding the 7a-dehydroxylating enzyme has been codon-optimized for use in Lactococcus.

[0200] The present disclosure further comprises genes encoding functional fragments of a 7a-dehydroxylating enzyme or functional variants of a 7a-dehydroxylating enzyme. As used herein, the term "functional fragment thereof or "functional variant thereof of a 7a- dehydroxylating enzyme relates to an element having qualitative biological activity in common with the wild-type 7a-dehydroxylating enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated 7a- dehydroxylating enzyme is one which retains essentially the same ability to metabolize secondary bile salts as the 7a-dehydroxylating enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having 7a-dehydroxylating enzyme activity may be truncated at the N-terminus or C-terminus and the retention of 7a- dehydroxylating enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a 7a-dehydroxylating enzyme functional variant. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a 7a-dehydroxylating enzyme functional fragment.

[0201] The present disclosure provides nucleic acid comprising gene sequence encoding a functional fragment of a 7a-dehydroxylating enzyme or a functional variant of a 7a-dehydroxylating enzyme. In some embodiments, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme that is truncated at the N-terminus. In some embodiments, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme that is truncated at the C-terminus. In some embodiments, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme that is mutanagized.

[0202] In some embodiments, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme that is mutagenized. Mutants exhibiting increased activity are selected; and the mutagenized gene encoding the 7a-dehydroxylating enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome.

[0203] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene having at least about 80% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. Accordingly, in one embodiment, the 7a-dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO:80. In yet another embodiment the 7a-dehydroxylating enzyme gene consists of the sequence of SEQ ID NO:80.

[0204] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence comprises SEQ ID NO:80. In yet another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence consists of SEQ ID NO:80.

[0205] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID NO: 97. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO:97. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO:97. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO:97. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:97. Accordingly, in one embodiment, the 7a-dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:97. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO:97. In yet another embodiment the 7a-dehydroxylating enzyme gene consists of the sequence of SEQ ID NO:97.

[0206] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO:97. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO:97. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO:97. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO:97. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:97. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:97. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence comprises SEQ ID NO:97. In yet another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence consists of SEQ ID NO:97.

[0207] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID NO: 99. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO:99. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO:99. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO:99. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:99. Accordingly, in one embodiment, the 7a-dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:99. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO:99. In yet another embodiment the 7a-dehydroxylating enzyme gene consists of the sequence of SEQ ID NO:99.

[0208] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme gene, wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO:99. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO:99. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO:99. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO:99. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:99. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:99. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence comprises SEQ ID NO:99. In yet another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence consists of SEQ ID NO:99.

[0209] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 101. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 101. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 101. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 101. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 101. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 101. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 101. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 101.

[0210] In one embodiment, the disclosure provides a nucleic acid that comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 101. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 101. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 101. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 101. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 101. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 101. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme, wherein the gene sequence comprises SEQ ID NO: 101. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence consists of SEQ ID NO: 101.

[0211] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. Accordingly, in one embodiment, the 7a-dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO:80. In yet another embodiment the 7a-dehydroxylating enzyme gene consists of the sequence of SEQ ID NO:80.

[0212] In one embodiment, the disclosure provides a nucleic acid that comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO:80. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme, wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO:80. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence comprises SEQ ID NO:80. In yet another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme, wherein the gene sequence consists of SEQ ID NO:80.

[0213] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 103. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 103. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 103. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 103. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 103. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 103. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 103. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 103.

[0214] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 103. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 103. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 103. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 103. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 103. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 103. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO:103. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 103.

[0215] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 105. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 105. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 105. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 105. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 105. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 105. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 105. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 105.

[0216] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 105. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 105. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 105. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 105. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 105. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 105. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 105. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 105.

[0217] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 107. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 107. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 107. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 107. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 107. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 107. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 107. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 107.

[0218] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 107. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 107. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 107. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 107. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 107. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 107. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 107. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 107.

[0219] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 109. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 109. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 109. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 109. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 109. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 109. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 109. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 109.

[0220] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 109. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 109. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 109. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 109. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 109. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 109. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 109. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 109. [0221] In one embodiment, the recombinant bacteria comprises a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 112. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 112. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 112. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 112. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 112. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 112. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 112. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 112.

[0222] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 112. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 112. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 112. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 112. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 112. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 112. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO:l 12. In yet another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 112.

[0223] In one embodiment, the recombinant bacteria comprise a7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 114. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 114. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 114. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 114. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 114. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 114. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 114. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 114.

[0224] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 114. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 114. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 114. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 114. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 114. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 114. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO:l 14. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 114.

[0225] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 116. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 116. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 116. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 116. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 116. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 116. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 116. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 116.

[0226] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 116. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 116. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 116. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 116. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 116. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 116. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO:l 16. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 116.

[0227] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 118. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 118. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 118. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 118. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 118. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 118. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 118. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 118.

[0228] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 118. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 118. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 118. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 118. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 118. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 118. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO:l 18. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 118.

[0229] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 120. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 120. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 120. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 120. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 120. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 120. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 120. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 120.

[0230] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 120. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 120. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 120. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 120. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 120. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 120. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 120. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 120.

[0231] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 122. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 122. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 122. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 122. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 122. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 122. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 122. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 122.

[0232] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 122. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 122. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 122. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 122. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 122. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 122. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 122. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 122.

[0233] In one embodiment, the recombinant bacteria comprise a 7a-dehydroxylating enzyme gene that has at least about 80% identity with the entire sequence of SEQ ID

NO: 124. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 85% identity with the entire sequence of SEQ ID NO: 124. In one embodiment, the 7a- dehydroxylating enzyme gene has at least about 90% identity with the entire sequence of SEQ ID NO: 124. In one embodiment, the 7a-dehydroxylating enzyme gene has at least about 95% identity with the entire sequence of SEQ ID NO: 124. In another embodiment, the 7a-dehydroxylating enzyme gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 124. Accordingly, in one embodiment, the 7a- dehydroxylating enzyme gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 124. In another embodiment, the 7a-dehydroxylating enzyme gene comprises the sequence of SEQ ID NO: 124. In yet another embodiment the 7a- dehydroxylating enzyme gene consists of the sequence of SEQ ID NO: 124.

[0234] In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80% identity with the entire sequence of SEQ ID NO: 124. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 124. In one embodiment, the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 124. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 124. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 124. Accordingly, in one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 124. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a- dehydroxylating enzyme wherein the gene sequence comprises SEQ ID NO: 124. In yet another embodiment the nucleic acid comprises gene sequence encoding a 7 a- dehydroxylating enzyme wherein the gene sequence consists of SEQ ID NO: 124.

[0235] In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s) wherein the gene sequence comprises the nucleotide sequence(s) of any one or more of the following: SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124. In some embodiments, the recombinant bacteria produce one or more polypeptide(s) wherein the one or more polypeptide(s) comprises amino acid sequence(s) selected from any one or more of the following: SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125.

[0236] In one embodiment, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is directly operably linked to a promoter. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is indirectly operably linked to a promoter. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is operably linked to an inducible promoter. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is operably linked to a constitutive promoter. In one embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is operably linked to a promoter that it is not naturally linked to in nature. In one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is directly operably linked to a promoter. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is indirectly operably linked to a promoter. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene seauence is operably linked to a promoter that it is not naturally linked to in nature. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is operably linked to an inducible promoter. In another embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is operably linked to a constitutive promoter. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is operably linked to a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the nucleic acid comprises gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is operably linked to a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions.

[0237] In some embodiments, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is expressed under the control of a constitutive promoter. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene sequence encoding the 7a-dehydroxylating enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous

environmental conditions. In one embodiment, the gene sequence encoding the 7a- dehydroxylating enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the 7a-dehydroxylating enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Other inducible promoters are described in more detail infra.

[0238] In some embodiments, the recombinant bacteria comprise gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequenve is present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene sequence encoding the 7a- dehydroxylating enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the 7a- dehydroxylating enzyme is located in the chromosome of the bacterial cell, and a gene encoding a 7a-dehydroxylating enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the 7a-dehydroxylating enzyme is located on a plasmid in the bacterial cell, and a gene encoding the 7a-dehydroxylating enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the 7a-dehydroxylating enzyme is located in the chromosome of the bacterial cell, and a gene encoding the 7a-dehydroxylating enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example, E. coli comprises a native 7a- dehydroxylating enzyme gene.

[0239] In some embodiments, the gene encoding the 7a-dehydroxylating enzyme is expressed on a low-copy plasmid. In some embodiments, the gene encoding the 7a- dehydroxylating enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the 7a-dehydroxylating enzyme, thereby increasing the production of secondary bile salts.

[0240] In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme wherein the gene sequence is present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene sequence encoding the 7a-dehydroxylating enzyme is located in the chromosome of the bacterial cell. In some embodiments, the gene sequence encoding the 7a-dehydroxylating enzyme is expressed on a low-copy plasmid. In some embodiments, the gene sequence encoding the 7a-dehydroxylating enzyme is expressed on a high-copy plasmid.

[0241] In one embodiment, the recombinant bacteria comprises a gene encoding a 7 a- dehydroxylating enzyme from a different organism, e.g., a different species of bacteria. In one embodiment, the recombinant bacteria comprises at least one gene from a different bacterium encoding a 7a-dehydroxylating enzyme. In one embodiment, the recombinant bacteria comprises more than one copy of at least one gene from a different bacterium encoding a 7a-dehydroxylating enzyme. In one embodiment, the recombinant bacteria comprise two or more genes encoding 7a-dehydroxylating enzyme, wherein the two or more genes are from more than one bacterial strain or species. In one embodiment, the

recombinant bacteria comprises at least one native gene encoding a 7a-dehydroxylating enzyme. In some embodiments, the at least one native gene encoding a 7a-dehydroxylating enzyme is not modified. In another embodiment, the recombinant bacteria comprises more than one copy of at least one native gene encoding a 7a-dehydroxylating enzyme. In yet another embodiment, the bacterial cell comprises a copy of a gene encoding a native 7a- dehydroxylating enzyme, as well as at least one copy of a heterologous gene encoding a 7a- dehydroxylating enzyme from a different bacterial species. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of the heterologous gene encoding a 7a-dehydroxylating enzyme. In one embodiment, the bacterial cell comprises multiple copies of the heterologous gene encoding a 7a-dehydroxylating enzyme.

[0242] In some embodiments, the recombinant bacteria comprise two or more bai operons. In some embodiments, the two or more bai operons are multiple copies of the same bai operon. In some embodiments, the two or more bai operons comprise bai operons from different bacterial species or strains. In some embodiments, the recombinant bacteria comprise at least one copy of a bai operon and at least one heterologous gene encoding a 7a- dehydroxylating enzyme.

[0243] In some embodiments, the recombinant bacteria optionally further comprise a heterologous gene encoding a bile salt hydrolase enzyme and are thus capable of

deconjugating bile salts, as described herein. In some embodiments, the recombinant bacteria comprise at least one heterologous gene encoding 7a-dehydroxylating enzyme and at least one heterologous gene encoding a bile salt hydrolase enzyme. In some embodiments, the recombinant bacteria comprise at least one bai operon and at least one heterologous gene encoding a bile salt hydrolase enzyme. Thus, in some embodiments, the recombinant bacteria have an increased rate of bile salt catabolism. In some embodiments, the

recombinant bacteria have an increased rate of metabolism of bile salts into primary bile acids. In some embodiments, the recombinant bacteria have an increased rate of metabolism of primary bile acids into secondary bile acids. In some embodiments, the recombinant bacteria have increased production of primaty bile acids, in some embodiments, the recombinant bacteria have increased production of secondary bile acids, in some

embodiments, the recombinant bacteria have increased production of primary bile acids and secondary bile acids. In some embodiments, the recombinant bacteria are capable of decreasingthe levels of bile salts in a cell or in a subject. In some embodiments, the recombinant bacteria are capable of decreasing the levels of taurocholic acid (TCA) and/or glycochenodeoxycholic acid (GCDCA) in a cell or in a subject. In some embodiments, the recombinant bacteria are capable of increasing the levels of cholic acid (CA) and/or chenodeoxycholic acid (CDCA) in the cell or subject. In some embodiments, the recombinant bacteria are capable of increasing the levels of deoxycholic acid (DCA) and/or lithocholic acid (LCA) in a cell or subject. In some embodiments, the recombinant bacteria are capable of decreasing the levels of taurocholic acid (TCA) and/or glycochenodeoxycholic acid (GCDCA) in a cell or in a subject, thereby increasing the levels of cholic acid (CA) and/or chenodeoxycholic acid (CDCA) in the cell or subject, and increase the metabolism of CA and/or CDC A into deoxycholic acid (DC A) and/or lithocholic acid (LCA). Thus, the recombinant bacteria comprising a heterologous gene encoding a 7a-dehydroxylating enzyme and a heterologous gene encoding a bile salt hydrolase enzyme may be used to catabolize excess bile salts and metabolize primary bile acids into secondary bile acids to treat a disorder associated with bile salts and bile acids, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH, inflammatory and autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancer, and/or C. difficile infection.

[0244] In some embodiments, the recombinant bacteria comprise gene sequence encoding one or more 7a-dehydroxylating enzyme(s) and one or more bile salt hydrolase enzyme(s) wherein the gene sequence comprises nucleotide sequence(s) selected from one or more of: SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124, and nucleotide sequence(s) selected from one or more of: SEQ ID NOs: 1, 3, 15,

17, 19, 21, 23, 25, 27, and/or 29. In some embodiments, the recombinant bacteria produce one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125, and one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 2, 4, 16,

18, 20, 22, 24, 26, 28, and/or 30. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding one or more 7a-dehydroxylating enzyme(s) and one or more bile salt hydrolase enzyme(s) wherein the gene sequence comprises nucleotide sequence(s) selected from one or more of: SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124, and nucleotide sequence(s) selected from one or more of: SEQ ID NOs: 1, 3, 15, 17, 19, 21, 23, 25, 27, and/or 29. In some embodiments, the nucleic acid comprises gene sequence encoding one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125, and one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 2, 4, 16, 18, 20, 22, 24, 26, 28, and/or 30.

[0245] In some embodiments, the recombinant bacteria optionally further comprise a heterologous gene encoding a bile salt transporter, e.g., a transporter that imports a bile salt and that may be used to import bile salts into the bacteria, as described herein. Thus, in some embodiments, the recombinant bacteria comprise at least one heterologous genes encoding a 7a-dehydroxylating enzyme and at least one heterologous gene encoding a bile salt transporter, in some embodiments, the recombinant bacteria comprise at least one heterologous genes encoding a 7a-dehydroxylating enzyme, at least one gene encoding a bile salt hydrolase, and at least one heterologous gene encoding a bile salt transporter. In some embodiments, the recombinant bacteria comprise at least one bai operon, at least one heterologous gene encoding a bile salt hydrolase, and at least one heterologous gene encoding a bile salt transporter. Thus, in some embodiments, the recombinant bacteria import more bile salts into the cell when the bile salt transporter is expressed, and have also an increased rate of metabolism of primary bile acids into secondary bile acids.

[0246] In some embodiments, the recombinant bacteria comprise one or more of gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124, and one or more of gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 11, 13, and/or 38. In some embodiments, the recombinant bacteria produce one or more of polypeptide(s) having the amino acid sequence(s) of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125, and one or more polypeptide(s) having the amino acid sequence(s) of SEQ ID NOs: 12, 14, and/or 39. In some embodiments, the disclosure provides a nucleic acid comprising one or more gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124, and one or more gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 11, 13, and/or 38. In some embodiments, the disclosure provides a nucleic acid that encodes one or more polypeptide(s) having the amino acid sequence(s) of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125, and one or more polypeptide(s) having the amino acid sequence(s) of SEQ ID NOs: 12, 14, and/or 39. In one embodiment, the bacterial cell comprises at least one heterologous gene encoding a 7a-dehydroxylating enzyme, at least one heterologous gene encoding a bile salt hydrolase enzyme, and at least one heterologous gene encoding a bile salt transporter. Thus, in some embodiments, the recombinant bacteria have an increased rate of bile salt catabolism, an increased rate of metabolism of primary bile acids into secondary bile acids, and import more bile salts into the cell when the transporter of bile salts is expressed.

[0247] For example, in some embodiments, the recombinant bacteria comprise one or more of gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 80, 97, 99, 101, 103, 105, 107, 109, 112, 114, 116, 118, 120, 122, and/or 124, and one or more gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 1, 3, 15, 17, 19, 21, 23, 25, 27, and/or 29, and one or more gene(s) having the nucleotide sequence(s) of SEQ ID NOs: 11, 13, and/or 38. In some embodiments, the recombinant bacteria produce one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 113, 115, 117, 119, 121, 123, and/or 125, and one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 2, 4, 16, 18, 20, 22, 24, 26, 28, and/or 30, and one or more polypeptide(s) having an amino acid sequence(s) selected from SEQ ID NOs: 12, 14, and/or 39.

Transporters of Bile Salts and Bile Acids

[0248] The uptake of bile salts into the Lactobacillus and Bifidobacterium has been found to occur via the bile salt transporters CbsTl and CbsT2 (see, e.g., Elkins et ah, Microbiology, 147(Pt. 12):3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference). The uptake of bile acids into the Neisseria meningitides has been found to occur via the bile acid sodium symporter ASBT {see, e.g., Hu et ah, Nature, 478(7369):408-411 (2011), the contents of which are expressly incorporated herein by reference. Other proteins that mediate the import of bile salts or acids into cells are well known to those of skill in the art. For the purposes of this invention, a bile salt transporter includes bile salt importers and bile acid symporters. However, in one embodiment, a bile salt transporter may be a bile salt importer, only, and not import bile acids. In another embodiment, a bile salt transporter may be a bile acid importer, only, and not import bile salts. In another embodiment, a bile salt transporter may import both bile salts and bile acids.

[0249] Transporters, e.g., bile salt importers or bile acid symporters, may be expressed or modified in the bacteria in order to enhance bile salt or bile acid transport into the cell. For example, when a transporter of bile salts is expressed in the recombinant bacterial cells, the bacterial cells import more bile salts and/or bile acids into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a transporter of bile salts may be used to import bile salts and/or bile acids into the bacteria so that any gene encoding a bile salt hydrolase (BSH) enzyme and/or a 7a- dehydrolase enzyme expressed in the organism can be used to treat disorders associated with bile salts, such as cardiac disease, metabolic disease, liver disease, cancer, and C. difficile infection. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile acid. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt and a bile acid. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt,a heterologous gene encoding a bile salt hydrolase (BSH) enzyme, and/or a heterologous gene encoding a 7a-dehydrolase enzyme.

[0250] Thus, in some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a bile salt and/or bile acid transporter operably linked to a second promoter . In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a bile salt and/or bile acid transporter operably linked to the first promoter, in some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a 7a-dehydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a bile salt and/or bile acid transporter operably linked to a second promoter. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a 7a- dehydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a bile salt and/or bile acid transporter operably linked to the first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme and/or a heterologous gene encoding at least one 7a-dehydrolase enzyme, and a heterologous gene encoding a bile salt and/or bile acid transporter, wherein the gene encoding the at least one bile salt hydrolase enzyme and the gene encoding the at least one 7a-dehydrolase enzyme are operably linked to a first promoter and the heterologous gene encoding the bile salt and/or bile acid transporter is operably linked to a second promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme and/or a heterologous gene encoding at least one 7a-dehydrolase enzyme, and a heterologous gene encoding a bile salt and/or bile acid transporter, wherein the gene encoding the at least one bile salt hydrolase enzyme and the gene encoding the at least one 7a-dehydrolase enzyme and the gene encoding the bile salt and/or bile acid transporter are operably linked to a first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme and/or a heterologous gene encoding at least one 7a-dehydrolase enzyme, and a heterologous gene encoding a bile salt and/or bile acid transporter, wherein the gene encoding the at least one bile salt hydrolase enzyme is operably linked to a first promoter, the gene encoding the at least one 7a- dehydrolase enzyme is operably linked to a second promoter, and the gene encoding the bile salt and/or bile acid transporter is operably linked to a third promoter. In one embodiment, the first promoter is the same copy of the same promoter. In one embodiment, the first promoter is a different copy of the same promoter. In one embodiment, the first promoter and the second promoter are different promoters. In one embodiment, the first promoter, the second promoter, and the third promoters are different promoters. In any of these embodiments, the first promoter, second promoter, and/or third promoter is an inducible promoter. In any of these embodiments, the first promoter, second promoter, and/or third promoter is a constitutive promoter.

[0251] In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a bile salt hydrolase enzyme and/or gene sequence encoding a 7a- dehydroxylating enzyme and/or gene sequence encoding a bile salt and/or bile acid transporter. In some embodiments, any of the gene sequences is present on a plasmid. In some embodiments, any of the gene sequences is present on a chromosome in the bacterial cell. In some embodiments, any of the gene sequences is operably linked to an inducible promoter. In some embodiments, any of the gene sequences is operably linked to a constitutive promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a bile salt hydrolase enzyme operably linked to an inducible promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a bile salt hydrolase enzyme operably linked to a constitutive promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme operably linked to an inducible promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a 7a-dehydroxylating enzyme operably linked to a constitutive promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a a bile salt and/or bile acid transporter operably linked to an inducible promoter. In some embodiments, the disclosure provides a nucleic acid comprising gene sequence encoding a a bile salt and/or bile acid transporter operably linked to a constitutive promoter, and/or gene sequence encoding a bile salt and/or bile acid transporter. In some embodiments, any of the gene sequences and operably linked promoter are present on a plasmid. In some

embodiments, any of the gene sequences and operably linked promoter are present on a chromosome in the bacterial cell. [0252] In one embodiment, the bacterial cell comprises a gene encoding a transporter of a bile salt and/or bile acid from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding transporter of a bile salt and/or bile acid. In some embodiments, the at least one native gene encoding a transporter of a bile salt and/or bile acid is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding a transporter of a bile salt and/or bile acid. In yet another embodiment, the bacterial cell comprises a copy of a gene encoding a native transporter of a bile salt and/or bile acid, as well as at least one copy of a heterologous gene encoding a transporter of a bile salt and/or bile acid from a different bacterial species. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of the heterologous gene encoding a transporter of a bile salt and/or bile acid. In one embodiment, the bacterial cell comprises multiple copies of the heterologous gene encoding a transporter of a bile salt and/or bile acid.

[0253] In some embodiments, the transporterof a bile salt and/or bile acid is encoded by a transporter of a bile salt gene derived from a bacterial genus or species, including but not limited to, Lactobacillus. In some embodiments, the transporterof a bile salt and/or bile acid gene is derived from a bacteria of the species Lactobacillus johnsonni strain 100-100.

[0254] The present disclosure further comprises genes encoding functional fragments of a transporter of a bile salt and/or bile acid or functional variants of a transporter of a bile salt and/or bile acid. As used herein, the term "functional fragment thereof or "functional variant thereof of a transporter of a bile salt and/or bile acid relates to an element having qualitative biological activity in common with the wild-type transporter of a bile salt and/or bile acid from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated transporter of bile salt and/or bile acid protein is one which retains essentially the same ability to import the bile salt and/or bile acid into the bacterial cell as does the transporter protein from which the functional fragment or functional variant was derived. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional fragment of a transporter of a bile salt and/or bile acid. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional variant of a transporter of a bile salt and/or bile acid.

[0255] Assays for testing the activity of a transporter of a bile salt and/or bile acid, a functional variant of a transporter of a bile salt and/or bile acid, or a functional fragment of a transporter of a bile salt and/or bile acid are well known to one of ordinary skill in the art. For example, bile salt and/or bile acid import can be assessed as described in Elkins et ah, Microbiology, 147:3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference.

[0256] In one embodiment the gene(s) encoding the transporter of a bile salt and/or bile acid have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the transporter of a bile salt and/or bile acid have been codon-optimized for use in Escherichia coli.

[0257] The present disclosure also encompasses genes encoding a transporter of a bile salt and/or bile acid comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.

[0258] In some embodiments, the gene encoding a transporter of a bile salt and/or bile acid is mutagenized; mutants exhibiting increased bile salt and/or bile acid transport are selected; and the mutagenized gene encoding a transporter of a bile salt and/or bile acid is isolated and inserted into the bacterial cell. In some embodiments, the gene encoding a transporter of a bile salt and/or bile acid is mutagenized; mutants exhibiting decreased bile salt and/or bile acid transport are selected; and the mutagenized gene encoding a transporter of the bile salt and/or bile acid is isolated and inserted into the bacterial cell. The transporter modifications described herein may be present on a plasmid or chromosome.

[0259] In one embodiment, the bacteria comprise gene sequence encoding one or more bile salt transporters wherein the bile salt importer is CbsTl. In the one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt transporters wherein the bile salt importer is CbsTl. In one embodiment, the cbsTl gene has at least about 80% identity to SEQ ID NO: 11. Accordingly, in one embodiment, the cbsTl gene has at least about 90% identity to SEQ ID NO: 11. Accordingly, in one embodiment, the cbsTl gene has at least about 95% identity to SEQ ID NO: 11. Accordingly, in one embodiment, the cbsTl gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: l l. In another embodiment, the cbsTl gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the cbsTl gene consists of the sequence of SEQ ID NO: 11. [0260] In one embodiment, the bacteria comprise gene sequence encoding one or more bile salt transporters wherein the bile salt importer is CbsT2. In the one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile salt transporters wherein the bile salt importer is CbsT2. In one embodiment, the cbsT2 gene has at least about 80% identity to SEQ ID NO: 13. Accordingly, in one embodiment, the cbsT2 gene has at least about 90% identity to SEQ ID NO: 13. Accordingly, in one embodiment, the cbsT2 gene has at least about 95% identity to SEQ ID NO: 13. Accordingly, in one embodiment, the cbsT2 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13. In another embodiment, the cbsT2 gene comprises the sequence of SEQ ID NO: 13. In yet another embodiment the cbsT2 gene consists of the sequence of SEQ ID NO: 13.

[0261] In one embodiment, the bacteria comprise gene sequence encoding one or more bile acid transporters wherein the bile acid transporter is the bile acid sodium symporter ASBT_NM. In the one embodiment, the disclosure provides a nucleic acid comprising gene sequence encoding one or more bile acid transporters wherein the bile acid transporter is the bile acid sodium symporter ASBT_NM- In one embodiment, the the bile acid sodium symporter ASBT_NM NMB0705 gene of Neisseria meningitides has at least about 80% identity to SEQ ID NO:38. Accordingly, in one embodiment, the NMB0705 gene has at least about 90% identity to SEQ ID NO:38. Accordingly, in one embodiment, the NMB0705 gene has at least about 95% identity to SEQ ID NO:38. Accordingly, in one embodiment, the NMB0705 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:38. In another embodiment, the NMB0705 gene comprises the sequence of SEQ ID NO:38. In yet another embodiment the NMB0705 gene consists of the sequence of SEQ ID NO:38.

[0262] In some embodiments, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt and/or bile acid. In some embodiments, the heterologous gene encoding a transporter of the bile salt and/or bile acid is operably linked to the first promoter. In other embodiments, the heterologous gene encoding a transporter of the bile salt and/or bile acid is operably linked to a second promoter. In one embodiment, the gene encoding a transporter of the bile salt and/or bile acid is directly operably linked to the second promoter. In another embodiment, the gene encoding a transporter of the bile salt and/or bile acid is indirectly operably linked to the second promoter. [0263] In some embodiments, expression of a gene encoding a transporter of a bile salt and/or bile acid is controlled by a different promoter than the promoter that controls expression of the gene encoding the bile salt hydrolase enzyme. In some embodiments, expression of the gene encoding a transporter of a bile salt and/or bile acid is controlled by the same promoter that controls expression of the bile salt hydrolase enzyme. In some embodiments, a gene encoding a transporter of a bile salt and/or bile acid and the bile salt hydrolase enzyme are divergently transcribed from a promoter region. In some

embodiments, expression of each of genes encoding the gene encoding a transporter of a bile salt and/or bile acid and the gene encoding the bile salt hydrolase enzyme is controlled by different promoters.

[0264] In one embodiment, the the gene encoding a transporter of a bile salt and/or bile acid is operably linked to a promoter that is not its natural promoter. In some

embodiments, the gene encoding the transporter of the bile salt and/or bile acid is controlled by its native promoter. In some embodiments, the gene encoding the transporter of the bile salt and/or bile acid is controlled by an inducible promoter. In some embodiments, the gene encoding the transporter of the bile salt and/or bile acid is controlled by a promoter that is stronger than its native promoter. In some embodiments, the gene encoding the transporter of the bile salt and/or bile acid is controlled by a constitutive promoter.

[0265] In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra.

[0266] In one embodiment, the gene encoding a transporter of a bile salt and/or bile acid is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding a transporter of a bile salt and/or bile acid is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt and/or bile acid is located in the chromosome of the bacterial cell, and a copy of a gene encoding a transporter of a bile salt and/or bile acid from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt and/or bile acid is located on a plasmid in the bacterial cell, and a copy of a gene encoding a transporter of a bile salt f and/or bile acid rom a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt and/or bile acid is located in the chromosome of the bacterial cell, and a copy of the gene encoding a transporter of a bile salt and/or bile acid from a different species of bacteria is located in the chromosome of the bacterial cell.

[0267] In some embodiments, the at least one native gene encoding the transporter of a bile salt and/or bile acid in the bacterial cell is not modified, and one or more additional copies of the native transporter of a bile salt and/or bile acid are inserted into the genome. In one embodiment, the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the bile salt hydrolase enzyme, or a constitutive promoter. In alternate embodiments, the at least one native gene encoding the transporter is not modified, and one or more additional copies of the transporter from a different bacterial species is inserted into the genome of the bacterial cell. In one embodiment, the one or more additional copies of the transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the gene encoding the bile salt hydrolase enzyme, or a constitutive promoter.

[0268] In one embodiment, when the transporter of a bile salt and/or bile acid is expressed in the recombinant bacterial cells, the bacterial cells import 10% more bile salt and/or bile acid into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the transporter of a bile salt and/or bile acid is expressed in the recombinant bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more bile salt and/or bile acid into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt and/or bile acid is expressed in the recombinant bacterial cells, the bacterial cells import two-fold more bile salt and/or bile acid into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the transporter of a bile salt and/or bile acid is expressed in the recombinant bacterial cells, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more bile salt and/or bile acid into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Generation of Bacterial Strains with Enhance Ability to Transport Bile Salts and/or Bile Acids

[0269] Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g. , temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population.

[0270] This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite.

[0271] For example, if the biosynthetic pathway for producing an amino acid is disrupted a strain capable of high- affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid - at growth-limiting

concentrations - will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid.

[0272] Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream

metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways. [0273] A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate. Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth.

[0274] Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures.

[0275] Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations "screened" throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 10 11 2 CCD 1. This rate can be accelerated by the addition of chemical mutagens to the cultures - such as N-methyl-N-nitro-N- nitrosoguanidine (NTG) - which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted.

[0276] At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. & Palsson, B. 0. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).

[0277] Similar methods can be used to generate E.coli Nissle mutants that consume bile salts and/or over-produce bile salt hydrolase and/or 7a-dehydroxylating enzyme and/or a bile salt and/or bile acid transporter.

Exporters of Bile Salts and/or Bile Acids

[0278] The export of bile salts and bile acids is mediated by proteins well known to those of skill in the art. For example, the ATP-binding cassette, sub-family B member 11 (ABCB 11, also called BSEP or "bile salt export pump") is responsible for the export of taurochoate and other cholate conjugates from hepatocytes to the bile in mammals, and mutations in this gene have been associated with progressive familial intrahepatic cholestasis type 2 (PFIC2) and hepatocellular carcinoma (see Strautnieks et al., Nature Genetics, 20(3):233-238, 1998; Knisely et al, Hepatology, 44(2):478-486, 2006; and Ho et al, Pharmacogenet. Genomics, 20(l):45-57, 2010; SEQ ID NO:33). In bacteria, Streptococcus thermophilus comprises a bile salt export pump (Msba subfamily ABC transporter ATP- binding protein; accession F8LYG6; SEQ ID NO:35), and Nostoc spp. are known to comprise a bile salt export pump (Asll293; accession Q8YXC2; SEQ ID NO:36). Multiple other bile salt exporters are known in the art.

-I l l- [0279] For the purposes of this invention, a "bile salt exporter" includes bile salt exporters and bile acid exporters. However, in one embodiment, a bile salt exporter may be a bile salt exporter, only, and will not export bile acids. In another embodiment, a bile salt exporter may be a bile acid exporter and will not exporter bile salts. In another embodiment, a bile salt exporter may export both bile salts and bile acids.

[0280] Thus, in one embodiment of the invention, when the recombinant bacterial cell comprises an endogenous bile salt exporter gene, the recombinant bacterial cells may comprise a genetic modification that reduces export of one or more bile salts and/or bile acids from the bacterial cell. In another embodiment, the recombinant bacterial cell comprises a genetic modification that reduces export of one or more bile salts and/or bile acids from the bacterial cell and a heterologous gene encoding a bile salt catabolism enzyme. When the recombinant bacterial cells comprise a genetic modification that reduces export of a bile salt and/or bile acid, the bacterial cells retain more bile salts and/or bile acids in the bacterial cell than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a genetic modification that reduces export of a bile salt may be used to retain more bile salts and/or bile acids in the bacterial cell so that any bile salt catabolism enzyme and/or 7a-dehydrolase enzyme expressed in the organism can catabolize the bile salt(s) and/or bile acid(s) to treat diseases associated with bile salts, including cardiovascular disease. In one embodiment, the recombinant bacteria further comprise a heterologous gene encoding a transporter of one or more bile salts.

[0281] In one embodiment, the recombinant bacterial cell comprises a genetic modification in a gene encoding a bile salt exporter wherein said bile salt exporter comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a bile salt exporter gene disclosed herein. In one embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:35. In another embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO:36. [0282] In one embodiment, the genetic modification reduces export of a bile salt from the bacterial cell. In one embodiment, the bacterial cell is from a bacterial genus or species that includes but is not limited to, Streptococcus thermophilics or Nostoc spp.

[0283] In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in an exporter having reduced activity as compared to a wild-type exporter protein. In one embodiment, the activity of the exporter is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the exporter is reduced at least twofold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in an exporter having no activity, i.e., results in an exporter which cannot export one or more bile salts or bile acids from the bacterial cell.

[0284] It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of the exporter of an amino acid. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; U.S. Pat. No. 6,586,182; U.S. Pat. No. 6,117,679; and Ling, et al, 1999,

"Approaches to DNA mutagenesis: an overview," Anal. Biochem., 254(2): 157-78; Smith, 1985, "In vitro mutagenesis," Ann. Rev. Genet, 19:423-462; Carter, 1986, "Site-directed mutagenesis," Biochem. J., 237: 1-7; and Minshull, et al., 1999, "Protein evolution by molecular breeding," Current Opinion in Chemical Biology, 3:284-290. For example, the lambda red system can be used to knock-out genes in E. coli (see, for example, Datta et ah, Gene, 379: 109-115 (2006)).

[0285] The term "inactivated" as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). The term "inactivated" encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be

accomplished, for example, by gene "knockout," inactivation, mutation {e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs {e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene's coding sequence. The term "knockout" refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome.

[0286] Assays for testing the activity of an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, export of one or more bile salts may be determined using the methods described by Telbisz and Homolya, Expert Opinion Ther. Targets, 1-14, 2015, the entire contents of which are expressly incorporated herein by reference.

[0287] In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in decreased expression of the exporter gene. In one

embodiment, exporter gene expression is reduced by about 50%, 75%, or 100%. In another embodiment, exporter gene expression is reduced about two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation completely inhibits expression of the exporter gene.

[0288] Assays for testing the level of expression of a gene, such as an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, reverse- transcriptase polymerase chain reaction may be used to detect the level of mRNA expression of a gene. Alternatively, Western blots using antibodies directed against a protein may be used to determine the level of expression of the protein.

[0289] In another embodiment, the genetic modification is an overexpression of a repressor of an exporter of one or more bile salts. In one embodiment, the overexpression of the repressor of the exporter is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the exporter is caused by the insertion of an inducible promoter in front of the repressor so that the expression of the repressor can be induced. Inducible promoters are described in more detail herein.

In one embodiment, the recombinant bacterial cells described herein comprise at least one genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise two genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise three genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise four genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise five genetic modifications that reduce export of one or more bile salts from the bacterial cell.

Inducible Promoters

[0290] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.

[0291] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.

[0292] In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the promoter may be tissue-specific.

[0293] In certain embodiments, the bacterial cell comprises a gene encoding a payload, e.g., a bile salt hydrolase and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter, expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In E. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et ah, 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In alternate embodiments, the genetically engineered bacteria comprise a bile salt hydrolase enzyme 7a-dehydrolase enzyme and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter expressed under the control of an alternate oxygen level-dependent promoter, e.g., an anaerobic regulation of arginine deiminiase and nitrate reduction ANR promoter (Ray et ah, 1997), a dissimilatory nitrate respiration regulator DNR promoter (Trunk et ah, 2010). In these embodiments, expression of the bile salt hydrolase enzyme is particularly activated in a low-oxygen or anaerobic environment.

Table 3. FNR Promoter Sequences

ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTT

ATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAA

SEQ ID NO: 7

ACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGT TACGTGGGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT

GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGC

GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTA

CATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAA

SEQ ID NO: 8 ACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACA

AATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTTGA

TTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGATCCCTCT

AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACT

TATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAA

SEQ ID NO: 9 ACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGG

ATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

CAT

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAA

ATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAA

SEQ ID NO: 10 ACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTC

AGGGCAATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTA

ACTTTAAGAAGGAGATATACAT

Table 4. FNR Promoter sequences

CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTA

CAGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGT

TAGGTTTCGTCAGCCGTCACCGTCAGCATAACACCCTGACCTCT

CATTAATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCTTTT

nirB2

CCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCA SEQ ID NO: 43

TTTTGTCTATTTTTTGCACAAACATGAAATATCAGACAATTCCGT

GACTTAAGAAAATTTATACAAATCAGCAATATACCCATTAAGGA

GTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTTGCT

G A ATC GTT A AGGT AGGC GGT A AT AG A A A AG A A ATC G AGGC A A A

Aatgtttgtttaactttaagaaggagatatacat

GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGAC

GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTG

nirB3 CATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAA SEQ ID NO: 44 ACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACA

AATCAGCAATATACCCATTAAGGAGTATATAAAGGTGAATTTGA

TTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGT

A AT AG A A A AG A A ATC G AGGC A A A A

ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTT

ydfZ ATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAA SEQ ID NO: 45 ACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGT

TACGTGGGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT

GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGC

GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTA

CATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAA

nirB+RBS

ACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACA SEQ ID NO: 46

AATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTTGA

TTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGATCCCTCT

AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACT

TATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAA

ydfZ+RBS

ACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGG SEQ ID NO: 47

ATCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

CAT

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAA

ATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAA

fnrSl

ACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTC SEQ ID NO: 48

AGGGCAATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTA

ACTTTAAGAAGGAGATATACAT AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAA ATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAA

fnrS2

ACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTC SEQ ID NO: 49

AGGGCAATATCTCTCTTGGATCCAAAGTGAACTCTAGAAATAAT

TTTGTTTAACTTTAAGAAGGAGATATACAT

TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACC

GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGAC

GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTG

CATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAA

nirB+crp

ACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATACA SEQ ID NO: 50

AATCAGCAATATACCCATTAAGGAGTATATAAAGGTGAATTTGA

TTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGaaatgtgat ctagttcacatttGCGGTAATAGAAAAGAAATCGAGGCAAAAa¾ii¾iitaac tttaagaaggagatatacat

AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAA

ATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAA

fnrS+crp

ACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTC SEQ ID NO: 51

AGGGCAATATCTCTCaaatgtgatctagttcacatttiiigiitoaciitoagaaggagatotoc at

[0294] FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.

[0244] Non-limiting FNR promoter sequences are provided in Table 4 Table 4 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 40, SEQ ID NO: 41, nirB l promoter (SEQ ID NO: 42), nirB2 promoter (SEQ ID NO: 43), nirB3 promoter (SEQ ID NO: 44), ydfZ promoter (SEQ ID NO: 45), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 46), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 47), fnrS, an anaerobically induced small RNA gene (fnrS l promoter SEQ ID NO: 48 or fnrS2 promoter SEQ ID NO: 49), nirB promoter fused to a crp binding site (SEQ ID NO: 50), and fnrS fused to a crp binding site (SEQ ID NO: 51). [0245] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51, or a functional fragment thereof.

[0246] In one embodiment, the FNR responsive promoter comprises SEQ ID NO:6. In another embodiment, the FNR responsive promoter comprises SEQ ID NO:7. In another embodiment, the FNR responsive promoter comprises SEQ ID NO:8. In another

embodiment, the FNR responsive promoter comprises SEQ ID NO:9. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 10.

[0247] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g. , DNR (Trunk et al. , 2010) or ANR (Ray et al. , 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.

[0248] In other embodiments, the one or more gene sequence(s) for producing a payload, e.g. , bile salt hydrolase and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter,are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g. , CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al. , 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Gorke and Stiilke, 2008). In some embodiments, the gene or gene cassette for producing the payload is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload, e.g. , a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g. , an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g. , by adding glucose to growth media in vitro.

[0249] In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen-level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non- native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae ( see, e.g., Isabella et at , 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.

[0250] In some embodiments, the genetically engineered bacteria comprise a wild- type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et ah, (2006). In some embodiments, both the oxygen level-sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.

[0251] In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some

embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.

Inflammation-dependent regulation

RNS -dependent regulation

[0252] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload, e.g. , a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload, e.g. , a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g. , a reactive nitrogen species or RNS promoter.

[0253] As used herein, "reactive nitrogen species" and "RNS" are used

interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO*), peroxynitrite or peroxynitrite anion (ONOO-), nitrogen dioxide (·Ν02), dinitrogen trioxide (N203), peroxynitrous acid

(ONOOH), and nitroperoxycarbonate (ONOOC02-) (unpaired electrons denoted by ·).

Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.

[0254] As used herein, "RNS -inducible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g. , a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme sequence(s), and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, e.g. , any of the bile salt hydrolase and/or 7a-dehydrolase enzymes, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter,described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.

[0255] As used herein, "RNS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g. , a bile salt hydrolase enzyme and/or 7a- dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.

[0256] As used herein, "RNS-repressible regulatory region" refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS- repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.

[0257] As used herein, a "RNS -responsive regulatory region" refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS -responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS -responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 5.

Table 5. Examples of RNS-sensing transcription factors and RNS -responsive genes

[0258] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a bile salt hydrolase enzyme, a bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, thus controlling expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, , and/or 7a-dehydrolase enzyme, such as any of the bile salt hydrolase enzymes, bile saltand/or bile acid transporter , and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzymes provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing

transcription factor binds to and/or activates the regulatory region and drives expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene or genes. Subsequently, when

inflammation is ameliorated, RNS levels are reduced, and production of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is decreased or eliminated.

[0259] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0260] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR "is an NO- responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide" (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011;

Karlinsey et al., 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exportergene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the protein(s) of interest, e.g., bile salt hydrolase enzyme and/or 7a- dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter.

[0261] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) "promotes the expression of the nir, the nor and the nos genes" in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more proteins of interest, e.g., bile salt hydrolase enzymes and/or 7a-dehydrolase enzymes, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.

[0262] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0263] In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is "an Rrf2- type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism" (Isabella et ah, 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et ah, 2009; Dunn et ah, 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked gene or genes and producing the protein(s) of interest.

[0264] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria. [0265] In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0266] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a gene of interest, e.g., a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g. , bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g. , a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter gene or genes is expressed.

[0267] A RNS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and

corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g. , NsrR, and one corresponding regulatory region sequence, e.g. , from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS- sensing transcription factor, e.g. , NsrR, and two or more different corresponding regulatory region sequences, e.g. , from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS -responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et ah, 2009; Dunn et ah, 2010; Vine et al, 2011; Karlinsey et al, 2012).

[0268] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0269] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0270] In some embodiments, the genetically engineered bacteria comprise a RNS- sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g. , NsrR, is deleted or mutated to reduce or eliminate wild-type activity.

[0271] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g. , the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.

[0272] In some embodiments, the genetically engineered bacteria comprise a wild- type gene encoding a RNS-sensing transcription factor, e.g. , the NsrR gene, and a

corresponding regulatory region, e.g. , a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the gene of interest, e.g., a bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS -responsive regulatory region, e.g. , the norB regulatory region, and a corresponding transcription factor, e.g. , NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme, and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter in the presence of RNS.

[0273] In some embodiments, the gene or gene cassette for producing the anti- inflammation and/or gut barrier function enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0274] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the protein(s) of interest and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[0275] In some embodiments, the genetically engineered bacteria of the invention produce at least one bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter, in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g. , counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g. , by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen). [0276] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the bile salt hydrolase enzyme and/or 7a-dehydrolase enzyme and/or bile salt and/or bile acid transporter and/or bile salt and/or bile acid exporter. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.

ROS-dependent regulation

[0277] In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g. , a reactive oxygen species or ROS promoter.

[0278] As used herein, "reactive oxygen species" and "ROS" are used

interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H202), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (·ΟΗ), superoxide or superoxide anion (·02-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical (·02-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO*), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ·). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et ah , 2014).

[0279] As used herein, "ROS -inducible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating

downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g. , a sequence or sequences encoding one or more bile salt hydrolase enzyme(s), bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme(s). For example, in the presence of ROS, a transcription factor, e.g. , OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.

[0280] As used herein, "ROS-derepressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g. , one or more genes encoding one or more bile salt hydrolase enzyme(s), bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme(s). For example, in the presence of ROS, a transcription factor, e.g. , OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.

[0281] As used herein, "ROS-repressible regulatory region" refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS- repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.

[0282] As used herein, a "ROS -responsive regulatory region" refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS -responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS -responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 6.

Table 6. Examples of ROS-sensing transcription factors and ROS-responsive genes

ROS-sensing Primarily capable of Examples of responsive genes, transcription factor: sensing: promoters, and/or regulatory regions:

SoxR •0₂ ^~ soxS

NO

(also capable of

sensing H₂0₂)

RosR H₂0₂ rbtT; tnpl6a; rluCl; tnp5a; mscL;

tnp2d; phoD; tnpl5b; pstA; tnp5b; xylC; gabDl; rluC2; cgtS9; azlC; narKGHJI; rosR

[0283] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, thus controlling expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme relative to ROS levels. For example, the tunable regulatory region is a ROS -inducible regulatory region, and the molecule is a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme; when ROS is present, e.g., in an inflamed tissue, a ROS- sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, thereby producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is decreased or eliminated.

[0284] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.

[0285] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR "functions primarily as a global regulator of the peroxide stress response" and is capable of regulating dozens of genes, e.g., "genes involved in H202 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)" and "OxyS, a small regulatory RNA" (Dubbs et ah, 2012). The genetically engineered bacteria may comprise any suitable ROS -responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et ah, 2001; Dubbs et ah, 2012; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a bile salt hydrolase enzyme, bile salt transporter, and/or 7a-dehydrolase enzyme gene. In the presence of ROS, e.g., H202, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme gene and producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS- inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.

[0286] In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is "activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression" (Koo et ah, 2003). "SoxR is known to respond primarily to superoxide and nitric oxide" (Koo et ah, 2003), and is also capable of responding to H202. The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et ah, 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS- inducible regulatory region from soxS that is operatively linked to a gene, e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene and producing the a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme.

[0287] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.

[0288] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR "binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event," but oxidized OhrR is "unable to bind its DNA target" (Duarte et al., 2010). OhrR is a "transcriptional repressor [that] ... senses both organic peroxides and NaOCl" (Dubbs et al., 2012) and is "weakly activated by H202 but it shows much higher reactivity for organic hydroperoxides" (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012 Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene and producing the a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. [0289] OhrR is a member of the MarR family of ROS-responsive regulators. "Most members of the MarR family are transcriptional repressors and often bind to the -10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding" (Bussmann et ah , 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g. , Dubbs et ah , 2012).

[0290] In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is "a MarR-type transcriptional regulator" that binds to an "18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA" and is "reversibly inhibited by the oxidant H202" (Bussmann et ah , 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to "a putative polyisoprenoid-binding protein (cgl322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgl l50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)" (Bussmann et ah , 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g. , Bussmann et ah , 2010; Table 1). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In the presence of ROS, e.g. , H202, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene and producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. [0291] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.

[0292] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.

[0293] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR "when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho et ah, 2014). PerR is a "global regulator that responds primarily to H202" (Dubbs et ah, 2012) and "interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes" (Marinho et ah, 2014). PerR is capable of binding a regulatory region that "overlaps part of the promoter or is immediately downstream from it" (Dubbs et ah, 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al, 2012; Table 1). [0294] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g. , PerR, and a second repressor, e.g. , TetR, which is operatively linked to a gene or gene cassette, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is expressed.

[0295] A ROS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although "OxyR is primarily thought of as a

transcriptional activator under oxidizing conditions... OxyR can function as either a repressor or activator under both oxidizing and reducing conditions" (Dubbs et ah , 2012), and OxyR "has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)" (Zheng et ah , 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS- responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g. , Zheng et ah , 2001 ; Table 1). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g. , the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by RosR. In addition, "PerR-mediated positive regulation has also been observed...and appears to involve PerR binding to distant upstream sites" (Dubbs et al. , 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by PerR.

[0296] One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, "OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both" (Dubbs et al. , 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g. , OxyR, and one corresponding regulatory region sequence, e.g. , from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g. , OxyR, and two or more different corresponding regulatory region sequences, e.g. , from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g. , OxyR and PerR, and two or more corresponding regulatory region sequences, e.g. , from oxyS and katA, respectively. One ROS -responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.

[0297] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 7. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 52, 53, 54, or 55, or a functional fragment thereof. Table 7. Nucleotide sequences of exemplary OxyR-regulated regulatory regions

[0298] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.

[0299] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.

[0300] In some embodiments, the genetically engineered bacteria comprise a ROS- sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from

Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g. , OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS- sensing transcription factor, e.g. , OxyR, is deleted or mutated to reduce or eliminate wild- type activity.

[0301] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g. , the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS- sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome. [0302] In some embodiments, the genetically engineered bacteria comprise a wild- type gene encoding a ROS-sensing transcription factor, e.g. , the soxR gene, and a

corresponding regulatory region, e.g. , a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS- responsive regulatory region, e.g. , the oxyS regulatory region, and a corresponding transcription factor, e.g. , OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme in the presence of ROS, as compared to the wild- type transcription factor under the same conditions. In some embodiments, both the ROS- sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme in the presence of ROS.

[0303] In some embodiments, the gene or gene cassette for producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.

[0304] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a bile salt hydrolase enzyme(s), bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme(s). In some embodiments, the gene(s) capable of producing a bile salt hydrolase enzyme(s), bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme(s) is present on a plasmid and operatively linked to a ROS- responsive regulatory region. In some embodiments, the gene(s) capable of producing a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is present in a chromosome and operatively linked to a ROS -responsive regulatory region.

[0305] Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more bile salt hydrolase enzymes, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzymes under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS) -dependent promoter, and a corresponding transcription factor.

[0306] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, such that the bile salt hydrolase enzyme, bile salt transporter, and/or 7a-dehydrolase enzyme can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme. In some embodiments, the gene encoding the bile salt hydrolase enzyme, bile salt transporter, and/or 7a-dehydrolase enzyme is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In some embodiments, the gene encoding the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is expressed on a chromosome.

[0307] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g. , circuits producing multiple copies of the same product (e.g. , to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene encoding a particular bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene encoding a particular bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme inserted at three different insertion sites and three copies of the gene encoding a different bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme inserted at three different insertion sites.

[0308] In some embodiments, under conditions where the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15- fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100- fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

[0309] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene(s). Primers specific for the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene(s).

[0310] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene(s). Primers specific for payload the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90- 100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme gene(s).

Other Inducible Promoters

[0311] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an arabinose inducible system. The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub-saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L-arabinose transporter (Zhang et ah, Development and Application of an Arabinose-Inducible

Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ. Microbiol. August 2012 vol. 78 no. 16 5831-5838). Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

[0312] In one embodiment, expression of one or more protein(s) of interest, e.g., one or more bile salt hydrolase enzyme(s), bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme(s), is driven directly or indirectly by one or more arabinose inducible promoter(s).

[0313] In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., arabinose. [0314] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the pay load prior to administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown arerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically.

[0315] In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non- limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s).

[0316] In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. [0317] In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter

[0318] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 56. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 57. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 58.

[0319] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. [0320] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.

[0321] In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., rhamnose

[0322] In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically.

[0323] In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non- limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest, e.g., bile salt hydrolase enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporterand/or 7a-dehydroxylating enzyme, and/or transcriptional regulator(s), e.g.,

FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).

[0324] In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest, from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest, from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0325] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 59.

[0326] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-l- thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydrolyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacl) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli, its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate Lacl, can be used instead of IPTG in a similar manner.

[0327] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s). [0328] In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., IPTG.

[0329] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to

administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.

[0330] In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s). [0331] In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0332] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 60 In some embodiments, the IPTG inducible construct further comprises a gene encoding lacl, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 62. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 63.

[0333] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline -responsive promoters. Gossen M & Bujard H.PNAS, 1992 Jun 15;89(12):5547- 51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline-controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP 16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline- controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system. [0334] In one embodiment, expression of one or more protein(s) of interest, e.g., a bile salt hydrolase enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporterand/or a 7a-dehydroxylating enzyme, is driven directly or indirectly by one or more tetracycline inducible promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a tetracycline inducible promoter.

[0335] In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest, e.g., bile salt hydrolase enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporterand/or 7a-dehydroxylating enzyme and/or transcriptional regulator(s), e.g.,

FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., tetracycline

[0336] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the pay load prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.

[0337] In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non- limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

[0338] In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0339] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 129 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is

divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 129 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 129 in italics (Tet repressor is in italics).

[0340] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et ah, Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the λ promoters can then be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage λ. At temperatures below 37 °C, cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in Fig. 12A. Inducible expression from the ParaBad can be controlled or further fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.

[0341] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). In one embodiment, expression of PAL is driven directly or indirectly by a thermoregulated promoter.

[0342] In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature.

[0343] In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shut off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or

manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.

[0344] In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).

[0345] In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0346] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 64 In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 65. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the

polypeptide encoded by any of the sequences of SEQ ID NO: 65. [0347] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.

[0348] This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. Fig. 13A depicts a schematic of the gene organization of a PssB promoter.

[0349] In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s).

[0350] In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest .In some embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo

administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.

[0351] In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.

[0352] In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dap A or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 117.

Induction of Payloads During Strain Culture

[0353] In some embodiments, it is desirable to pre-induce expression of a payload or protein of interest, e.g., a bile salt hydrolase enzyme, a bile salt transporter, and/or 7a- dehydrolase enzyme, and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term "bacterial culture" or bacterial cell culture" or "culture" refers to bacterial cells or microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term "fermentation" refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like.

[0354] Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. In some embodiments, phenylalanine is added to the media, e.g., to boost cell health. Without wishing to be bound by theory, addition of phenylalanine to the medium may prevent bacteria from catabolizing endogenously produced phenylalanine required for cell growth.

[0355] In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is "wasted", in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites).

[0356] In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, a bile salt tranbile salt and/or bile acid transporter, and/or bile salt and/or bile acid exportersporter, and/or 7a-dehydrolase enzyme, is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is driven from the one or more different promoters.

[0357] In some embodiments, the strains are administered without any pre-induction protocols during strain growth prior to in vivo administration. Anaerobic induction

[0358] In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g. , ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^A8 to 1X10^A11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more FNR promoters.

[0359] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR

promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion,

fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.

[0360] In one embodiment, expression of two or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR

promoter(s) and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), e.g. , ba bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under anaerobic or low oxygen conditions. [0361] Without wishing to be bound by theory, strains that comprise one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut.

[0362] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, under the control of one or more FNR promoter(s) and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and/or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more FNR promoter(s), and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of an FNR promoter and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more thermoregulated promoter(s) described herein.

[0363] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[0364] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions.

[0365] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g. , an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. , a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload, e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Aerobic induction

[0366] In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g. , for 1.5 to 3 hours) until they have reached a certain OD, e.g. , ODs within the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10^A8 to 1X10^A11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.

[0367] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[0368] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.

[0369] In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. [0370] In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.

[0371] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.

[0372] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.

[0373] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one

embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.

[0374] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., one or more genes encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, and /or transcriptional regulator gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s), e.g., one or more genes encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and /or transcriptional regulator gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload, e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more constitutive promoter(s) active under aerobic conditions.

[0375] In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. [0376] In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[0377] In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[0378] In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.

[0379] As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation.

Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.

Microaerobic Induction

[0380] In some embodiments, viability, growth, and activity are optimized by pre- inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to "strike a balance" between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, are driven by a anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^A8 to 1Χ10^Λ11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours.

[0381] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

[0382] In one embodiment, expression of one or more payload(s), , e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR

promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.

[0383] Without wishing to be bound by theory, strains that comprise one or more payload(s), e.g., ba bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and/or transcriptional regulator(s) under the control of an FNR promoter, may allow expression of payload(s) and/or transcriptional regulator(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut.

[0384] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more FNR promoter(s), and one or more payload gene sequence(s), e.g. , gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of an FNR promoter and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more thermoregulated promoter(s) described herein.

[0385] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.

[0386] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.

[0387] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g. , an anaerobic/low oxygen promoter or microaerobic promoter, e.g. , FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of an FNR promoter and one or more payload gene sequence(s), e.g. , gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions.

Induction of Strains using Phasing, Pulsing and/or Cycling

[0388] In some embodiments, cycling, phasing, or pulsing techniques are emplyed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficienty induce and grow the strains prior to in vivo administration. This method is used to "strike a balance" between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g. , for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g. , ODs within the range of 0.1 to 10, indicating a certain density e.g. , ranging from 1X10^A8 to lX10^Al l,and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g., reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.

[0389] In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.

[0390] In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non-limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g., for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1X10^A8 to 1X10^A11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.

[0391] In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. For example, lowering the temperature during culture may improve the proper folding of the payload, e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. In such instances, cells are first grown at a temperature optimal for growth (e.g. , 37 C). In some embodiments, the cells are then induced, e.g. , by a chemical inducer, to express the payload, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload, e.g., PAL.

[0392] In some embodiments, payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, are under the control of different inducible promoters, for example two different chemical inducers. In other embodiments, the payload, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, is induced under low oxygen conditions or microaerobic conditions and a second payload, e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is induced by a chemical inducer.

[0393] In one embodiment, expression of one or more payload(s), e.g. , ba bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR

promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques. [0394] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR

promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), e.g., a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques.

[0395] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of one or more FNR promoter(s) and one or more payload gene sequence(s), e.g. , gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, under the control of one or more FNR promoter(s), and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, and /or transcriptional regulator gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a- dehydrolase enzyme, under the control of an FNR promoter and one or more payload gene sequence(s), e.g., gene(s) encoding a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques.

[0396] Any of the strains described herein can be grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions.

[0397] In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme, is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), e.g. , a bile salt hydrolase enzyme, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.

[0398] In one embodiment, strains may comprise a combination of gene

sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g. , an anaerobic/low oxygen promoter, e.g. , FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen

conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques. Aerobic induction of the FNR promoter

[0399] FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, oxygen bypass system shown and described in Fig. 9A is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of a gene of interest by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in high levels of expression of the gene of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the protein of interest.

[0400] In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non-limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g., from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.

[0401] In some embodiments, a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.

[0402] Sequences useful for expression from inducible promoters are listed in Table

8 Table 8. Inducible promoter construct sequences

AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT

Lac Promoter ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG 60 region CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG

CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA

TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG

TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG

TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT

CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA

CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

CAT

LacO GGAATTGTGAGCGCTCACAATT 61

Lacl (in reverse TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC 62 orientation) TGC ATT A ATG A ATC GGCC A AC GCGC GGGG AG AGGC GGTTT

GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA

GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA

GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA

ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG

ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC

GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT

CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT

TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT

CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG

CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA

TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG

ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG

AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC

AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC

AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC

AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG

CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC

ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG

CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG

AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG

TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC

ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG

GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG

ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT

TCAT

Lacl MKP VTLYD V AE Y AG VS YQT VS R V VNQ AS H VS AKTREKVE A 63 polypeptide AMAELNYIPNRVAQQLAGKQSLLIG VATS S LALHAPS QIVAA sequence IKS RAD QLG AS V V VS M VERS G VE ACKA A VHNLLAQRVS GLI

INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN QIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQ M ALG AMR AITES GLR VG ADIS V VG YDDTED S S C YIPPLTTIK QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA PNTQTASPRALADSLMQLARQVSRLESGQ

Region ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC 64 comprising GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG Temperature CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCG sensitive TGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAAC promoter AACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAAC

TTTAAGAAGGAGATATACAT

mutant cI857 TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT 65 repressor TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG

GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT

ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAA

GTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCTC

AGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGGC

TTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTC

AAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTA

CCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTTA

GGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGG

TACTCATACTCACTTCTAAGTGACGGCTGCATACTAACCGC

TTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAA

ATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCAATGCG

GCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAG

CACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACA

GATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATA

AATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTG

TTAATGGTTTCTTTTTTGTGCTCAT

RBS and leader CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA 66 region CAT

mutant cI857 MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM 67 repressor GMGQS G VG ALFNGIN ALN A YN A ALLTKILK VS VEEFS PS IAR polypeptide EIYEM YE A VS MQPS LRS E YE YP VFS H VQ AGMFS PKLRTFTKG sequence D AERW VS TTKKAS DS AFWLE VEGNS MT APTGS KPS FPDGML

ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP QYPMIPCNESCSVVGKVIASQWPEETFG

Constitutive promoters

[0295] In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.

[0296] In some embodiments, the constitutive promoter is active under in vivo conditions, e.g. , the gut, or in the presence of metabolites associated with certain bile salt diseases, as described herein. In some embodiments, the promoter is active under in vitro conditions, e.g. , various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoter is active under in vivo conditions, e.g. , the gut and/or in the presence of metabolites associated with certain diseases, such as bile salt associated diseases and conditions, as described herein, and under in vitro conditions, e.g. , various cell culture and/or cell production and/or manufacturing conditions, as described herein.

[0297] In some embodiments, the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g. , in vivo and/or in vitro and/or production/manufacturing conditions).

[0298] In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoter is active in low- oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.

[0299] Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables. The strength of the constitutive promoter can be further fine-tuned through the selection of ribosome binding sites of the desired strengths.

[0300] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a Escherichia coli σ70 promoter.

Exemplary E. coli σ70 promoters are listed in Table 8A.

[0301] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a Escherichia coli σ70 promoter.

Exemplary E. coli σ70 promoters are listed in Table 8A.

Table 8A. Constitutive E. coli σ70 promoters

174 family member agctagctcagtcctagggactatgctagc

35 elements SEQ ID NO: optimized (TA) repeat

193 constitutive promoter with

BBa_K137085 31

13 bp between -10 and - tgacaatatatatatatatataatgctagc 35 elements

SEQ ID NO: optimized (TA) repeat

194 constitutive promoter with

BBa_K137086 33

15 bp between -10 and - acaatatatatatatatatataatgctagc 35 elements

SEQ ID NO: optimized (TA) repeat

195 constitutive promoter with

BBa_K 137087 . . . aatatatatatatatatatataatgctagc 35

17 bp between -10 and - 35 elements

SEQ ID NO: optimized (TA) repeat

196 constitutive promoter with

BBa_K137088 . . . tatatatatatatatatatataatgctagc 37

19 bp between -10 and - 35 elements

SEQ ID NO: optimized (TA) repeat

197 constitutive promoter with

BBa_K 137089 . . . tatatatatatatatatatataatgctagc 39

21 bp between -10 and - 35 elements

SEQ ID NO: optimized (A) repeat

198 constitutive promoter with

BBa_K 137090 35

17 bp between -10 and - aaaaaaaaaaaaaaaaaatataatgctagc 35 elements

SEQ ID NO: optimized (A) repeat

199 constitutive promoter with

BBa_K 137091 36

18 bp between -10 and - aaaaaaaaaaaaaaaaaatataatgctagc 35 elements

SEQ ID NO: Anderson Promoter with

BBa_K1585100 78 200 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585101 78 201 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585102 78 202 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585103 78

203 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585104 78 204 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585105 78 205 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585106 78 206 lacl binding site ggaattgtgagcggataacaatttcacaca

SEQ ID NO: Anderson Promoter with

BBa_K1585110 78 207 lacl binding site ggaattgtgagcggataacaatttcacaca

227 J23103 agctagctcagtcctagggattatgctagc

[0302] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a E. coli oS promoters. Exemplary E. coli GS promoters are listed in Table 8B. Table 8B. Constitutive E. coli σ promoters

[0303] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a E. coli σ³² promoters. Exemplary E. coli σ³² promoters are listed in Table 8C.

32

Table 8C. Constitutive E. coli σ promoters

[0304] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a B. subtilis σ^Α promoters. Exemplary B. subtilis σ^Α promoters are listed in Table 8D.

Table 8D. Constitutive B. subtilis σ^Α promoters

aatgggctcgtgttgtacaataaatgtagt

[0305] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to a B. subtilis σΒ promoters. Exemplary B subtilis σΒ promoters are listed in Table 8E.

Table 8E. Constitutive B. subtilis σ promoters

[0306] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to promoters from Salmonella. Exemplary Salmonella promoters are listed in Table 8F.

Table 8F. Constitutive promoters from miscellaneous prokaryotes

[0307] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to promoters from bacteriophage T7. Exemplary promoters from bacteriophage T7 are listed in Table 8G. Table 8G. Constitutive promoters from bacteriophage T7

[0308] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to promoters bacteriophage SP6. Exemplary promoters from bacteriophage SP6 are listed in Table 8H. Table 8H. Constitutive promoters from bacteriophage SP6

[0309] In some embodiments, the gene sequence(s) encoding a bile salt hydrolase, and/or 7a-dehydroxylating enzyme and/or bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter is operably linked to promoters from yeast. Exemplary promoters from yeast are listed in Table 81.

Table 81. Constitutive promoters from yeast

SEQ ID NO: 291 Yeast CLB 1 promoter

BBa M31201 region, G2/M cell cycle 500 accatcaaaggaagctttaatcttctcata specific

[0310] In some embodiments, the gene sequence(s) encoding a detoxification molecule, anti-inflammatory molecule, and/or gut-barrier-enhancing molecule is operably linked to promoters from eukaryotes. Exemplary promoters from eukaryotes are listed in Table 8J.

Table 8J. Constitutive promoters from miscellaneous eukaryotes

Other exemplary promoters are listed in Table 8K.

Table 8K. Other Constitutive Promoters

SEQ ID PSYN231 ggaaaatttttttaaaaaaaaaacTT UP element at 5' end; consensus -10 NO: 298 19 GACAGCTAGCTCAGTC region is TATAAT; the consensus -35 is

CTTGGTATAATGCTAG TTGACA; the extended -10 region is CACGAA generally TGNTATAAT

(TGGTATAAT in this sequence)

[0312] In some embodiments, the constitutive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ

ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ

ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ

ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ

ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ

ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ

ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ

ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ

ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ

ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ

ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ

ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ

ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ

ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ

ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ

ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ

ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ

ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ

ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ

ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ

ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ

ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ

ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ

ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ

ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ

ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ

ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, and/or SEQ ID NO: 298.

Essential Genes and Auxotrophs

[0403] As used herein, the term "essential gene" refers to a gene that is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et ai, Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).

[0404] An "essential gene" may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.

[0405] An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient (see Figures 9 and 10). In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thy A. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil, as long as the corresponding wild-type gene product is not produced in the bacteria.

[0406] Table 9 lists exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis. Table 9. Non-limiting Examples of Bacterial Genes Useful for Generation of an

Auxotroph

[0407] Table 10 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.

Table 10. Survival of amino acid auxotrophs in the mouse gut

pheA Phenylalanine Present Present Present proA Proline Present Present Absent

serA Serine Present Present Present

thrC Threonine Present Present Present

trpC Tryptophan Present Present Present

tyrA Tyrosine Present Present Present

ilvD Valine/Isoleucin Present Present Absent

e/Leucine

thyA Thiamine Present Absent Absent

uraA Uracil Present Absent Absent

flhD FlhD Present Present Present

[0408] For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thymidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0409] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro, or in the presence of high DAP levels found naturally in the human gut in vivo. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).

[0410] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et ah, 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g. , by adding uracil to growth media in vitro, or in the presence of high uracil levels found naturally in the human gut in vivo. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product {e.g., outside of the gut).

[0411] In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non- auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph.

Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[0412] Other examples of essential genes include, but are not limited to, yhbV, yagG, hemB, secD, secF, ribD, ribE, ML, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, UgA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, Igt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, IpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, IpxD, fabZ, IpxA, IpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, IpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.

[0413] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson "Synthetic

Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, "ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).

[0414] .In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnciN, tyrS, metG, and adk. In some embodiments, the essential gene is dnciN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnciN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some

embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A, and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A, and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I, and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I, and L6G.

[0415] In some embodiments, the genetically engineered bacterium is

complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole- 3 -butyric acid, indole-3- acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2- aminobenzothiazole, indole- 3 -butyric acid, indole-3-acetic acid, or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole, or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2- aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole.

[0416] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).

[0417] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system shown in Fig 19...

[0418] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill switch circuitry, such as any of the kill switch components and systems described herein. For example, the genetically engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synth Biol, 2015;4(3):307-316, the entire contents of which are expressly incorporated herein by reference). In some

embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill switch circuitry, such as any of the kill switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et ah, 2015). In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the bile salt hydrolase enzyme.

[0419] In one embodiment, a genetically engineered bacterium, comprises one or more biosafety constructs integrated into the bacterial chromosome in combination with one or more biosafety plasmid(s). In some embodiments, the plasmid comprises a conditional origin of replication (COR), for which the plasmid replication initiator protein is provided in trans, i.e., is encoded by the chromosomally integrated biosafety construct. In some embodiments, the chromosomally integrated construct is further introduced into the host such that an auxotrophy results {e.g., dapA or thyA auxotrophy), which in turn is complemented by a gene product expressed from the biosafety plasmid construct. In some embodiments, the biosafety plasmid further encodes a broad- spectrum toxin {e.g., Kis), while the integrated biosafety construct encodes an anti-toxin {e.g., anti-Kis), permitting propagation of the plasmid in the bacterial cell containing both constructs. Without wishing to be bound by theory, this mechanism functions to select against plasmid spread by making the plasmid DNA itself disadvantageous to maintain by a wild-type bacterium. A non-limiting example of such a biosafety system is shown in Fig. 26A, Fig. 26B, Fig. 26C, and Fig. 26D.

[0420] In some embodiments, the genetically engineered bacteria comprise a chromosomally inserted biosafety construct nucleic acid sequence (to be combined with a plasmid based biosafety construct) that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 81, 82, 83, 84, 85, or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a chromosomally inserted biosafety construct nucleic acid sequence (to be combined with a plasmid based biosafety construct) encoding a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of SEQ ID NO: 86, 87, 88, or a functional fragment thereof.

[0421] In some embodiments, the genetically engineered bacteria comprise a chromosome based biosafety construct nucleic acid sequence (to be combined with a plasmid based biosafety construct) that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 89, 90, 91, 92, 93, 94 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a chromosome based biosafety construct nucleic acid sequence (to be combined with a plasmid based biosafety construct) encoding a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence encoded by the DNA sequence of SEQ ID NO: 89, 90, 91, 92, 93, 94 or a functional fragment thereof.

[0422] In some embodiments, the genetically engineered bacteria comprise a plasmid based biosafety construct nucleic acid sequence (to be combined with a chromosome based biosafety construct) that comprises a construct for bile salt hydrolase, bile salt and/or bile acid transporter, and/or bile salt and/or bile acid exporter, and/or 7a-dehydrolase enzyme.

Genetic Regulatory Circuits

[0313] In some embodiments, the genetically engineered bacteria comprise multi- layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811, incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that produce a bile salt hydrolase enzyme or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest.

[0314] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a bile salt hydrolase enzyme, 7a- dehydrolase enzyme, and/or 7a-dehydrolase enzyme and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to an FNR-responsive promoter; a second gene or gene cassette for producing a bile salt hydrolase enzyme, 7a- dehydrolase enzyme, and/or 7a-dehydrolase enzyme, wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression.

[0315] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a a bile salt hydrolase enzyme, 7a- dehydrolase enzyme, and/or 7a-dehydrolase enzyme and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to an FNR-responsive promoter; a second gene or gene cassette for producing a a bile salt hydrolase enzyme, 7a- dehydrolase enzyme, and/or 7a-dehydrolase enzyme operably linked to a Tet regulatory region (TetO); and a third gene encoding an mf-lon degradation signal linked to a Tet repressor (TetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the TetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the TetR, and the a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme is expressed.

[0316] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.

[0317] Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191).

[0318] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to an FNR- responsive promoter, and a second gene or gene cassette for producing a payload. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.

[0319] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to an FNR-responsive promoter; a second gene or gene cassette for producing a payload, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed. [0320] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to an FNR-responsive promoter, and a second gene or gene cassette for producing a payload operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR- responsive promoter, the recombinase is not expressed, the payload remains in the 3' to 5' orientation, and no functional payload is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5' to 3' orientation, and a functional payload is produced.

[0321] In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a payload and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to an FNR-responsive promoter; a second gene or gene cassette for producing a payload operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload. The third gene encoding the T7 polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5' to 3'). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5' to 3' orientation, and the payload is expressed.

[0322] Synthetic gene circuits expressed on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et ah , 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a bile salt hydrolase enzyme and further comprise a toxin- anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et ah, 2015). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et ah, 2015).

Kill Switches

[0423] In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935 and 62/263,329 incorporated herein by reference in their entireties). The kill switch is intended to actively kill genetically engineered bacteria in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.

[0435] Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of the bile salt hydrolase enzyme. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the bile salt hydrolase enzyme. Alternatively, the bacteria may be engineered to die after the bacteria have spread outside of a disease site, e.g., the gut. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the blood or stool of the subject). Examples of such toxins that can be used in kill switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g. , Gardner et al. , 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl β-D- l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al. , 2010). In some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of the bile salt hydrolase enzyme. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the bile salt hydrolase enzyme.

[0436] Kill switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g. , the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.

[0437] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low-oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill switch systems, once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable. [0438] In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[0439] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.

[0440] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.

[0441] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.

[0442] In one embodiment, the first recombinase further flips an inverted

heterologous gene encoding a second excision enzyme. In one embodiment, the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.

[0443] In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.

[0444] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: Bxbl, PhiC31, TP901, Bxbl, PhiC31, TP901, HK022, HPl, R4, Intl, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, IntlO, Intl l, Intl2, Intl3, Intl4, Intl5, Intl6, Intl7, Intl8, Intl9, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof. [0445] In the above-described kill switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill switch circuitry, a toxin may be repressed in the presence of an environmental factor (i.e., not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) are shown in Figs. 20-23. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter (ParaBAD). In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example TetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the TetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.

[0446] Thus, in some embodiments, in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more

heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an anti-toxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.

[0447] Arabinose inducible promoters are known in the art, including Para, ParaB, Parac, and ParaBAD. In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the Parac promoter and the ParaBAD promoter operate as a bidirectional promoter, with the ParaBAD promoter controlling expression of a heterologous gene(s) in one direction, and the Parac (in close proximity to, and on the opposite strand from the ParaBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.

[0448] In one exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contain a kill switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding a tetracycline repressor protein (TetR), a Parac promoter operably linked to a heterologous gene encoding the AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the TetR protein. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the TetR protein, which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the the ParaBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.

[0449] In one embodiment of the disclosure, the genetically engineered bacterium further comprises an anti-toxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the anti-toxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.

[0450] In another embodiment of the disclosure, the genetically engineered bacterium further comprises an anti-toxin under the control of the ParaBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.

[0451] In another exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contain a kill switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a Parac promoter operably linked to a heterologous gene encoding the AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill switch system described directly above. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the

TetR/toxin/anti-toxin kill switch system described directly above.

[0452] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.

[0453] In some embodiments, the engineered bacteria of the present disclosure that are capable of producing an bile salt hydrolase enzyme further comprise the gene(s) encoding the components of any of the above-described kill switch circuits.

[0454] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B 17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin El, colicin K, colicin N, colicin U, colicin B, colicin la, colicin lb, colicin 5, colicinlO, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.

[0455] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNAl, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccECTD, MccF, Cai, ImmEl, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, ImlO, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.

[0456] In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.

[0457] In some embodiments, the engineered bacteria provided herein are capable of producing an bile salt hydrolase enzyme, wherein the gene or gene cassette for producing the bile salt hydrolase enzyme is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter.

[0458] In some embodiments, the genetically engineered bacteria of the present disclosure is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a AthyA and AdapA auxotroph.

[0459] In some embodiments, the genetically engineered bacteria of the present disclosure further comprises a kill switch circuit, such as any of the kill switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD- In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin.

[0460] In some instances, basal or leaky expression from an inducible promoter may result in the activation of the kill switch, thereby creating strong selective pressure for one or more mutations that disable the switch and thus the ability to kill the cell. In some embodiments, an environmental factor, e.g. arabinose, is present during manufacturing, and activates the production of a repressor that shuts down toxin production. Mutations in this circuit, with the exception of the toxin gene itself, will result in death with reduced chance for negative selection. When the environmental factor is absent, the repressor stops being made, and the toxin is produced. When the toxin concentration overcomes that of the antitoxin, the cell dies. In some embodiments, variations in the promoter and ribosome binding sequences of the antitoxin and the toxin allow for tuning of the circuit to produce variations in the timing of cell death. In alternate embodiments, the circuit comprises recombinases that are repressed by tetR and produced in the absence of tetR. These recombinases are capable of flipping the toxin gene or its promoter into the active configuration, thereby resulting in toxin production.

[0461] Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term. In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest, e.g. , a bile salt hydrolase enzyme, over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a bile salt hydrolase enzyme and further comprise a toxin- antitoxin system that simultaneously produces a toxin (hok) and a short-lived antitoxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et ah , 2015; Fig. 24). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et ah , 2015). [0462] In some embodiments, the genetically engineered bacteria of the present disclosure is an auxotroph comprising a gene encoding a bile salt hydrolase enzyme and further comprises a kill switch circuit, such as any of the kill switch circuits described herein.

[0463] In some embodiments of the above described genetically engineered bacteria, the gene encoding the bile salt hydrolase enzyme is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding the bile salt hydrolase enzyme is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.

Secretion

[0464] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting a molecule, e.g., a bile salt hydrolase enzyme, 7a-dehydrolase enzyme, and/or 7a-dehydrolase enzyme, from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.

[0465] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane- spanning secretion system. Membrane- spanning secretion systems include, but are not limited to, the type I secretion system (TISS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation- division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007;

Collinson et al, 2015; Costa et al, 2015; Reeves et al, 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in FIG. 27, FIG. 28, FIG. 29, FIG. 30, and FIG. 31 Mycobacteria, which have a Gram-negativelike cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et ah, 2003). With the exception of the T2SS, double membrane- spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al, 2015).

[0467] In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type Ill-like secretion system (T3SS) from Shigella,

Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest, e.g., a bile sale hydrolase enzyme and/or a 7a-dehydroxylating enzyme, from the bacterial cytoplasm. In some embodiments, the secreted molecule, , e.g., a bile sale hydrolase enzyme and/or a 7a-dehydroxylating enzyme, comprises a type III secretion sequence that allows the molecule of interest, e.g., a bile sale hydrolase enzyme and/or a 7a- dehydroxylating enzyme, be secreted from the bacteria.

[0468] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest, e.g., a bile sale hydrolase enzyme and/or a 7a-dehydroxylating enzyme. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173-bp region, is fused to the gene encoding the polypeptide of interest can be used to secrete heterologous polypeptides (See, e.g., Majander et ah, Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005

Apr;23(4):475-81). In some cases, the untranslated region from the fliC loci, may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et ah, Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437- 7438).

[0469] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g. , therapeutic peptide. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in FIG. 28 a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex ('Beta-barrel assembly machinery') where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane- associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide, e.g., a bile sale hydrolase enzyme and/or a 7a-dehydroxylating enzyme, comprises an N-terminal secretion signal, a linker, and beta- domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.

[0470] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g. , therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. FIG. 29shows the alpha-hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC , an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C- terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide. [0471] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane- spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g. , the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii,

Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et ah, 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest, e.g. , a bile sale hydrolase enzyme and/or a 7a-dehydroxylating enzyme, from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different pay loads.

[0472] In order to translocate a protein, e.g. , therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g. , therapeutic polypeptides) - particularly those of eukaryotic origin - contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.

[0473] One way to secrete properly folded proteins in gram-negative bacteria- particularly those requiring disulphide bonds - is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These "leaky" gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a "leaky" or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at -500,000 copies per cell and functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan. 1. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes, in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.

[0474] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g. , selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl, under the control of an inducible promoter. For example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., over expression of colicins or the third topological domain of TolA, which peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.

[0475] The Table 11 and Table 12 below lists secretion systems for Gram positive bacteria and Gram negative bacteria.

Table 11 Secretion systems for gram positive bacteria

Table 12. Secretion Systems for Gram negative bacteria

None (IISP and 1.B.2

IISP) 2

OmpIP Outer 1.B.3 + + >4 None

membrane 3 (mitochon ?

insertion dria;

porin chloroplast

s)

[0476] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006 "Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently", the contents of which is herein incorporated by reference in its entirety.

[0477] Any of the secretion systems described herein may according to the disclosure be employed to secrete the protein(s) of interest. Non-limiting examples of the protein(s) of interest includes bile salt hydrolase enzyme(s), bile salt transporter(s), and/or 7a-dehydrolase enzyme(s), as described herein. These proteins may be mutated to increase stability, resistance to protease digestion, and/or activity.

Table 13. Comparison of Secretion systems for secretion of polypeptide from engineered bacteria

[0477] In some embodiments, the protein(s) of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a protein(s) of interest, such as a bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is assembled behind a fliC-5'UTR (e.g. , 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the protein(s) of interest is secreted using components of the flagellar type III secretion system, and is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g. , FNR-inducible promoter), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g. , arabinose is used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g. , a medium copy plasmid). In some embodiments, the protein(s) of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g. , the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.

[0478] In some embodiments, the protein(s) of interest, e.g. , bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the protein(s) of interest, e.g. , bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the protein(s) of interest is fused to a Tat-dependent secretion signal. Exemplary Tat-dependent tags include TorA, FdnG, and DmsA. In some embodiments, expression of the secretion-tagged protein(s) is driven by a tet promoter or an inducible promoter, such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter), or by promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g. , arabinose. In some embodiments, the secretion-tagged protein(s) of interest, e.g. , bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is expressed from a plasmid (e.g. , a medium copy plasmid). In other embodiments, the protein(s) of interest, e.g. , bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is expressed from a construct which is integrated into the bacterial chromosome, e.g. , at one or more of the integration sites shown in FIG. 14. In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include lpp, pal, tolA, and/or nlpl. In some embodiments, lpp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, tolA is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non-limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.

[0479] In some embodiments, the protein(s) of interest, e.g., bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodimetns, the protein(s) of interest is expressed as a fusion protein with the native Nissle auto-secreter E. coli _01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest.

[0480] In some embodiments, the protein(s) of interest, e.g., bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is secreted via Type I Hemolysin Secretion. In one embodiment, protein(s) of interest, e.g., bile salt hydrolase(s) and/or 7a-dehydroxylating enzyme(s), is expressed as fusion protein with the 53 amino acids of the C terminus of alpha- hemolysin (hlyA) of E. coli CFT073.

Isolated Plasmids

[0466] In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a bile salt hydrolase enzyme operably linked to a first inducible promoter. In another embodiment, the disclosure provides an isolated plasmid comprising a second nucleic acid encoding at least one additional bile salt hydrolase enzyme. In one embodiment, the first nucleic acid and the second nucleic acid are operably linked to the first promoter. In another embodiment, the second nucleic acid is operably linked to a second inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In another embodiment, the first inducible promoter and the second inducible promoter are different inducible promoters. In one embodiment, the first promoter, the second promoter, or the first promoter and the second promoter, are each directly or indirectly induced by low-oxygen or anaerobic conditions. In another embodiment, the first promoter, the second promoter, or the first promoter and the second promoter, are each a fumarate and nitrate reduction regulator (FNR) responsive promoter. In another embodiment, the first promoter, the second promoter, or the first promoter and second promoter are each a ROS-inducible regulatory region. In another embodiment, the first promoter, the second promoter, or the first promoter and second promoter are each a RNS-inducible regulatory region.

[0467] In one embodiment, the heterologous gene encoding the bile salt hydrolase is operably linked to a constitutive promoter. In one embodiment, the constitutive promoter is a lac promoter. In another embodiment, the constitutive promoter is a tet promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ 32 promoter. In another embodiment, the constitutive promoter is a constitutive Escherichia coli σ 70 promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis σ^Α promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis σ promoter. In another embodiment, the constitutive promoter is a Salmonella promoter. In another embodiment, the constitutive promoter is a bacteriophage T7 promoter. In another embodiment, the constitutive promoter is and a bacteriophage SP6 promoter. In any of the above-described embodiments, the plasmid further comprises a heterologous gene encoding a transporter of a bile salt and/or a kill switch construct, either or both of which may be operably linked to a constitutive promoter or an inducible promoter.

[0468] In one embodiment, the isolated plasmid comprises at least one heterologous bile salt hydrolase enzyme gene operably linked to a first inducible promoter; a heterologous gene encoding a TetR protein operably linked to a ParaBAD promoter, a heterologous gene encoding AraC operably linked to a Parac promoter, a heterologous gene encoding an antitoxin operably linked to a constitutive promoter, and a heterologous gene encoding a toxin operably linked to a Px_etR promoter. In another embodiment, the isolated plasmid comprises at least one heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first inducible promoter; a heterologous gene encoding a TetR protein and an antitoxin operably linked to a ParaBAD promoter, a heterologous gene encoding AraC operably linked to a Parac promoter, and a heterologous gene encoding a toxin operably linked to a PietR promoter.

[0469] In any of the above-described embodiments, the plasmid is a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid.

[0470] In another aspect, the disclosure provides a recombinant bacterial cell comprising an isolated plasmid described herein. In another embodiment, the disclosure provides a pharmaceutical composition comprising the recombinant bacterial cell. Host-Plasmid Mutual Dependency

[0471] In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et ah , 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad- spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the antitoxin {e.g. , a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the

GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.

[0472] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein {e.g. , kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.

[0473] In some embodiments, the vector comprises a conditional origin of replication. In some embodiments, the conditional origin of replication is a R6K or ColE2- P9. In embodiments where the plasmid comprises the conditional origin of replication R6K, the host cell expresses the replication initiator protein π. In embodiments where the plasmid comprises the conditional origin or replication ColE2, the host cell expresses the replication initiator protein RepA. It is understood by those of skill in the art that the expression of the replication initiator protein may be regulated so that a desired expression level of the protein is achieved in the host cell to thereby control the replication of the plasmid. For example, in some embodiments, the expression of the gene encoding the replication initiator protein may be placed under the control of a strong, moderate, or weak promoter to regulate the expression of the protein.

[0474] In some embodiments, the vector comprises a gene encoding a protein required for complementation of a host cell auxotrophy, preferably a rich-media compatible auxotrophy. In some embodiments, the host cell is auxotrophic for thymidine (AthyA), and the vector comprises the thymidylate synthase (thyA) gene. In some embodiments, the host cell is auxotrophic for diaminopimelic acid (AdapA) and the vector comprises the 4-hydroxy- tetrahydrodipicolinate synthase (dapA) gene. It is understood by those of skill in the art that the expression of the gene encoding a protein required for complementation of the host cell auxotrophy may be regulated so that a desired expression level of the protein is achieved in the host cell.

[0475] In some embodiments, the vector comprises a toxin gene. In some embodiments, the host cell comprises an anti-toxin gene encoding and/or required for the expression of an anti-toxin. In some embodiments, the toxin is Zeta and the anti-toxin is Epsilon. In some embodiments, the toxin is Kid, and the anti-toxin is Kis. In preferred embodiments, the toxin is bacteriostatic. Any of the toxin/antitoxin pairs described herein may be used in the vector systems of the present disclosure. It is understood by those of skill in the art that the expression of the gene encoding the toxin may be regulated using art known methods to prevent the expression levels of the toxin from being deleterious to a host cell that expresses the anti-toxin. For example, in some embodiments, the gene encoding the toxin may be regulated by a moderate promoter. In other embodiments, the gene encoding the toxin may be cloned adjacent to ribosomal binding site of interest to regulate the expression of the gene at desired levels (see, e.g. , Wright et al. (2015)).

Integration

[0476] In some embodiments, any of the gene(s) or gene cassette(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. One or more copies of the bile salt hydrolase gene cassette may be integrated into the bacterial chromosome. Having multiple copies of the bile salt hydrolase gene cassette integrated into the chromosome allows for greater production of the bile salt hydrolase enzyme and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the kill-switch circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.

[0477] For example, Fig. 14 depicts map of integration sites within the E. coli Nissle chromosome. Fig. 15 depicts three bacterial strains wherein the RFP gene has been successfully integrated into the bacterial chromosome at an integration site.

In vivo Models

[0478] The recombinant bacteria may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with bile salts may be used. For example, an in vivo mouse model of weight gain and lipid metabolism was described by Joyce et al, PNAS, l l l(20):7421-7426 (2014), the entire contents of which are expressly incorporated herein by reference. To briefly summarize, C57BL/6J mice and germ-free Swiss Webster mice can be fasted and fed either a normal low-fat diet or a high-fat diet for ten weeks. After ten weeks, the mice can be inoculated with recombinant bacteria comprising a bile salt hydrolase enzyme (as described herein) or control bacteria. Body weight, plasma samples, and fecal samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and internal organs (liver, spleen, intestines) and fat pads can be removed and assayed.

Pharmaceutical Compositions and Formulations

[0479] Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a diseases associated with bile salts or symptom(s) associated with diseases or disorders associated with bile salts. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers are provided.

[0480] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic

modifications described herein, e.g., to express a bile salt hydrolase enzyme. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein, e.g., to express a bile salt hydrolase enzyme. [0481] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into

compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of

administration.

[0482] The genetically engineered bacteria described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delayed- release, or sustained release). Suitable dosage amounts for the genetically engineered

5 12 5

bacteria may range from about 10 to 10 bacteria, e.g. , approximately 10 bacteria,

6 7 8 approximately 10 bacteria, approximately 10 bacteria, approximately 10 bacteria, approximately 10⁹ bacteria, approximately 10¹⁰ bacteria, approximately 10¹¹ bacteria, or approximately 10¹¹ bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[0483] The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g. , 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

[0484] The genetically engineered bacteria disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

[0485] The genetically engineered bacteria disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0486] Tablets or capsules can be prepared by conventional means with

pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., /actose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L- leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A- PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly

pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co- glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

[0487] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.

[0488] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with

pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The

preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria described herein.

[0489] In one embodiment, the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al. , Pediatrics,

134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to- swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

[0490] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors. [0491] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[0492] In certain embodiments, the genetically engineered bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject' s diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0493] In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the

recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. See, e.g., US 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese. [0494] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g. , conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[0495] The genetically engineered bacteria described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g. , dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g. , of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0496] The genetically engineered bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g. , as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g. , as a sparingly soluble salt).

[0497] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g. , single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g. , by infusion.

[0498] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[0499] In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g. , U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0500] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD₅₀, ED₅₀, EC₅₀, and IC₅₀ may be determined, and the dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[0501] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water- free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0502] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

[0503] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl

methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[0504] The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Methods of Treatment

[0505] Further disclosed herein are methods of treating a disease or disorder associated with bile salts. In some embodiments, disclosed herein are methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders. In some embodiments, the disease or disorder associated with bile salts is cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH, inflammatory and autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), gastrointestinal cancer, and/or C. difficile infection.

[0506] In some embodiments, the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to chest pain, heart failure, or weight gain. In some embodiments, the disease is secondary to other conditions, e.g., cardiovascular disease or liver disease.

[0507] In certain embodiments, the bacterial cells are capable of deconjugating bile salts in a subject in order to treat a disorder associated with bile salts. In these embodiments, a patient suffering from a disorder associated with bile salts, e.g., obesity, may be able to resume a substantially normal diet, or a diet that is less restrictive.

[0508] In certain embodiments, the bacterial cells are capable of metabolizing primary bile acids into secondary bile acids in a subject in order to treat or prevent a disorder associated with bile salts and/or bile acids, such as C. difficile infection. In these

embodiments, a subject at risk of suffering from C. difficile infection will have enhanced resistance to infection, and a subject having C. difficile infection will have enhanced resistance and recover more quickly. For example, a hospital patient receiving treatment will be less likely to become infected with C. difficile.

[0509] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria disclosed herein are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria are lyophilized in a gel cap and administered orally. In some

embodiments, the genetically engineered bacteria are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically.

[0510] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. The genetically engineered microorganisms may be administered locally, e.g., injected directly into a tissue or supplying vessel, or systemically, e.g., intravenously by infusion or injection. In some embodiments, the genetically engineered bacteria are administered intravenously, intra-arterially, intramuscularly, intraperitoneally, orally, or topically. In some embodiments, the genetically engineered microorganisms are

administered intravenously, i.e., systemically.

[0511] In certain embodiments, administering the pharmaceutical composition to the subject reduces the level of bile salts in a subject. In some embodiments, the methods of the present disclosure may reduce the level of bile salts in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the bile salts concentration in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a disease or disorder associated with bile salts allows one or more symptoms of the disease or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. Bile salt levels, bile acid levels, and/or secondary bile acid levels may be measured by methods known in the art (see bile salt hydrolase enzyme section, supra).

[0512] Before, during, and after the administration of the pharmaceutical

composition, ammonia concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions to reduce bile salt concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject' s bile salts concentrations prior to treatment.

[0513] In certain embodiments, administering the pharmaceutical composition to the subject increases the level of secondary bile acids, e.g. , DCA and/or LCA, in a subject. In some embodiments, the methods of the present disclosure may increase the level of secondary bile acids in a subject by at least about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, increase is measured by comparing the bile acid concentration in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a disease or disorder associated with bile acids allows one or more symptoms of the disease or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. Bile salt levels, bile acid levels, and/or secondary bile acid levels may be measured by methods known in the art.

[0514] The methods disclosed herein may further comprise isolating a sample from the subject prior to administration of a composition and determining the level of the bile salts and/or bile acids in the sample. In some embodiments, the methods may further comprise isolating a sample from the subject after to administration of a composition and determining the level of bile salts and/or bile acids in the sample.

[0515] In certain embodiments, the genetically engineered bacteria comprising a bile salt hydrolase is E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the tissues or blood serum (Sonnenborn et ah, 2009), or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the gene or gene cassette for producing bile salt hydrolase enzyme may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice can be determined. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after administration.

[0516] The pharmaceutical composition may be administered alone or in

combination with one or more additional therapies, e.g., high blood pressure medicines or high cholesterol medicines. The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents.

[0517] An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the bacteria, e.g., the agent(s) must not interfere with or kill the bacteria. In one embodiment, the composition is administered in combination with a bile salt binding agent, such as cholestyramine. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria disclosed herein, e.g., the agent(s) must not kill the bacteria. In some embodiments, the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet and amino acid supplementation.

[0518] In some embodiments the genetically administered bacteria are administered with one or more enzyme replacement therapies. In some embodiments, the bacteria are administered in combination with other therapies, such as gene therapy delivering a therapeutic polynucleotide through viral vectors.

[0519] The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of

administration can be selected by a treating clinician.

Treatment in vitro and in vivo

[0520] The genetically engineered bacteria may be evaluated in vitro and/or in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with bile salts may be used. The genetically engineered bacteria may be administered to the animal systemically or locally, e.g., via oral administration (gavage), intravenous, or subcutaneous injection or via intrathecal administration, and treatment efficacy determined. [0521] In some embodiments, these animal models of diseases or disorders associated with bile salts are used to determine efficacy and to identify potential unwanted side effects.

Examples

[0522] The present disclosure is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto are also expressly incorporated herein by reference.

Development of recombinant bacterial cells

[0523] Example 1. Construction of Plasmids Encoding Bile Salt Hydrolase Enzymes

[0524] The bile salt hydrolase genes from Lactobacillus plantarum (SEQ ID NO: l) is synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57- Kan by Gibson assembly, and transformed into E. coli DH5a as described herein to generate the plasmid pTet-BSH.

[0525] Example 2. Generation of Recombinant Bacteria Comprising a Bile Salt Hydrolase Enzyme

[0526] The pTet-BSH plasmid described above is transformed into E. coli Nissle, DH5a, or PIR1. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli (Nissle, DH5a or PIR1) is diluted 1: 100 in 4 mL of LB and grown until it reaches an OD₆oo of 0.4-0.6. lmL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40uL pre-chilled 10% glycerol. The electroporator is set to 1.8kV. luL of a pTet-BSH miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500uL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing 50ug/mL Kanamycin for pTet-BSH. Functional assays using recombinant bacterial cells

[0527] Example 3. Functional Assay Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration

[0528] For in vitro studies, all incubations will be performed at 37° C. Cultures of E. coli Nissle containing pTet-BSH are grown overnight in LB and then diluted 1: 100 in LB. The cells are grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of bile salt hydrolase, and bacteria are grown for another 3 hours. Culture broths are then inoculated at 20% in flasks containing fresh LB culture media containing excess bile salts (either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile) and grown for 16 hours with shaking (250 rpm). A "medium blank" for each culture condition broth is also prepared whereby the "medium blank" is not inoculated with bacteria but treated under the same conditions as the inoculated broths.

Following the 16 hour incubation period, broth cultures are pasteurized at 90°C for 15 minutes, centrifuged at 5,000 rpm for 10 minutes, and supernatants filtered with a 0.45 micron filter.

[0529] Bile salt levels and activity in the supernatants is determined. Briefly, bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and

Feighner, Applied Environ. Microbiol., 55: 11-16 (1989) and Christiaens et al., Appl. Environ. Microbiol., 58:3792-3798 (1992). BSH activity can also be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al, PNAS, 105(36): 13580-13585 (2008)). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al., Gut Microbes, 3(3): 186-202 (2012)).

[0530] Example 4. In vivo Studies Demonstrating that the Recombinant

Bacterial Cells Decrease Bile Salt Concentration

[0531] For in vivo studies, a mouse model of weight gain and lipid metabolism (as described by Joyce et al, PNAS, 111(20):7421-7426 (2014)) is used. To briefly summarize, C57BL/6J mice and germ-free Swiss Webster mice can be fasted and fed either a normal low-fat diet or a high-fat diet for ten weeks. After ten weeks, the mice can be inoculated with recombinant bacteria comprising a bile salt hydrolase enzyme (as described herein) or control bacteria. Body weight, plasma samples, and fecal samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and internal organs (liver, spleen, intestines) and fat pads can be removed and assayed. Treatment efficacy is determined, for example, by measuring levels of bile salts and bile acids. A decrease in levels of bile salts after treatment with the recombinant bacterial cells indicates that the recombinant bacterial cells described herein are effective for treating disorders associated with bile salts.

[0532] Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with disorders associated with bile salts, for example, weight loss, further indicates the efficacy of the recombinant bacterial cells described herein for treating disorders associated with bile salts.

[0533] Example 5. Lambda red recombination

[0534] Lambda red recombination is used to make chromosomal modifications, e.g., to express CPD G₂ in E. coli Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli. A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1 : 100 in 5 mL of LB media and grown until it reaches an OD₆oo of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.

[0535] DNA sequences comprising the desired CPD G₂ sequences shown above were ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. In some embodiments, the construct is in the E. coli Nissle genome at the malP/T site (Fig. 14). To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene.

Alternatively, the homologous sequences may be added by PCR. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting construct comprises approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the CPD G₂ gene, and approximately 50 bases of homology downstream.

[0536] Example 6. Transforming E. coli Nissle

[0537] The CPD G₂ construct above is transformed into E. coli Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1: 100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD₆oo of 0.1. 0.05 mL of 100X L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD₆oo of 0.4-0.6. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 0.5 μg of the mutated ARG box construct is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing kanamycin and incubated overnight.

[0538] Example 7. Verifying mutants

[0539] The presence of the CPD G₂ is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 μΐ of cold ddH₂0 by pipetting up and down. 3 μΐ of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C overnight. A PCR master mix is made using 5 μΐ of 10X PCR buffer, 0.6 μΐ of 10 mM dNTPs, 0.4 μΐ of 50 mM Mg₂S0₄, 6.0 μΐ of 10X enhancer, and 3.0 μΐ of ddH₂0 (15 μΐ of master mix per PCR reaction). A 10 μΜ primer mix is made by mixing 2 μΐ_^ of primers unique to the CPD G₂ construct (100 μΜ stock) into 16 μΐ_^ of ddH₂0. For each 20 μΐ reaction, 15μί of the PCR master mix, 2.0 μΐ_^ of the colony suspension (template), 2.0 μΐ_^ of the primer mix, and 1.0 μΐ_^ of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C at 5:00 min., 2) 94° C at 0: 15 min., 3) 55° C at 0:30 min., 4) 68° C at 2:00 min., 5) 68° C at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μΐ_^ of each amplicon and 2.5 μΐ_^ 5X dye. The PCR product only forms if the mutation has inserted into the genome.

[0540] Example 8. Removing selection marker

[0541] The antibiotic resistance gene is removed with pCP20. Each strain with the CPD G₂ is grown in LB media containing antibiotics at 37° C until it reaches an OD₆oo of 0.4- 0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the

supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pCP20 plasmid DNA is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette was placed into the sample chamber, and the electric pulse was applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1-3 hrs. The cells are spread out on an LB plate containing kanamycin and incubated overnight. Colonies that do not grow to a sufficient OD₆oo overnight are further incubated for an additional 24 hrs. 200 μΐ_^ of cells are spread on ampicillin plates, 200 μL· oΐ cells are spread on kanamycin plates, and both are grown at 37° C overnight. The ampicillin plate contains cells with pCP20. The kanamycin plate provides an indication of how many cells survived the electroporation. Transformants from the ampicillin plate are purified non-selectively at 43° C and allowed to grow overnight.

[0542] Example 9. Verifying transformants

[0543] The purified transformants are tested for sensitivity to ampicillin and kanamycin. A colony from the plate grown at 43° C is picked and and resuspended in 10 μΐ_^ of LB media. 3 μΐ_^ of the cell suspension is pipetted onto each of three plates: 1) an LB plate with kanamycin incubated at 37° C, which tests for the presence or absence of the KanR gene in the genome of the host strain; 2) an LB plate with ampicillin incubated at 30° C, which tests for the presence or absence of the AmpR gene from the pCP20 plasmid; and 3) an LB plate without antibiotic incubated at 37° C. If no growth is observed on the kanamycin or ampicillin plates for a particular colony, then both the KanR gene and the pCP20 plasmid were lost, and the colony is saved for further analysis. The saved colonies are restreaked onto an LB plate to obtain single colonies and grown overnight at 37° C. The presence of the CPD G₂ is confirmed by sequencing the genome.

[0544] Example 10. Measuring the activity of an FNR promoter

[0545] To determine the kinetics of FNR promoter-driven gene expression, E. coli strains harboring a low-copy fnrS-lacZ fusion gene (Fig. 7A) were grown aerobically with shaking at 250 rpm. Cultures were split after 1 hr., and then incubated either aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N₂, 5% C0₂, and 5%H₂) at 37 °C. Promoter activity was measured as a function of β-galactosidase activity using a standard colorimetric assay (Miller, 1972). Fig. 7B demonstrates that the fnrS promoter begins to drive high-level gene expression within 1 hr. under anaerobic conditions. Growth curves of bacterial cell cultures expressing lacZ are shown in Fig. 7C, both in the presence and absence of oxygen.

[0546] Example 11. Nissle Residence

[0547] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non-limiting example using a streptomycin- resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.

[0548] C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation {i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 14. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted. Table 14: CFU administered via oral gavage

[0549] On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1-6; Table 15). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37°C overnight, and colonies were counted.

[0550] Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 15.

[0551] Fig. 16 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.

Table 15. Nissle residence in vivo

[0552] Example 12. Intestinal Residence and Survival of Bacterial Strains in vivo

[0553] Localization and intestinal residence time of streptomycin resistant Nissle, FIG. 17, was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.

[0554] Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10¹⁰ CFU/mL. Mice

(C57BL6/J, 10-12 weeks old) were gavaged with 100 μL· of bacteria (approximately 10⁹ CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post- gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.

[0555] In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37°C overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in Fig. 17. [0556] Example 13. FNR promoter activity

[0557] In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9,

Cambridge, MA) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 3. The nucleotide sequences of these constructs are shown in Tables 16-20 (SEQ ID NOs 74-78). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity. Alternatively, beta-galactosidase may be used as a reporter, exemplary results are shown in Fig. 6.

[0558] Table 16 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf_nrl (SEQ ID NO: 74). The construct comprises a translational fusion of the Nissle nirBl gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The Pfnj-i sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0559] Table 17 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf^ (SEQ ID NO: 75). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8^th codon of the lacZ coding region. The Pfnj-2 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0560] Table 18 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pf^ (SEQ ID NO: 76). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The Pf^ sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0561] Table 19 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P_fnr4 (SEQ ID NO: 77). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The P_fnr4 sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

[0562] Table 20 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS (SEQ ID NO: 78). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrSl, fused to lacZ. The P_fnrs sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case.

Table 16

Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 74)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtcgtccggccttttcctctcttac tctgctacgtacatctatttctataaatccgttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgacttaag aaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggttgctgaat cgttaaggtaggcggtaatagaaaagaaatcgaggcaaaaATGagcaaagtcagactcgcaattatGGATCCTCT

GGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT

CGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCA

CCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTG

GTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGA

CGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCT

ATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGG

AGAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACA

GGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGG

TGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTG

ACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCG

CTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCAT

TTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA

GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTC

AGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGG

GTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATG

AGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGG

AACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACAC

CGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGT Nucleotide sequences of Pfnrl-lacZ construct, low-copy (SEQ ID NO: 74)

GCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGC

GTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGA

TGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTC

GCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTAT

GTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTG

ACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTG

CAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCA

GGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTT

CCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTA

TTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAA

ATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTT

TGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGC

AGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGA

TCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGT

GATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTG

CCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATT

TCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCG

TCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCT

GGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGA

ACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACG

CGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTG

GCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCAC

GCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATA

AGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGA

TGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGAT

AACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAA

CGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACG

GCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCAT

CAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGT

GAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCG

CGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTC

GGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACC

GCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAA

CGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGG

CGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAG

CCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTC

CATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCC

AGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 17

Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 75)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatgcatgcatcaaaaaagatgtg agcttgatcaaaaacaaaaaatatttcactcgacaggagtatttatattgcgcccgttacgtgggcttcgactgtaaatcagaa aggagaaaacacctATGacgacctacgatcgGGATCCTCTGGCCGTCGTATTACAACGTCGTGA

CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTC

GCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTG Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 75)

CGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGC

CGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTC

AAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCC

ATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGC

TCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTT

TGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTAC

GGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCG

GAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGG

AAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCA

TAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAATGATGAT

TTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGAT

GAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGC

ACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCG

TCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGA

ATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGC

AGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTG

CTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTC

TGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGA

AGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTG

GTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAA

ACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCG

CGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTG

TGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGC

TGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGG

CGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGAT

GAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCG

CTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGGGTA

ACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTT

ACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGA

AAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGA

TCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCG

CTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGA

ACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGC

ACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGG

ATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGG

AGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCG

CATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAA

ACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAG

CGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAG

TCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGC

TGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAG

CGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATT

ACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGG

TGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAG

CCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGT

TGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCT

GGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTA

TCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGAC Nucleotide sequences of Pfnr2-lacZ construct, low-copy (SEQ ID NO: 75)

ATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCG

AATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCG

CTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGA

AGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGA

CTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCAT

TACCAGTTGGTCTGGTGTCAAAAATAA

Table 18

Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 76)

GGTACCgtcagcataacaccctgacctctcattaattgttcatgccgggcggcactatcgtcgtccggccttttcctctcttac tctgctacgtacatctatttctataaatccgttcaatttgtctgttttttgcacaaacatgaaatatcagacaattccgtgacttaag aaaatttatacaaatcagcaatataccccttaaggagtatataaaggtgaatttgatttacatcaataagcggggttgctgaat cgttaaGGATCCctctagaaataattttgtttaactttaagaaggagatatacatATGACTATGATTACGGA

TTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAA

CTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG

CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTT

TGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTT

CCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATG

CGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCC

GCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGC

TACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCT

GTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGA

ATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTG

CTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGC

GGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATT

TCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGA

AGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGG

CAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATC

GATGAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATC

CGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCA

CACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGA

GGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGC

GGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGA

CGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCT

GTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCT

GTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCG

TCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGAT

GGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGA

ATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGAT

CCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGAT

ATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGC

CGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGAT

CCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATAC

TGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGG

TGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACG Nucleotide sequences of Pfnr3-lacZ construct, low-copy (SEQ ID NO: 76)

GCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGT

CTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCA

GTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTG

TTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGC

CGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGA

TTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGG

TACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCG

CCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTC

CCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGT

AATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTG

GCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCT

GGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGT

CGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTG

CACGGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCA

GCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCA

CGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCC

GGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTG

GCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTT

GACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCG

AAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGC

GCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAA

CCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACG

GTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGA

ATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

Table 19

Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 77)

GGTACCcatttcctctcatcccatccggggtgagagtcttttcccccgacttatggctcatgcatgcatcaaaaaagatgtg agcttgatcaaaaacaaaaaatatttcactcgacaggagtatttatattgcgcccGGATCCctctagaaataattttgttta actttaagaaggagatatacatATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAAC

GTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCACATCC

CCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA

CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAG

CGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTCGT

CCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCAACGTGACC

TATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTT

ACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAA

TTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTC

GGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTAC

GCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTT

ATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTT

GCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAAT

GATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTG

CGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCC

AGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCG Nucleotide sequences of Pfnr4-lacZ construct, low-copy (SEQ ID NO: 77)

ATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAA

TCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGAT

TGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCT

GCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCAT

CATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGC

TGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCC

GCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAAT

ATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGC

TACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACC

CGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACG

ACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGA

AGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCG

CGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATG

GCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCG

ATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACC

CCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATA

TGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCC

GAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCAT

CCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCG

GGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGT

TCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGC

CTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCA

GCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGC

GACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGC

GGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACC

ACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACC

GCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGAC

CCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGT

GAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGC

CATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGAC

GCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTT

ATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTG

GATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCTGC

CAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAA

AACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTC

AGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGAC

GCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATC

AGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCAC

GCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGC

GACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCT

ACCATTACCAGTTGGTCTGGTGTCAAAAATAA Table 20

Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 78)

GGTACCagttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgtaacaaaagcaatttttccggctgtct gtatacaaaaacgccgtaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctcttGGATCCctcta gaaataattttgtttaactttaagaaggagatatacatATGCTATGATTACGGATTCTCTGGCCGTCGT

ATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCG

GCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCC

CTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGC

ACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATAC

TGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACC

AACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGA

CAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCA

GACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGG

CGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCG

CATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGA

CGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGAC

GTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTC

TCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGG

CGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCA

GGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGG

TTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGC

GCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCA

CGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAA

ATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCA

CGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGA

TATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCG

AACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATG

AAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATC

CGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATC

GTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCG

CTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGT

ACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGAT

GTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCAT

CAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATAT

GCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTC

GTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCT

GATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGC

GATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCA

CGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCC

GTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGA

TAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGG

TGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAA

CTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAA

CCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGG

CGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTC

AACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGC

AATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACA

ACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATT Nucleotide sequences of Pfnrs-lacZ construct, low-copy (SEQ ID NO: 78)

GGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAG

GCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACA

CTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAA

ACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCA

TCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCT

GACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC

GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTG

CCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCT

GCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGT

TCAACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATC

TGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGA

TTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGC

CGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA

[0563] Each of the plasmids was transformed into E. coli Nissle, as described above. Cultures of transformed E. coli Nissle were grown overnight and then diluted 1:200 in LB. The cells were grown with shaking at 250 rpm either aerobically or anaerobically in a Coy anaerobic chamber supplied with 90% N₂, 5% C0₂, and 5% H₂. After 4-6 hrs of incubation, samples were collected, and promoter activity was analyzed by performing β-galactosidase assays (Miller, 1972). As shown in Fig. 6, the activities of the FNR promoters were greatly enhanced under anaerobic conditions compared to aerobic conditions.

[0564] Example 14. Nitric oxide-inducible reporter constructs

[0558] ATC and nitric oxide-inducible reporter constructs were synthesized

(Genewiz, Cambridge, MA). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an

excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR- GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations; promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS Table 21. SEQ ID NO: 95

SEQ ID NO: 95 ttattatcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgttgagcaggtcttgcagcgtgaaaccgt ccagatacgtgaaaaacgacttcattgcaccgccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttc gggcccatacactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgttcgggcggcgcggcca gcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccgcctttgaccagcgcggtaaccactttcatcaaatggctttt aaat cc ta tc a c at t c atatt acca c c tc tc tt ac c t ta at a ac c ca c

gacaattaatcatcggctcgtataatgtataacattcatattttgtgaattttaaactctagaaataattttgtttaactttaagaaggag atotocatotggctagcaaaggcgaagaattgttcacgggcgttgttcctattttggttgaattggatggcgatgttaatggccat aaattcagcgttagcggcgaaggcgaaggcgatgctacgtatggcaaattgacgttgaaattcatttgtacgacgggcaaatt gcctgttccttggcctacgttggttacgacgttcagctatggcgttcaatgtttcagccgttatcctgatcatatgaaacgtcatga tttcttcaaaagcgctatgcctgaaggctatgttcaagaacgtacgattagcttcaaagatgatggcaattataaaacgcgtgct gaagttaaattcgaaggcgatacgttggttaatcgtattgaattgaaaggcattgatttcaaagaagatggcaatattttgggc cataaattggaatataattataatagccataatgtttatattacggctgataaacaaaaaaatggcattaaagctaatttcaaa attcgtcataatattgaagatggcagcgttcaattggctgatcattatcaacaaaatacgcctattggcgatggccctgttttgtt gcctgataatcattatttgagcacgcaaagcgctttgagcaaagatcctaatgaaaaacgtgatcatatggttttgttggaattc gttacggctgctggcattacgcatggcatggatgaattgtataaataataa

[0559] These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600= -0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units.

[0560] NO-GFP constructs (the dot blot) E. coli Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1: 100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR- regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS-treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation.

[0561] Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR- inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1: 100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice.

[0562] Example 15. Measuring the activity of an FNR promoter

[0565] To determine the kinetics of FNR promoter-driven gene expression, E. coli strains harboring a low-copy fnrS-lacZ fusion gene (Fig. 7A) were grown aerobically with shaking at 250 rpm. Cultures were split after 1 hr., and then incubated either aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N₂, 5% C0₂, and 5%H₂) at 37 °C. Promoter activity was measured as a function of β-galactosidase activity using a standard colorimetric assay (Miller, 1972). Fig. 7B demonstrates that the fnrS promoter begins to drive high-level gene expression within 1 hr. under anaerobic conditions. Growth curves of bacterial cell cultures expressing lacZ are shown in Fig. 7C, both in the presence and absence of oxygen.

[0566] Example 16. Wild Type clbA and clbA knock out

[0567] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thy A, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.

[0568] A thyA:: cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a lOOum concentration are found in Table 426

Table 22. Primer Sequences

[0569] For the first PCR round, 4x50ul PCR reactions containing lng pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:

[0570] step 1: 98c for 30s

[0571] step2: 98c for 10s

[0572] step3: 55c for 15s

[0573] step4: 72c for 20s

[0574] repeat step 2-4 for 30 cycles

[0575] step5: 72c for 5min

[0576] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer's instructions and eluted in 30ul nuclease free water.

[0577] For the second round of PCR, lul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.

[0578] For the third round of PCR, lul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by fit sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. {thyA auxotrophs will only grow in media supplemented with thy 3mM).

[0579] Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. lmL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in lmL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with lng pCP20 plasmid DNA, and lmL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for lhours. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in lOOul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and lOOug/ml carbeniciUin and grown at 30°C for 16-24 hours. Next, transformants were colony purified non- selectively (no antibiotics) at 42°C.

[0580] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in ΙΟμΙ_^ LB. 3μί of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.

Table 23. wild Type clbA and clbA knock out

[0581] Example 18. Repeat-Dose Pharmacokinetic and Pharmacodynamic Study of Genetically Engineered Bacteria Following Daily Nasogastric Gavage Dose Administration for 28-days in Cynomolgus Monkeys (non-GLP)

[0582] To evaluate any potential toxicities arising from administration of the genetically engineered bacteria or E. coli Nissle alone, the pharmacokinetics and

pharmacodynamics of the genetically engineered bacteria and an E. coli Nissle are studied following daily nasogastric gavage (NG) dose administration for 28-days to female cynomolgus monkeys. Cynomolgus monkeys is selected because this species is closely related, both phylogenetically and physiologically, to humans and is a species commonly used for nonclinical toxicity evaluations. The genetically engineered bacteria are

administered by nasal gastric gavage, consistent with the proposed route of administration in humans. Animals overall well-being (clinical observations), weight clinical pathology (serum chemistry, hematology, and coagulation) are tracked. Plasma is analyzed for ammonia levels, and fecal samples examined for bacterial load.

[0583] The genetically engineered strain comprises one or more copies of a bile salt hydrolase enzyme(s) integrated into the chromosome, each of which are under the control of an FNRS promoter. In some embodiments, the genetically engineered strain also comprises one or more copies of a bile sale transporter(s), driven by an arabinose inducible promoter, e.g., ParaBAD. In some embodiments, the strain further comprises a auxotrophy mutation, e.g., deltaThyA. In some embodiments, the genetically engineered bacteria further comprise an antibiotic resistance, e.g., kanamycin. In some embodiments, the genetically engineered bacteria do not comprise an auxotrophy mutation. In some embodiments, the genetically engineered bacteria do not comprise an antibiotic resistance.

Materials, animals and dosing regimen:

[0584] The study is conducted in compliance with nonclinical Laboratory Studies Good Laboratory Practice Regulations issued by the U.S. Food and Drug Administration (Title 21 of the Code of Federal Regulations, Part 58; effective June 20, 1979) and the OECD Principles on Good Laboratory Practice (C [97] 186/Final; effective 1997). The animals are individually housed based on the recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).

[0585] Animals used in the study are Female Purpose-bred, non-naive cynomolgus monkey (Macaca fascicularis) with 3 to 6 kg (at initial physical exam) 3 to 8 years (at initial physical exam) of age (SNBL USA stock, Origin: Cambodia). [0586] For the duration of the study, animals are offered PMI LabDiet® Fiber- Plus® Monkey Diet 5049 biscuits twice daily. Animal are fasted for at least 2 hours prior to dose administration and fed within 1-hour post dose. Animals also are fasted as required by specific procedures (e.g., prior to blood draws for serum chemistry, fecal collection). The diet is routinely analyzed for contaminants and found to be within manufacturer's specifications. No contaminants are expected to be present at levels that would interfere with the outcome of the study. Food analysis records are maintained in the testing facility records.

[0587] Fresh drinking water is provided ad libitum to all animals. The water is routinely analyzed for contaminants. No contaminants are present at levels that would interfere with the outcome of the study. Animals are given fruits, vegetables, other dietary supplements, and cage enrichment devices throughout the course of the study.

[0588] Previously quarantined animals are acclimated to the study room for 7 days prior to initiation of dosing (day 1). The last dosing occurs on day 28. A stratified

randomization scheme incorporating body weights is used to assign animals to study groups. Animals are assigned to groups and treated as indicated in Table 24.

Table 24. Group Assignments

[0589] Nissle control and genetically engineered bacterial stocks are prepared at 1x109 cfu/mL and 1x1011 cfu/mL in 15% glycerol in IX PBS with 2.2% glucose and 3 niM thymidine and are kept at 86 to -60 °C (see Table 24). PBS made in 20% glycerol with sodium bicarbonate is used as a control vehicle. Carbonate concentration is 0.36M and 0.12M for sodium bicarbonate. On the day of each dosing, bacteria and vehicle control are removed from the freezer and put on ice and thawed and placed on ice until dosing.

[0590] Animals are dosed at 0, 1x10 9 , or 1x1012 cfu/animal. All animals are dosed via nasal gastric gavage (NG) followed by control/vehicle flush once daily for 28-days. The concentration of bicarbonate and volume for each group is specified in Table 24 Vials are inverted at least 3 times prior to drawing the dose in the syringe. The dose site and dose time (end of flush time) is recorded.

Analysis

[0591] Overall condition: Clinical observations are performed twice daily beginning on the second day of acclimation for each animal. The first observation is in the AM, prior to room cleaning. The second observation is no sooner than 4 hours after the AM observation. During the dosing phase, the second observation is performed 4 hour (+10 minutes) post dose administration. Additional clinical observations are performed, as necessary.

[0592] Weight: Each animal is weighed on Day -6, 1, 8, 15, 22, and 29 prior to the first feeding and also prior to dose administration. Additional body weights are taken as needed if necessary.

[0593] Blood Collection: Blood is collected from a peripheral vein of restrained, conscious animals. Whenever possible, blood is collected via a single draw and then divided appropriately. Specimen collection frequency is summarized in Table 25.

Table 25. Specimen collection frequency

Dosing

- - - Day28 Day 28- Week 4 (Predose) 30

Dosing Day 30 Day 30 Day Day 30 Day 35, 40 Weeks 5

30

- = Not applicable

x = Number of times procedure performed within the week

[0594] Hematology: Approximately 1.3 mL of blood is tested in 2 mL K2EDTA tubes using an Advia automated analyzer. Parameters measured are White Blood Cells, Red Blood Cells, Hemoglobin, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Red Cell Distribution Width, Platelets, Mean Platelet Volume, Differential leukocyte count (absolute): Neutrophils Absolute Lymphocytes Absolute Monocytes Absolute Eosinophils Absolute_^Basophils Absolute Reticulocyte Percent, and Reticulocyte Absolute Count.

[0595] Coagulation: Approximately 1.3 mL of blood is tested in 1.8 mL 3.2% sodium citrate tubes. The following Coagulation parameters are determined using a

STACompact automated analyzer: Activated Partial Thromboplastin Time, Fibrinogen, and Prothrombin Time. Sodium citrate-treated plasma is stored at_-60 to -86 °C prior to analysis and discarded after analysis.

[0596] Serum Chemistry: Animals are fasted for 4 hours prior to removal of sample. The following parameters are tested in approximately 1 mL of blood in 4 mL serum separator tubes using a AU680 analyzer: Albumin, Alkaline Phosphatase, Alanine Aminotransferase Aspartate Aminotransferase, Total Bilirubin, Calcium, Total Cholesterol, Creatine Kinase, Creatinine, Glucose, Inorganic Phosphorus, Total Protein, Triglyceride, Sodium, Potassium, Chloride Globulin, Albumin/Globulin Ratio, Blood Urea Nitrogen, and Gamma

Glutamyltransferase.

[0597] Residual serum is stored at-60 to -86 °C and disposed of prior to study finalization.

[0598] Plasma Samples: Animals are fasted for 4 hours prior to removal of the sample. Blood samples are collected from the femoral vein at the target time points listed in Table 25 After aliquotting the target volume of blood in the blood tube, approximately 0.05 mL of mineral oil is added covering the surface of blood. Tubes are not inverted and placed on a rack and wet ice. Blood sample collection dates and times were recorded. The minimum sample volume is 1 ml of blood collected in a 2 ml lithium heparin tube. Within 15 minutes of collection, the samples are centrifuged at 2 to 8 °C to obtain plasma. Plasma is transferred to a vial and stored at-60 to -86 °C. Specimens are stored on dry ice prior to analysis.

Analysis of specimens is conducted using a blood ammonia analyzer instrument.

[0599] Phenylalanine, trans-cinnamic acid, and hippuric acid is measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer.

[0600] Fecal Sample Collection: Two fecal samples per animal are collected at the target time points listed in Table 25 Sample collection dates and times are recorded. 50 mL falcon tube with approximately 5mL PBS are used as the container (If feces is liquid, no PBS is added). To get the fecal sample weight, pre- and post- sampling weight of container was taken. Samples are collected from the bottom of the cage from each animal. To get fresh and un-contaminated samples, remaining food is removed and the cage pan was cleaned and squeegeed to remove debris and/or water before the collection. Sample is put on wet ice immediately after the collection. Samples are stored at -20 to -15 °C until analysis. Analysis of specimens is conducted using a PCR analytical method.

[0601] Example 19. 4- Week Toxicity Study in Cynomolgus Monkeys with a 4- Week Recovery (GLP)

[0602] To evaluate any potential toxicities arising from administration of the genetically engineered bacteria, the pharmacokinetics and pharmacodynamics of the genetically engineered bacteria is studied following daily nasogastric gavage (NG) dose administration for 28-day s to female cynomolgus monkeys under GLP conditions.

[0603] The genetically engineered strain comprises one or more copies of bile hydrolase enzyme(s), each of which are under the control of an FNRS promoter. In some embodiments, the genetically engineered strain also comprises one or more copies of a bile transporter(s), driven by an arabinose inducible promoter, e.g., ParaBAD. In some embodiments, the strains further comprise a auxotrophy mutation, e.g. , deltaThyA. In some embodiments, the genetically engineered bacteria further comprise an antibiotic resistance, e.g. , kanamycin. In some embodiments, the genetically engineered bacteria do not comprise an auxotrophy mutation. In some embodiments, the genetically engineered bacteria do not comprise an antibiotic resistance.

[0604] The study is conducted in compliance with nonclinical Laboratory Studies Good Laboratory Practice Regulations issued by the U.S. Food and Drug Administration (Title 21 of the Code of Federal Regulations, Part 58; effective June 20, 1979) and the OECD Principles on Good Laboratory Practice (C[97] 186/Final; effective 1997). The animals are individually housed based on the recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).

[0605] Animals are administered the genetically engineered bacteria or control vehicle essentially as described in Example 18 except that all materials are manufactured under GMP standards. Dosing is tabulated in Table 26. Additionally, animals are acclimated for 14 days and the dosing period is daily for 28 days followed by a recovery period of 28 days. Additionally, animals are euthanized at the end of the study to conduct histological analysis.

Table 26. Dosing Period and Regimen

Terminal Necropsy, Day 29

bRecovery Necropsy, Day 56

[0606] Study Analysis is conducted as described in Table 27. Hematology, Coagulation, Serum Chemistry and Plasma Samples parameters are essentially as described in Example 18 and are analyzed using the methods described in Example 18 Collection and analysis of fecal samples is essentially conducted as described in Example 18 Table 27. Study Analysis

[0607] Example 20. Assessment of in vitro and in vivo activity of Biosafety System Containing Strain

[0608] The activity of thebiosafety system containing strains are tested.

[0609] Cells are grown overnight in LB and diluted 1: 100. After 1.5 hrs of growth, Cells are grown for 4 hours in the presence of 1 mM IPTG ImM to turn on expression of the Plac promoters controlling expression of a bile salt hydrolase enzyme (and in some cases a bile salt transporter). Bacteria are spun down and are resuspended in assay buffer containing 50 mM phenylalanine. Aliquots are removed from cell assays every 20 min for 1.5 hrs for trans-cinnamate quantification by absorbance at 290 nm. In another study the same constructs as above are employed except that the strains further comprise chromosomally integrated Para-FNRS24Y.

[0610] Sequences for the construction of these constructs are shown in Table 28, Table 29, and Table 30 In some embodiments, a bile salt hydrolase gene for use in the above strains is codon optimized. In other embodiments, an original bile salt hydrolase sequence as described herein is used in any of the constructs described above.

[0611] In vivo studies are conducted as described herein.

Table 28. Biosafety System Constructs and Sequence Components

GATTATCATGTCGCCAGCGGTACTTCGGCGATCGTT

TCTGTTGGCACCACTGGCGAGTCCGCTACCTTAAAT

CATGACGAACATGCTGATGTGGTGATGATGACGCT

GGATCTGGCTGATGGGCGCATTCCGGTAATTGCCGG

GACCGGCGCTAACGCTACTGCGGAAGCCATTAGCC

TG AC GC AGC GCTTC A ATG AC AGTGGT ATC GTC GGCT

GCCTGACGGTAACCCCTTACTACAATCGTCCGTCGC

AAGAAGGTTTGTATCAGCATTTCAAAGCCATCGCTG

AGCATACTGACCTGCCGCAAATTCTGTATAATGTGC

CGTCCCGTACTGGCTGCGATCTGCTCCCGGAAACGG

TGGGCCGTCTGGCGAAAGTAAAAAATATTATCGGA

ATCAAAGAGGCAACAGGGAACTTAACGCGTGTAAA

CCAGATCAAAGAGCTGGTTTCAGATGATTTTGTTCT

GCTGAGCGGCGATGATGCGAGCGCGCTGGACTTCA

TGCAATTGGGCGGTCATGGGGTTATTTCCGTTACGG

CTAACGTCGCAGCGCGTGATATGGCCCAGATGTGC

AAACTGGCAGCAGAAGGGCATTTTGCCGAGGCACG

CGTTATTAATCAGCGTCTGATGCCATTACACAACAA

ACTATTTGTCGAACCCAATCCAATCCCGGTGAAATG

GGCATGTAAGGAACTGGGTCTTGTGGCGACCGATA

CGCTGCGCCTGCCAATGACACCAATCACCGACAGT

GGCCGTGAGACGGTCAGAGCGGCGCTTAAACATGC

CGGTTTGCTGTAAGACTTTTGTCAGGTTCCTACTGT

GACGACTACCACCGATAGACTGGAGTGTTGCTGCG

AAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGAA

GTCACCACGATTGTGCTTTACACGGAGTAGTCGGCA

GTTCCTTAAGTCAGAATAGTGGACAGGCGGCCAAG

AACTTCGTTCATGATAGTCTCCGGAACCCGTTCGAG

TCGTTTTCCGCCCCGTGCTTTCATATCAATTGTCCGG

GGTTGATCGCAACGTACAACACCTGTGGTACGTATG

CCAACACCATCCAACGACACCGCAAAGCCGGCAGT

GCGGGCAAAATTGCCTCCGCTGGTTACGGGCACAA

CAACAGGCAGGCGGGTCACGCGATTAAAGGCCGCC

GGTGTGACAATCAGCACCGGCCGCGTTCCCTGCTGC

TCATGACCTGCGGTAGGATCAAGCGAGACAAGCCA

GATTTCCCCTCTTTCCATCTAGTATAACTATTGTTTC

TCTAGTAACATTTATTGTACAACACGAGCCCATTTT

TGTCAAATAAATTTTAAATTATATCAACGTTAATAA

GACGTTGTCAATAAAATTATTTTGACAAAATTGGCC

GGCCGGCGCGCCGATCTGAAGATCAGCAGTTCAAC

CTGTTGATAGTACGTACTAAGCTCTCATGTTTCACG

TACTAAGCTCTCATGTTTAACGTACTAAGCTCTCAT

GTTTAACGAACTAAACCCTCATGGCTAACGTACTAA

GCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCA

CGTACTAAGCTCTCATGTTTGAACAATAAAATTAAT

ATAAATCAGCAACTTAAATAGCCTCTAAGGTTTTAA

GTTTTATAAGAAAAAAAAGAATATATAAGGCTTTT

AAAGCCTTTAAGGTTTAACGGTTGTGGACAACAAG

CCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCC

TCTCAAAGCAATTTTGAGTGACACAGGAACACTTA ACGGCTGACATGGGGCGCGCCCAGCTGTCTAGGGC

GGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGAC AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCT

Biosafety Plasmid ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG 82 System GGTTATTGTCTCATGAGCGGATACATATTTGAATGT Component - ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA ThyA AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG

Biosafety Plasmid CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC System Vector CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG sequences, CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC comprising ThyA, TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG Kid Toxin and AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC R6K minimal ori, GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC and promoter CCAATAATTACGATTTAAATCACAGCAAACACCAC elements driving GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT expression of these TGCTGCATAACTTGACAATTAATCATCCGGCTCGTA components, as GGGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGA shown in Fig. 61B GATCAACAATGAAAGCAATTTTCGTACTGAAACAT

CTTAATCATGCTGGGGAGGGTTTCTAATGAAACAGT

ATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGC

ACACAGAAAAACGACCGTACCGGAACCGGAACGCT

TTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCA

AGATGGATTCCCGCTGGTGACAACTAAACGTTGCC

ACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTC

TTCAGGGCGACACTAACATTGCTTATCTACACGAAA

ACAATGTCACCATCTGGGACGAATGGGCCGATGAA

AACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG

GCGTGCCTGGCCAACGCCAGATGGTCGTCATATTGA

CCAGATCACTACGGTACTGAACCAGCTGAAAAACG

ACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGA

ACGTAGGCGAACTGGATAAAATGGCGCTGGCACCG

TGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGC

AAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGAC

GTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTAC

GCGTTATTGGTGCATATGATGGCGCAGCAGTGCGAT

CTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGAC

ACGCATCTGTACAGCAACCATATGGATCAAACTCAT

CTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAA

GTTGATTATCAAACGTAAACCCGAATCCATCTTCGA

CT ACC GTTTC G A AG ACTTTG AG ATTG A AGGCT AC G A

TCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTA

AGACTTTTGTCAGGTTCCTACTGTGACGACTACCAC

CGATAGACTGGAGTGTTGCTGCGAAAAAACCCCGC

CGAAGCGGGGTTTTTTGCGAGAAGTCACCACGATT

GTGCTTTACACGGAGTAGTCGGCAGTTCCTTAAGTC

AGAATAGTGGACAGGCGGCCAAGAACTTCGTTCAT

GATAGTCTCCGGAACCCGTTCGAGTCGTTTTCCGCC

CCGTGCTTTCATATCAATTGTCCGGGGTTGATCGCA

ACGTACAACACCTGTGGTACGTATGCCAACACCATC CAACGACACCGCAAAGCCGGCAGTGCGGGCAAAAT

TGCCTCCGCTGGTTACGGGCACAACAACAGGCAGG

CGGGTCACGCGATTAAAGGCCGCCGGTGTGACAAT

CAGCACCGGCCGCGTTCCCTGCTGCTCATGACCTGC

GGTAGGATCAAGCGAGACAAGCCAGATTTCCCCTC

TTTCCATCTAGTATAACTATTGTTTCTCTAGTAACAT

TTATTGTACAACACGAGCCCATTTTTGTCAAATAAA

TTTTAAATTATATCAACGTTAATAAGACGTTGTCAA

TAAAATTATTTTGACAAAATTGGCCGGCCGGCGCGC

CGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTA

CGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTC

ATGTTTAACGTACTAAGCTCTCATGTTTAACGAACT

AAACCCTCATGGCTAACGTACTAAGCTCTCATGGCT

AACGTACTAAGCTCTCATGTTTCACGTACTAAGCTC

TCATGTTTGAACAATAAAATTAATATAAATCAGCAA

CTTAAATAGCCTCTAAGGTTTTAAGTTTTATAAGAA

AAAAAAGAATATATAAGGCTTTTAAAGCCTTTAAG

GTTTAACGGTTGTGGACAACAAGCCAGGGATGTAA

CGCACTGAGAAGCCCTTAGAGCCTCTCAAAGCAAT

TTTGAGTGACACAGGAACACTTAACGGCTGACATG

GGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTC

CTACTCAGGAGAGCGTTCACCGACAAACAACAGAT

AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG

TTTTATTTGATGCCT

Kid toxin (reverse TTAAGTCAGAATAGTGGACAGGCGGCCAAGAACTT 83 orientation) CGTTCATGATAGTCTCCGGAACCCGTTCGAGTCGTT

TTCCGCCCCGTGCTTTCATATCAATTGTCCGGGGTT

GATCGCAACGTACAACACCTGTGGTACGTATGCCA

ACACCATCCAACGACACCGCAAAGCCGGCAGTGCG

GGCAAAATTGCCTCCGCTGGTTACGGGCACAACAA

CAGGCAGGCGGGTCACGCGATTAAAGGCCGCCGGT

GTGACAATCAGCACCGGCCGCGTTCCCTGCTGCTCA

TGACCTGCGGTAGGATCAAGCGAGACAAGCCAGAT

TTCCCCTCTTTCCAT

dapA ATGTTCACGGGAAGTATTGTCGCGATTGTTACTCCG 84

ATGGATGAAAAAGGTAATGTCTGTCGGGCTAGCTT

GAAAAAACTGATTGATTATCATGTCGCCAGCGGTA

CTTCGGCGATCGTTTCTGTTGGCACCACTGGCGAGT

CCGCTACCTTAAATCATGACGAACATGCTGATGTGG

TGATGATGACGCTGGATCTGGCTGATGGGCGCATTC

CGGTAATTGCCGGGACCGGCGCTAACGCTACTGCG

GAAGCCATTAGCCTGACGCAGCGCTTCAATGACAG

TGGTATCGTCGGCTGCCTGACGGTAACCCCTTACTA

CAATCGTCCGTCGCAAGAAGGTTTGTATCAGCATTT

CAAAGCCATCGCTGAGCATACTGACCTGCCGCAAA

TTCTGTATAATGTGCCGTCCCGTACTGGCTGCGATC

TGCTCCCGGAAACGGTGGGCCGTCTGGCGAAAGTA

AAAAATATTATCGGAATCAAAGAGGCAACAGGGAA

CTTAACGCGTGTAAACCAGATCAAAGAGCTGGTTTC

AGATGATTTTGTTCTGCTGAGCGGCGATGATGCGAG CGCGCTGGACTTCATGCAATTGGGCGGTCATGGGGT

TATTTCCGTTACGGCTAACGTCGCAGCGCGTGATAT

GGCCCAGATGTGCAAACTGGCAGCAGAAGGGCATT

TTGCCGAGGCACGCGTTATTAATCAGCGTCTGATGC

CATTACACAACAAACTATTTGTCGAACCCAATCCAA

TCCCGGTGAAATGGGCATGTAAGGAACTGGGTCTT

GTGGCGACCGATACGCTGCGCCTGCCAATGACACC

AATCACCGACAGTGGCCGTGAGACGGTCAGAGCGG

CGCTTAAACATGCCGGTTTGCTGTAA

thyA ATGAAACAGTATTTAGAACTGATGCAAAAAGTGCT 85

CGACGAAGGCACACAGAAAAACGACCGTACCGGA

ACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGT

TTTAACCTGCAAGATGGATTCCCGCTGGTGACAACT

AAACGTTGCCACCTGCGTTCCATCATCCATGAACTG

CTGTGGTTTCTTCAGGGCGACACTAACATTGCTTAT

CTACACGAAAACAATGTCACCATCTGGGACGAATG

GGCC G ATG A A A AC GGC G ACCTC GGGCC AGTGT ATG

GTAAACAGTGGCGTGCCTGGCCAACGCCAGATGGT

CGTCATATTGACCAGATCACTACGGTACTGAACCAG

CTGAAAAACGACCCGGATTCGCGCCGCATTATTGTT

TCAGCGTGGAACGTAGGCGAACTGGATAAAATGGC

GCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGT

GGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCG

CTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT

TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCA

GCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGAC

CGGTGGCGACACGCATCTGTACAGCAACCATATGG

ATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTC

CGCTGCCGAAGTTGATTATCAAACGTAAACCCGAA

TCC ATCTTC G ACT ACC GTTTC G A AG ACTTTG AG ATT

GAAGGCTACGATCCGCATCCGGGCATTAAAGCGCC

GGTGGCTATCTAA

Kid toxin MERGEIWLVSLDPTAGHEQQGTRPVLIVTPAAFNRVT 86 polypeptide RLPVVVPVTSGGNFARTAGFAVSLDGVGIRTTGVVRC

DQPRTIDMKARGGKRLERVPETIMNEVLGRLSTILT* dapA polypeptide MFTGS IVAIVTPMDEKGNVCRASLKKLID YHVAS GTS 87

AIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVI AGTGANATAE AIS LTQRFNDS GIVGCLT VTPYYNRPS QEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVG RLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGD DAS ALDFMQLGGHG VIS VT AN V A ARDM AQMC KLA A EGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKEL GLVATDTLRLPMTPITDSGRETVRAALKHAGLL

ThyA polypeptide MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRF 88

NLQDGFPLVTTKRCHLRSIIHELLWFLQGDTNIAYLHE

NNVTIWDEWADENGDLGPVYGKQWRAWPTPDGRHI

DQITT VLNQLKNDPDS RRIIVS A WN VGELD KM ALAPC

H AFFQFY V AD GKLS C QLYQRS CD VFLGLPFNIAS Y AL

LVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTH

LQLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPG IKAPVAP

Table 29. Chromosomally Inserted Biosafety System Constructs

medium copy AAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGCG Rep (Pi) and Kis AGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAGA antitoxin (as ACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTGA shown in Fig. AGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTGC

61D) TTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTGC

CAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTTT

AGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACTC

CCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAGC

TTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCAC

CAGAACCATAGAACCATATATCTCTAGCCTTATTGGGAA

AAAAAATAAATTCACAACGCAATTGTTAACGGCAAGCTT

ACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAACTT

ATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATTAT

TTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAATA

GCTTATACTTTTGATAAAGATGGAAATATTGAGTACAAA

TACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAAATA

AAGCCATTGCTGAAATTAAAAAGAAAACAGAAATATCG

TTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGAAAA

ATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAAGAT

GAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTTATG

AATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTATTTG

ATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGAGGA

TCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA

GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACG

CTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC

CTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGACTtaaa gtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGGAGG

TAACAACCATGCGAGTGTTGAAGAAACATCTTAATCATG

CTAAGGAGGTTTTCTAATGCATACCACCCGACTGAAGAG

GGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGCACT

GCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGTTGG

CATGGTCATTGATAATGGCCGGCTGATTGTTGAGCCGTA

CAGACGCCCGCAATATTCACTGGCTGAGCTACTGGCACA

GTGTGATCCGAATGCTGAAATATCAGCTGAAGAACGAG

AATGGCTGGATGCACCGGCGACTGGTCAGGAGGAAATC

TGA

Rep (Pi) TGAGACTCAAGGTCATGATGGACGTGAACAAAAAAACG 91

AAAATTCGCCACCGAAACGAGCTAAATCACACCCTGGCT

CAACTTCCTTTGCCCGCAAAGCGAGTGATGTATATGGCG

CTTGCTCCCATTGATAGCAAAGAACCTCTTGAACGAGGG

CGAGTTTTCAAAATTAGGGCTGAAGACCTTGCAGCGCTC

GCCAAAATCACCCCATCGCTTGCTTATCGACAATTAAAA

GAGGGTGGTAAATTACTTGGTGCCAGCAAAATTTCGCTA

AGAGGGGATGATATCATTGCTTTAGCTAAAGAGCTTAAC

CTGCTCTTTACTGCTAAAAACTCCCCTGAAGAGTTAGAC

CTTAACATTATTGAGTGGATAGCTTATTCAAATGATGAA

GGATACTTGTCTTTAAAATTCACCAGAACCATAGAACCA

TATATCTCTAGCCTTATTGGGAAAAAAAATAAATTCACA

AC GC A ATTGTT A AC GGC A AGCTT AC GCTT A AGT AGCC AG

TATTCATCTTCTCTTTATCAACTTATCAGGAAGCATTACT CTAATTTTAAGAAGAAAAATTATTTTATTATTTCCGTTGA

TGAGTTAAAGGAAGAGTTAATAGCTTATACTTTTGATAA

AGATGGAAATATTGAGTACAAATACCCTGACTTTCCTAT

TTTTAAAAGGGATGTGTTAAATAAAGCCATTGCTGAAAT

TAAAAAGAAAACAGAAATATCGTTTGTTGGCTTCACTGT

TCATGAAAAAGAAGGAAGAAAAATTAGTAAGCTGAAGT

TCGAATTTGTCGTTGATGAAGATGAATTTTCTGGCGATA

AAGATGATGAAGCTTTTTTTATGAATTTATCTGAAGCTG

ATGCAGCTTTTCTCAAGGTATTTGATGAAACCGTACCTC

CCAAAAAAGCTAAGGGGTGA

Kis antitoxin CATACCACCCGACTGAAGAGGGTTGGCGGCTCAGTTATG 92

CTGACCGTCCCACCGGCACTGCTGAATGCGCTGTCTCTG

GGCACAGATAATGAAGTTGGCATGGTCATTGATAATGGC

CGGCTGATTGTTGAGCCGTACAGACGCCCGCAATATTCA

CTGGCTGAGCTACTGGCACAGTGTGATCCGAATGCTGAA

ATATCAGCTGAAGAACGAGAATGGCTGGATGCACCGGC

GACTGGTCAGGAGGAAATCTGA

RBS (low copy) GCTGGAACAGGTGG 93

RBS (medium TCCGGAAGACTAGG 94 copy)

Informal Sequence Listing

GTGTCAGTTTAAACCATGAGCACTTGGATACGACTGAA TTAATTTCTTATCCATTACGATCAGAAGCACAATACTAT GCAGTTAACTAA

Bile salt hydrolase MCT AIT YQS YNN YFGRNFD YEIS YNEM VTITPRKYPLVFR protein from KVENLDHHYAIIGITADVESYPLYYDAMNEKGLCIAGLNF Lactobacillus AGYADYKKYDADKVNITPFELIPWLLGQFSSVREVKKNIQ plantarum KLNLVNINFSEQLPLSPLHWLVADKQESIVIESVKEGLKIY

DNP VG VLTNNPNFD YQLFNLNN YRALS NS TPQNS FS EKVD LDS YS RGMGGLGLPGDLS SMS RFVR A AFTKLNS LS MQTES GSVSQFFHILGSVEQQKGLCEVTDGKYEYTIYSSCCDMDK GV Y Y YRT YDNS QINS VS LNHEHLDTTELIS YPLRS E AQ Y Y AVN

Bile salt hydrolase ATGTGTACTGCTGCAAATTATTTAACAAAATGCCATTAT from TTTGGCCGTAATTTTGACTATGAAATTTCATATAATGAA

Methanobrevibacte AGAGTAACGATAACTCCTAGAAACTATCCTTTAATATTC r smithii 3142 AGGGATACTGAGGACATTGAAAATCATTATGGGATTAT

TGGCATAGCTGCAGGTATTGATGAATATCCTTTGTATTA

TGATGCATGTAATGAGAAAGGATTAGCTATGGGGGGAT

TAAACTTTCCGGATTACTGTGACTACAAACCACTAGATA

AATCTAAAGTTAACATAGCTTCTTTTGAGATTATTCCAT

ATATATTATCTCAAGCAAAAACCATCAGTGATGCCGAA

AGGTTATTGGAAAACTTAAATATTTCAGATGAGAAATT

TTCCGCCCAGTTGCCTCCATCTCCACTTCATTGGATTATT

TCAGATAGGAATGCTTCAATTGTTGTAGAGGTTGTAGA

GGAAGGACTGGATATTTATGATAATCCTGTAGGAGTTTT

AACAAACAACCCTCCTTTTGATAAACAGCTATTTAATTT

AAATAATTATATGGCATTATCAAACAGAACGCCTGAAA

ATACCTTTGGAGGCAATTTGGATTTGGCAACTTATAGTC

GGGGAATGGGTTCAATTGGTCTTCCGGGGGATGTTTCTT

CACAGTCCCGTTTTGTAAAAGCAGCTTTTGTTAAAGAAA

ATTCCGTTTCCGGAGATTCTGAAAAAGAAAGTGTGTCTC

AGTTTTTCCATATTCTGGCATCTGTTGAACAGCAAAAAG

GATGTACGTTAGTGGAAGAACCTGATAAATTTGAGTAT

ACTATTTATTCAGACTGTTACAATACAGATAAGGGAAT

ATTGTATTATAAAACATATGATGGTCCTCAAACATCTGT

TAATATACATGATGAGGATTTGGAAACCAATCAGTTAA

TTAATTTTGAGTTGGTTGATTAA

Bile salt hydrolase MCT AANYLTKCHYFGRNFD YEIS YNERVTITPRNYPLIFRD protein from TEDIENHYGIIGIAAGIDEYPLYYDACNEKGLAMGGLNFPD Methanobrevibacte YCDYKPLDKSKVNIASFEIIPYILSQAKTISDAERLLENLNIS r smithii 3142 DEKFS AQLPPS PLHWIIS DRN AS IV VE V VEEGLDIYDNP VG

VLTNNPPFDKQLFNLNNYMALSNRTPENTFGGNLDLATYS RGMGS IGLPGD VS S QS RFVKA AF VKENS VS GDS EKES VS Q FFHILASVEQQKGCTLVEEPDKFEYTIYSDCYNTDKGILYY KTYDGPQTSVNIHDEDLETNQLINFELVD

Prp (Propionate) TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGA promoter CGCCACAGCGTGGTACGGCTGATCCCCAAATAACGT Bold: prpR GCGGCGGCGCGCTTATCGCCATTAAAGCGTGCGAGC Lower case: ACCTCCTGCAATGGAAGCGCTTCTGCTGACGAGGGC ribosome binding GTGATTTCTGCTGTGGTCCCCACCAGTTCAGGTAAT site AATTGCCGCATAAATTGTCTGTCCAGTGTTGGTGCG

ATG underlined: GGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCATC start of gene of ATATTCCGCAGTTCGCGAATATTACCGGGCCAATGA interest TAGTTCAGTAGAAGCGGCTGACACTGCGTCAGCCCA

TGACGCACCGATTCGGTAAAAGGGATCTCCATCGCG

GCCAGCGATTGTTTTAAAAAGTTTTCCGCCAGAGGC

AGAATATCAGGCTGTCGCTCGCGCAAGGGGGGAAG

CGGCAGACGCAGAATGCTCAAACGGTAAAACAGATC

GGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG

ATCGCAATGCGTGGCGCTGATCACCCGGACATCTAC

CGGGATCGGCTGATGCCCGCCAACGCGGGTGACGG

CTTTTTCCTCCAGTACGCGTAGAAGGCGGGTTTGTA

ACGGCAGCGGCATTTCGCCAATTTCGTCAAGAAACA

GCGTGCCGCCGTGGGCGACCTCAAACAGCCCCGCAC

GTCCACCTCGTCTTGAGCCGGTAAACGCTCCCTCCT

CATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGG

TAATCGCGCCGCAATTAACGGCGACAAAGGGCGGAG

AAGGCTTGTTCTGACGGTGGGGCTGACGGTTAAACA

ACGCCTGATGAATCGCTTGCGCCGCCAGCTCTTTCC

CGGTCCCTGTTTCCCCCTGAATCAGCACTGCCGCGC

GGGAACGGGCATAGAGTGTAATCGTATGGCGAACCT

GCTCCATTTGTGGTGAATCGCCGAGGATATCGCTCA

GCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTAT

GCTGGCTATACTGACGCCGTGTCAGGCGGGTCATAT

CCAGCGCATCATGGAAAGCCTGACGTACGGTGGCCG

CTGAATAAATAAAGATGGCGGTCATTCCTGCCTCTT

CCGCCAGGTCGGTAATTAGTCCTGCCCCAATTACAG

CCTCAATGCCGTTAGCTTTGAGCTCGTTAATTTGCCC

GCGAGCATCCTCTTCAGTGATATAGCTTCGCTGTTC

AAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG

AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCAT

TGAGGAAGTCAGCTTTCCCGCTTTTGCCAGAGCCTG

TAATACATCGAATCCGCTGGGTTTGATGAGGATGAC

AGGTACCGACAGTCGGCTTTTTAAATAAGCGCCGTT

GGAACCTGCCGCGATAATCGCGTCGCAGCGTTCGGT

TGCCAGTTTTTTGCGAATGTAGGCTACTGCCTTTTCA

AAACCGAGCTGAATAGGCGTGATCGTCGCCAGATGA

TCAAACTCCAGGCTGATATCCCGAAATAGTTCGAAC

AGGCGCGTTACCGAGACCGTCCAGATCACCGGTTTA

TCGCTATTATCGCGCGAAGCGCTATGCACAGTAACC

ATCGTCGTAGATTCATGTTTAAGGAACGAATTCTTGTTT

TATAGATGTTTCGTTAATGTTGCAATGAAACACAGGCCT

CCGTTTCATGAAACGTTAGCTGACTCGTTTTTCTTGTGA

CTCGTCTGTCAGTATTAAAAAAGATTTTTCATTTAACTG

ATTGTTTTTAAATTGAATTTTATTTAATGGTTTCTCGGTT

TTTGGGTCTGGCATATCCCTTGCTTTAATGAGTGCATCT

TAATTAACAATTCAATAACAAGAGGGCTGAATagtaatttcaa caaaataacgagcattcgaatg FNR responsive GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGC promoter #1 CGGGCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTAC

TCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTT

GTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCC

GTGACTTAAGAAAATTTATACAAATCAGCAATATACCC

CTTAAGGAGTATATAAAGGTGAATTTGATTTACATCAAT

AAGC GGGGTTGCTG A ATC GTT A AGGT AGGC GGT A AT AG

AAAAGAAATCGAGGCAAAA

FNR responsive ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCC promoter #2 CGACTTATGGCTCATGCATGCATCAAAAAAGATGTGAG

CTTGATCAAAAACAAAAAATATTTCACTCGACAGGAGT ATTTATATTGCGCCCGTTACGTGGGCTTCGACTGTAAAT CAGAAAGGAGAAAACACCT

FNR responsive GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGC promoter #3 CGGGCGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTAC

TCTGCTACGTACATCTATTTCTATAAATCCGTTCAATTT

GTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCC

GTGACTTAAGAAAATTTATACAAATCAGCAATATACCC

CTTAAGGAGTATATAAAGGTGAATTTGATTTACATCAAT

AAGCGGGGTTGCTGAATCGTTAAGGATCCCTCTAGAAA

TAATTTTGTTTAACTTTAAGAAGGAGATATACAT

FNR responsive CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCC promoter #4 CCGACTTATGGCTCATGCATGCATCAAAAAAGATGTGA

GCTTGATCAAAAACAAAAAATATTTCACTCGACAGGAG

TATTTATATTGCGCCCGGATCCCTCTAGAAATAATTTTG

TTTAACTTTAAGAAGGAGATATACAT

FNR responsive AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGT promoter #5 AGTAAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCT

GT AT AC A A A A AC GCC GT A A AGTTTG AGC G A AGTC A AT A AACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT

ACAT

cbsTl from ATGTCGACCACACCGACACAGCCATCATCACGAAAACA

Lactobacillus GGCTGTTTACCCGTACTTGATCGTGCTGTCGGGCATCGT johnsonii CTTCACGGCCATCCCGGTATCGCTGGTCTGCAGTTGCGC

AGGTATCTTCTTCACGCCTGTCAGCAGCTACTTCCATGT

TCCCAAGGCCGCATTCACCGGATATTTCAGCATATTCAG

CATCACCATGGTCGCCTTCCTGCCGGTGGCCGGATGGCT

GATGCACCGCTACGATCTGCGCATCGTACTGACCGCAA

GCACCGTCCTGGCTGGACTGGGCTGCCTGGGTATGTCCC

GATCATCCGCCATGTGGCAGTTCTATCTATGCGGAGTGG

TTCTGGGAATCGGCATGCCGGCCGTCCTCTATCTGTCAG

TGCCAACACTCATCAACGCCTGGTTCCGCAAGCGGGTC

GGGTTCTTCATCGGCCTGTGCATGGCCTTCACCGGCATA

GGCGGCGTGATCTTCAACCAGATAGGCACCATGATCAT

CAGATCCGCCCCTGATGGATGGAGGCGGGGATATCTGG

TTTTCGCTATTCTCATCCTGGTGATCACCCTGCCCTTCAC

CATTTTCGTCATTCGCAGCACACCCGAACAGATGGGTCT

GCATCCCTACGGCGCCGACCAGGAGCCTGATGCAGCTG

AGACGGCCACCAATAGTGCAGGCACCGGGAGCAAAGA CCAAAAGAGTCCTGAGCCTGCAGCGTCAACCGTAGGCA

TGACTGCCTCCCAGGCCTTGCGCTCCCCTGCCTTCTGGG

CGCTGGCGCTCTTCTGCGGTCTGATCACCATGAATCAGA

CCATTTACCAGTTCCTGCCCTCCTACGCGGCATCCCTGC

CATCCATGGCAGCCTACACGGGACTGATCGCCTCCTCCT

GCATGGCCGGCCAGGCCATCGGCAAGATCATCCTGGGC

ATGGTCAACGACGGCAGCATCGTAGGCGGTCTCTGTCT

GGGCATCGGCGGCGGCATTCTCGGCGTCTGCCTCATGG

TCGCCTTCCCCGGATTGCCCGTGCTCCTCCTGCTGGGAG

CCTTTGCCTTCGGCCTTGTCTACGCCTGCACTACTGTGC

AGACACCAATCCTGGTTACAGCGGTCTTCGGCTCGCGC

GACTACACCAACATCTATGCACGTATCCAGATGGTTGG

GTCCCTAGCCTCGGCCTTCGCAGCTCTCTTCTGGGGCGC

CATCGCTGACCAGCCCCACGGCTACATCATCATGTTCGG

TCTGAGCATCCTGATCATGGTTGTGGCCTTGTTCCTAGG

CATTATCCCTCTGAAAGGTACGCGCAAGTTGACCGATC

AGATCGCCTGA

CbsTl protein from MSTTPTQPSSRKQAVYPYLIVLSGIVFTAIPVSLVCSCAGI

Lactobacillus FF PVSSYFHVPKAAFTGYFSIFSITMVAFLPVAGWLMHR johnsonii YDLRIVLT AS T VL AGLGCLGMS RS S AMWQF YLC G V VLG

IGMPAVLYLSVPTL1NAWFRKRVGFFIGLCMAFTGIGGVI

F QIGTMIIRSAPDGWRRGYLVFAILILVITLPFTIFVIRSTP

EQMGLHPYGADQEPDAAETATNSAGTGSKDQKSPEPAA

STVGMTASQALRSPAFWALALFCGLITMNQTIYQFLPSY

AASLPSMAAYTGLIASSCMAGQAIGKIILGMVNDGSIVG

GLCLGIGGGILGVCLMVAFPGLPVLLLLGAFAFGLVYAC

TTVQTPILVTAVFGSRDYTNIYARIQMVGSLASAFAALF

WGAIADQPHGYIIMFGLSILIMVVALFLGIIPLKGTRKLTD

QIA

cbsT2 from atgtctactgatgccgctactaaagataaagtagtaagcaagggctataaatacttcatggtttt

Lactobacillus cctttgtatgttaacccaagctattccttatggaattgctcaaaacattcagcctttgtttatccac johnsonii cctttagttaatactttccactttaccttagcatcgtacacattaatttttacgtttggtgcggtttttg cttcagttgcttctccatttattggtaaggcattagaaaaagttaacttccgactaatgtatttaatt ggtattggtctttctgctattgcctacgtaatttttggaattagtacaaaactacccggtttctatat tgccgctatcatttgtatggttggttcaaccttttactccggccaaggtgttccctgggttattaac cactggttcccagcaaagggacgtggggctgccttaggaattgccttctgcggtggttctatt ggtaatatctttttacaaccagcaacccaagctattttaaaacactacatgacaggtaatactaa gaccggtcatttaacctctatggcaccattctttatctttgccgtagctttattagtaatcggtgta attatcgcctgcttcattagaacccctaagaaagacgaaattgttgtttctgatgcagaactagc tgaaagcaagaaagctgaagccgcagccaaagctaaagagtttaaaggctggactagtaa acaagtgttacaaatgaaatggttctggattttcagccttggtttcttaatcattggtttaggctta gcttctttaaatgaagactatgccgccttccttgatactaagctttctttaaccgatgttggtttagt tgggtcaatgtacggtgttggttgtttaatcggaaatatttctggtggtttcttatttgataaatttg gtacagcaaaatcaatgacctatgctggttgtatgtatattttatctattctgatgatgatctttatta gcttccagccatatggttcatctattagtaaggctgctggcattggctatgctatcttttgcggct tagctgtatttagttacatgtcaggcccagccttcatggcaaaagacctctttggttcaagagat caaggtgtcatgcttggatacgttggtttagcttatgcaattggctatgccattggtgctccacta tttgggattattaagggagcggcaagctttacagttgcttggtactttatgattgcctttgttgcaa ttggttttatcattttagtatttgccgttatccaaattaagagataccaaaagaaatacattgcaga gcaagcagcaaaagctaatgctaaataa CbsT2 protein from MSTDAATKDKVVSKGYKYFMVFLCMLTQAIPYGIAQNI

Lactobacillus QPLFIHPLVNTFHFTLASYTLIFTFGAVFASVASPFIGKAL johnsonii EKVNFRLMYLIGIGLSAIAYVIFGISTKLPGFYIAAIICMVG

STFYSGQGVPWVINHWFPAKGRGAALGIAFCGGSIGNIF

LQPATQAILKHYMTG TKTGHLTSMAPFFIFAVALLVIG

VnACFIRTPKKDEIVVSDAELAESKKAEAAAKAKEF G

WTS KQ VLQM WFWIFS LGFLIIG LGL A S LNED Y A AFLDT

KLSLTDVGLVGSMYGVGCLIGNISGGFLFDKFGTAKSMT

YAGCMYILS ILMMIFISFQPYGSSISKAAGIGYA1FCGLAV

FSYMSGPAFMAKDLFGSRDQGVMLGYVGLAYAIGYAIG

APLFGIIKGAASFTVAVVYFMIAFVAIGFIILVFAVIOIKRY

Q KYIAEQAAKANAK

Bile salt hydrolase ATGGTTATGAAAAAGATTTTGATAGCTTTGGCCTTATTG from Bacteroides CTGACAGGCATTGCAAGCGGATCGGCATGTACCGGTAT vulgatus TTCATTCCTCGCTGAAGATGGCGGATATGTGCAGGCAC

GTACTATAGAGTGGGGGAACAGTTATCTTCCGAGTGAA

TATGTTATTGTTCCCAGAGGACAGGATTTGGTATCTTAT

ACTCCAACGGGTGTAAATGGCTTGAGATTTCGGGCTAA

ATATGGTCTGGTAGGACTGGCTATCATTCAGAAAGAGT

TTGTGGCTGAAGGACTGAATGAAGTAGGGCTTTCGGCT

GGATTGTTTTATTTTCCCCATTATGGGAAGTATGAAGAA

TATGATGAGGCTCAAAATGCAATTACTTTGTCGGATTTG

CAGGTGGTGAACTGGATGCTTTCCCAATTTGCTACTATA

GACGAAGTGAGAGAAGCTATAGAAGGGGTGAAGGTGG

TGTCTCTTGATAAACCTGGTAAAAGTTCTACGGTACATT

GGCGCATTGGCGATGCTAAAGGAAATCAAATGGTGTTG

GAATTTGTAGGTGGTGTTCCTTATTTTTATGAAAATAAA

GTAGGAGTACTCACCAATTCTCCCGATTTTCCATGGCAG

GTGATTAACTTGAATAATTATGTAAATCTATATCCGGGA

GCTGTCACTCCACAGCAATGGGGTGGGGTGACTATTTTC

CCTTTTGGCGCAGGTGCCGGATTTCATGGTATTCCGGGG

GATGTAACTCCTCCATCCCGTTTTGTTCGTGTAGCGTTTT

ATAAGGCAACAGCTCCGGTGTGTCCTACAGCGTATGAC

GCTATATTACAAAGCTTTCATATCCTGAATAATTTTGAT

ATTCCTATTGGTATAGAATATGCGTTAGGGAAAGCACC

TGATATTCCTAGTGCCACACAATGGACTTCGGCTATTGA

TTTGACAAACAGGAAAGTGTATTATAAAACAGCATACA

ATAACAATATTCGTTGTATTAGTATGAAGAAGATTGATT

TTGATAAAGTGAAGTATCAGTCGTATCCATTGGATAAG

GAGTTGAAACAGCCTGTAGAAGAGATTATTGTGAAATA

G

Bile salt hydrolase MVMKKILIALALLLTGIASGSACTGISFLAEDGGYVQARTI protein from EWGNS YLPS E Y VIVPRGQDLVS YTPTG VNGLRFR AKYGL Bacteroides VGLAIIQKEFVAEGLNEVGLSAGLFYFPHYGKYEEYDEAQ vulgatus NAITLSDLQVVNWMLSQFATIDEVREAIEGVKVVSLDKPG

KSSTVHWRIGDAKGNQMVLEFVGGVPYFYENKVGVLTNS

PDFPWQVINLNNYVNLYPGAVTPQQWGGVTIFPFGAGAG

FHGIPGDVTPPSRFVRVAFYKATAPVCPTAYDAILQSFHIL

NNFDIPIGIEYALGKAPDIPSATQWTSAIDLTNRKVYYKTA

YNNNIRCISMKKIDFDKVKYQSYPLDKELKQPVEEIIVK Bile salt hydrolase ATGTGCACTGGTGTCCGTTTCTCCGATGATGAGGGCAAC from ACCTATTTCGGCCGTAATCTCGACTGGAGTTTCTCATAT

Bifidobacterium GGGGAGACCATCCTGGTTACTCCGCGCGGCTACCACTA longum TG AC AC GGTGTTTGGTGC GGGCGGC A AGGC G A AGCC G A

ACGCGGTGATCGGCGTGGGTGTGGTCATGGCCGATAGG

CCGATGTATTTCGACTGCGCCAATGAACATGGTCTGGCC

ATCGCCGGCTTGAATTTCCCCGGCTACGCCTCGTTCGTC

CACGAACCGGTCGAAGGCACGGAAAACGTCGCCACGTT

CGAATTTCCGCTGTGGGTGGCGCGTAATTTCGACTCCGT

CGACGAGGTCGAGGAGGCGCTCAGGAACGTGACGCTCG

TCTCCCAGATCGTGCCGGGACAGCAGGAGTCTCTGCTG

CACTGGTTCATCGGCGACGGCAAGCGCAGCATCGTCGT

CGAGCAGATGGCCGATGGCATGCACGTGCATCATGATG

ACGTCGATGTGCTGACCAATCAGCCGACGTTCGACTTCC

ATATGGAAAACCTGCGCAACTACATGTGCGTCAGCAAC

GAGATGGCCGAACCGACTTCATGGGGCAAGGCCTCCTT

GACCGCCTGGGGTGCGGGTGTGGGCATGCATGGCATCC

CGGGCGACGTGAGTTCCCCGTCGCGCTTCGTTCGTGTGG

CCTACACCAACGCGCATTACCCGCAGCAGAACGATGAA

GCCGCCAATGTGTCGCGCCTGTTCCACACCCTCGGCTCC

GTGCAGATGGTGGACGGCATGGCGAAGATGGGCGACG

GCCAGTTCGAACGCACGCTGTTCACCAGCGGATATTCG

TCCAAGACCAACACCTATTACATGAACACCTATGATGA

CCCCGCCATCCGTTCCTACGCCATGGCCGATTACGATAT

GGATTCCTCGGAGCTCATCAGCGTCGCCCGATGA

Bile salt hydrolase MCTG VRFS DDEGNT YFGRNLD WS FS YGETILVTPRG YH Y protein from DTVFGAGGKAKPNAVIGVGVVMADRPMYFDCANEHGLA Bifidobacterium IAGLNFPGYASFVHEPVEGTENVATFEFPLWVARNFDSVD longum EVEEALRNVTLVSQIVPGQQESLLHWFIGDGKRSIVVEQM

ADGMHVHHDDVDVLTNQPTFDFHMENLRNYMCVSNEM

AEPTSWGKASLTAWGAGVGMHGIPGDVSSPSRFVRVAYT

NAHYPQQNDEAANVSRLFHTLGSVQMVDGMAKMGDGQ

FERTLFTS GYS S KTNT YYMNTYDDPAIRS YAMAD YDMDS

SELISVAR

Bile salt hydrolase ATGTGTACGTCAATAACTTATACAACGAAGGATCACTA from Listeria TTTTGGAAGGAATTTCGATTATGAACTTTCTTACAAAGA monocytogenes AGTTGTGGTTGTTACGCCGAAAAATTACCCGTTCCATTT

TCGCAAGGTAGAGGATATAGAGAAGCATTATGCACTTA

TTGGTATTGCTGCTGTGATGGAAAACTACCCGTTGTATT

ACGATGCTACCAATGAAAAAGGCCTTAGTATGGCAGGA

CTCAATTTCTCAGGAAATGCGGATTACAAGGATTTTGCA

GAAGGTAAGGACAATGTGACCCCCTTTGAATTTATTCC

GTGGATTCTTGGTCAATGCGCTACTGTAAAAGAAGCAA

GAAGATTACTTCAGAGAATCAATCTCGTGAATATTAGTT

TTAGTGAAAATTTACCGCTGTCTCCATTACATTGGTTGA

TGGCTGATCAAACAGAATCTATTGTAGTGGAATGTGTG

AAAGATGGACTTCACATTTATGATAATCCTGTTGGCGTG

TTAACAAATAATCCAACATTTGATTACCAACTATTTAAT

TTAAACAATTATCGCGTTCTTTCGAGTGAAACCCCAGAA

AATAATTTTTCCAAAGAGATTGATTTGGATGCTTATAGT CGTGGGATGGGCGGAATTGGCTTACCTGGTGATTTATCT

TCTATGTCTCGTTTTGTGAAAGCAACTTTTACCAAATTG

AATTCTGTTTCAGGTGATTCTGAATCAGAAAGTATTAGC

CAATTTTTCCATATTTTAGGCTCGGTGGAACAACAAAAA

GGTCTTTGTGATGTTGGTGGGGGAAAATACGAGCATAC

TATTTATTCCTCGTGTTGCAATATCGATAAAGGAATTTA

TTATTATAGAACATACGGAAACAGTCAAATTACTGGTG

TGGATATGCACCAAGAGGATTTAGAGAGCAAAGAACTA

GCTATTTATCCACTCGTCAATGAGCAACGACTAAACATT

GTTAACAAATAA

Bile salt hydrolase MCTSITYTTKDHYFGRNFDYELSYKEVVVVTPKNYPFHFR protein from KVEDIEKHYALIGIAAVMENYPLYYDATNEKGLSMAGLN Listeria FSGNADYKDFAEGKDNVTPFEFIPWILGQCATVKEARPvLL monocytogenes QRINLVNIS FSENLPLS PLHWLM ADQTES IVVEC VKDGLHI

YDNP VG VLTNNPTFD YQLFNLNN YR VLS S ETPENNFS KEI DLDAYSRGMGGIGLPGDLSSMSRFVKATFTKLNSVSGDSE SESISQFFHILGSVEQQKGLCDVGGGKYEHTIYSSCCNIDK GIYYYRT YGNS QITG VDMHQEDLES KELAIYPLVNEQRLN IVNK

Bile salt hydrolase ATGTGTACAGGATTAGCCTTAGAAACAAAAGATGGATT from Clostridium ACATTTGTTTGGAAGAAATATGGATATTGAATATTCATT perfringens TAATCAATCTATTATATTTATTCCTAGGAATTTTAAATG

TGTAAACAAATCAAACAAAAAAGAATTAACAACAAAA

TATGCTGTTCTTGGAATGGGAACTATTTTTGATGATTAT

CCTACCTTTGCAGATGGTATGAATGAAAAGGGATTAGG

GTGTGCTGGCTTAAATTTCCCTGTTTATGTTAGCTATTCT

AAAGAAGATATAGAAGGTAAAACTAATATTCCAGTATA

TAATTTCTTATTATGGGTTTTAGCTAATTTTAGCTCAGTA

GAAGAGGTAAAGGAAGCATTAAAAAATGCTAATATAGT

GGATATACCTATTAGCGAAAATATTCCTAATACAACTCT

TCATTGGATGATAAGCGATATAACAGGAAAGTCTATTG

TGGTTGAACAAACAAAGGAAAAATTAAATGTATTTGAT

AATAATATTGGAGTATTAACTAATTCACCTACTTTTGAT

TGGCATGTAGCAAATTTAAATCAATATGTAGGTTTGAG

ATATAATCAAGTTCCAGAATTTAAGTTAGGAGATCAAT

CTTTAACTGCTTTAGGTCAAGGAACTGGTTTAGTAGGAT

TACCAGGGGACTTTACACCTGCATCTAGATTTATAAGA

GTAGCATTTTTAAGAGATGCAATGATAAAAAATGATAA

AGATTCAATAGACTTAATTGAATTTTTCCATATATTAAA

TAATGTTGCTATGGTAAGAGGATCAACTAGAACTGTAG

AAGAAAAAAGTGATCTTACTCAATATACAAGTTGCATG

TGTTTAGAAAAAGGAATTTATTATTATAATACCTATGAA

AATAATCAAATTAATGCAATAGACATGAATAAAGAAAA

CTTAGATGGAAATGAAATTAAAACATATAAATACAACA

AAACTTTAAGTATTAATCATGTAAATTAG

Bile salt hydrolase MCTGLALETKDGLHLFGRNMDIEYSFNQSIIFIPRNFKCV protein from NKSNKKELTTKYAVLGMGTIFDDYPTFADGMNEKGLGC Clostridium AG LNFP V Y VS YS KED IEGKTNIPV YNFLLW V LANFS S VE perfringens EVKEALK A IVDIPISENIPNTTLHWMISDITGKSIVVEQ

TKEKLNVFDNNIGVLTNSPTFDWHVANLNQYVGLRYNQ EFKLGDQSLTALGQGTGLVGLPGDFTPASRFIRVAFLR DAMI NDKDSIDLIEFFHILNNVAMVRGSTRTVEE SDLT QYTSCMCLEKGIYYYNTYENNQINAIDMNKENLDGNEIK TYK YNKTLSIN VN

Bile salt hydrolase ATGTGTACGTCTATTACTTATGTAACAAGTGATCATTAT from Enterococcus TTTGGAAGGAATTTTGATTATGAAATATCTTACAATGAA faecium GTAGTTACTGTTACTCCAAGAAATTATAAGTTGAATTTT

CGAAAGGTAAATGATTTGGATACTCATTATGCAATGAT

TGGTATTGCCGCTGGTATAGCTGACTACCCTCTTTATTA

CGATGCGACAAATGAAAAAGGATTGAGTATGGCTGGGC

TAAATTTTTCTGGGTATGCTGATTATAAAGAAATACAAG

AAGGGAAAGACAATGTATCTCCTTTTGAATTTATTCCTT

GGATTTTAGGACAATGCTCAACAGTAGGAGAAGCTAAA

AAATTGTTAAAAAATATCAATTTAGCAAATATAAATTA

TAGTGACGAACTTCCTTTATCCCCTTTACATTGGCTATT

AGCTGATAAAGAAAAATCAATTGTCATTGAAAGTATGA

AAGATGGACTTCATATATATGATAACCCTGTGGGCGTTC

TTACCAATAATCCTTCATTTGACTATCAATTATTTAATTT

AAACAATTATCGTGTCTTATCGAGTGAAACTCCTAAAA

ATAATTTTTCAAATCAAATAAGTTTGAATGCCTATAGCC

GCGGTATGGGAGGGATAGGCTTGCCTGGAGATTTATCC

TCAGTATCTCGTTTTGTTAAAGCGACTTTTACGAAGCTG

AATTCTGTATCTGGAGATTCAGAGTCAGAAAGTATTAG

TCAATTTTTCCATATCTTAGGTTCAGTAGAACAACAAAA

AGGTTTGTGTGATGTAGGTGATGGAAAATATGAATATA

CAATTTATTCTTCTTGTTGCAATGTTGACAAAGGAATCT

ATTATTATCGAACATATGAAGACAGTCAAATTACTGCA

ATTGATATGAATAAAGAAGACTTAGATAGTCATAAGTT

AATTAGTTATCCAATTATAGAAAAACAACAAATTAAAT

ATATAAATTAG

Bile salt hydrolase MCTSITYVTSDHYFGRNFDYEISYNEVVTVTPRNYKLNF protein from RKVNDLDTHYAMIGIAAGIADYPLYYDATNEKGLSMAG Enterococcus LNFSGYADYKEIQEGKDNVSPFEFIPWILGQCSTVGEAKK faecium LLKNINLANINYSDFT PLSPLHWLLADKEKS IVIES MKDG

LHIYDNPVGVLTNNPSFDYQLFNLNNYRVLSSETPKNNF

SNQISLNAYSRGMGGIGLPGDLSSVSRFVKATFTKLNSVS

GDSESESISQFFHILGSVEQQKGLCDVGDGKYEYTIYSSC

CNVD GIYYYRTYEDSQITAIDM EDLDSHKLISYPIIE QQIKYIN

Bile salt hydrolase AAGAGAAAAATATGTGTACATCAATTATATTCAGTCCC A from AAAGATCATTACTTTGGTCGTAACCTTGATTTAGAAATT Lacotbacillus ACTTTTGGTCAACAAGTTGTTATTACGCCACGCAATTAC acidophilus ACTTTTAAATTCCGTAAGATGCCCAGTTTAAAAAAGCA

CTATGCAATGATTGGTATCTCATTAGATATGGATGATTA

TCCCCTATATTTCGACGCTACAAATGAAAAAGGTTTAG

GTATGGCCGGACTCAACTATCCAGGAAATGCTACATAT

TATGAAGAAAAAGAAAATAAAGATAATATTGCTTCCTT

TGAATTCATCCCTTGGATTTTAGGACAGTGTAGCACTAT

TAGCGAAGTAAAGGATTTACTTAGCAGAATCAACATCG

CCGATTTAAATTTCAGCGAAAAAATGCAAGCCTCCTCTC TTCACTGGCTTATTGCAGATAAAACAGGTACATCATTAG

TTGTTGAAACAGACAAAGATGGAATGCATATTTATGAT

AATCCAGTTGGCTGCTTAACTAATAATCCACAATTTCCA

AAGCAATTATTCAATTTAAATAACTATGCTGACGTATCT

CCAAAAATGCCTAAAAATAACTTCTCAGATAAAGTAAA

TATGGCTGGCTACAGCCGTGGATTAGGGTCTCACAACTT

ACCAGGTGGAATGGATTCTGAATCACGTTTTGTCAGAG

TAGCTTTCAATAAATTTAATGCTCCAATTGCTGAAACCG

AAGAAGAAAATATTGATACTTACTTCCACATTTTACATT

CGGTTGAACAACAAAAGGGACTGGATGAAGTTGGTCCA

AACTCATTTGAATATACAATTTATTCTGATGGAACTAAC

TTAGACAAAGGTATTTTCTACTACACCACTTATTCAAAC

AAACAAATTAACGTTGTTGATATGAATAAAGAAGATCT

AGATAGCAGCAATTTGATCACTTATGATATGCTTGATAA

AACTAAATTTAACCATCAAAACTAA

Bile salt hydrolase MCTSIIFSPKDHYFG NLDLEITFGQQVVITP NYTFKFRK A protein from MPSLKKHYAMIGISLDMDDYPLYFDATNEKGLGMAGLN Lacotbacillus YPGNATYYEEKENKDNIASFtFiPWILGQCSTISEVKDLLS acidophilus RINIADLNFSEKMQASSLHWLIAD TGTSLVVETDKDGM

HIYDNPVGCLTNNPQFPKQLFNLNNYADVSPKMPKNNFS DKVNMAGYSRGLGSHNLPGGMDSESRFVRVAFNKFNAP IAETEEENIDTYFHILHSVEQQKGLDEVGPNSFFYTTYSDG T LDKGIF YYTTYSNKQIN WDMNKEDLDS SNLITYDML DKTKFNHQN

Bile salt hydrolase AGAAAGCGTGCAGTAAATGTGTACATCAATTTGTTATA B from ATCCTAACGATCATTATTTTGGTAGAAATCTTGACTATG Lacotbacillus AAATTGCTTATGGTCAAAAAGTAGTCATTGTACCAAGA acidophilus AACTACGAATTTAAGTATAGAGAAATGCCCTCTCAAAA

GATGCATTATGCTTTTATCGGAGTATCTGTAGTTAATGA

TGATTATCCATTATTATGTGATGCAATTAATGAAAAGGG

GCTTGGTATTGCAGGATTAAATTTTCAAGGTCCTAATCA

TTACTTTCCTAAAATCGAAGGTAAGAAGAATATTGCTTC

TTTTGAATTAATGCCATACTTATTAAGTAATTGTGAAAA

TACTGACGATGTTAAAGAAATCTTAGATAATGCAAATA

TTTTAAATATTAGCTTTTCAGCAAATTATCCTGCAGCTG

ATTTACATTGGATTTTAAGTGATAAAGCTGGTAAGAGT

ATCGTAGTTGAATCAACCAATTCAGGTTTACATATTTAT

GATAATCCAGTGAATGTCTTAACTAACAATCCTGAATTT

CCGGATCAATTAATTAAATTAAGTGACTACGCCGACGT

TACTCCACATAATCCTAAGAATACATTGGTTCCTAATGT

TGATCTTAATCTATATAGTAGAGGCTTAGGTACTCACCA

CTTACCTGGTGGAATGGATTCTAGCTCTCGATTTGTTAA

GGTAGCTTTTGTCTTGGCACACACTCCACAAGGAAAAA

ATGAAGTGGAAAATGTTACTAATTATTTCCATATTCTGC

ATTCAGTAGAACAACCTGATGGTTTAGATGAAGTAGAA

GATAATCGCTATGAATATACTATGTATACAGATTGTATG

AACTTAGATAAAGGTATTTTGTACTTTACTACTTATGAC

AATAATCGGATTAATGCAGTAGATATGCATAAAGCAGA

TTTAGATTCAGAAGATTTAATCTGCTACGATTTGTTTAA

GAAACAAGATATTGAATATATGAATTAA Bile salt hydrolase MCTSICYNPNDHYFGRNLDYEIAYGQKVVIVPRNYEF Y B protein from REMPSQKMHYAFIGVSVVNDDYPLLCDAINEKGLGIAGL Lacotbacillus NFQGPNHYFPKIEGKKNIASFELMPYLLSNCENTDDVKEI acidophilus LDNANILNISFSANYPAADLHWILSDKAG SIVVESTNSG

LHIYDNPVNVLTNNPEFPDQLIKLSDYADVTPHNPKNTL

VPNVDLNLYSRGLGTHHLPGGMDSSSRFVKVAFVLAHT

PQGKNEVENVTNYFHILHSVEQPDGLDEVEDNRYEYTM

YTDCMNLDKGILYFTTYD NRINAVDMHKADLDSEDLI

CYDLFK QDIEYMN

Bile salt hydrolase ATGGAAACGAAAAGCTCTCTCTGGAAATCATCGCGCCG from Brucella CGTGCTTGCACATGGGGCTGCAACTGTTCTGGTCGCGGC abortus GGGCCTTATCGTTCCCCAGGCGGCTATGGCTTGCACGA

GCTTCGTTCTGCCGACGAGCGACGGTGGTATGGTCTATG

GTCGCACGATGGAATTCGGGTTCAATCTCAAATCCGAC

ATGATTGCCATTCCGCGCAATTACACCATCACGGCAAG

CGGGCCGGACGGTGCTGCGGGCAAGAAATGGAAGGGC

AAATATGCCACGATCGGCATGAATGCTTTTGGTATCGTC

GCTCTCACCGACGGTATGAACGAGAAGGGGCTTGCAGG

CGGGCTTCTCTATTTCCCGGAATATGCCAAGTATCAGGA

CCCATCCACGGCGAAGCCGGAAGACAGCCTCGCTCCGT

GGGATTTCCTGACCTGGGCGCTGGCCAATTTTTCGACAG

TGGCCGAAGTCAAGGATGCTTTGAGCACCATTTCCATC

GTCGATGTGAAACAAAAGGACCTGGGATTTACCCCGCC

CGCTCACTACACGCTGCATGATGCGACCGGCGCATCCA

TCGTGATCGAACCGATCGACGGCAAGCTCAAGGTTTAC

GACAACAAGCTCGGTGTCATGACCAATTCGCCGTCTTTC

GACTGGCACATGACCAATCTGCGCAACTATGTCTATCTC

TCGCGTGAAAATCCGAAGCCGTTGCAGATCCTTGGCGA

GACGATCCAGTCATTCGGGCAAGGCGCCGGTATGCATG

GTATTCCGGGCGACACCACGCCGCCATCGCGTTTCGTGC

GTGCAAGCGCCTACGTCCTTTCCGCCAAGAAGGTGCCG

AGCGGCCTTGAAAGCGTGCGGCTGGCCGAGCATATTGC

CAATAACTTCGACATTCCAAAGGGATGGAGCGAAGAGC

AGAATATGTTTGAATATACCCAGTGGACCGCCTTTGCG

GACATGAAGAACGATGTCTATTACATCAAGACCTATGA

CGATCAGGTTCTGCGCAGCTTCAGCTTCAAGGATTTTGA

TGTCGATAGCAAAGATATTCTAACGATCAAGTTCGAGC

CAAAACTGGACGCGCCGTCACTGAAAAAGTAA

Bile salt hydrolase METKSSLWKSSRRVLAHGAATVLVAAGLIVPQAAMACT protein from SFVLPTSDGGMVYGRTMEFGFNLKSDMIAIPRNYTITASG Brucella abortus PDGAAG KW G YATIGMNAFGIVALTDGMNE GLAG

GLLYFPEYAKYQDPSTAKPEDSLAPWDFLTWALANFSTV

AE VKD A LS TXS I V D VKQ KD LGFTPP AH YT LHD ATG AS IVI

EPJDG L VYDNKLGVMTNSPSFDWHMTNLRNYVYLSR

ENPKPLQILGETIQSFGQGAGMHGIPGDTTPPSRFVRASA

YVLSAKKVPSGLESVRLAEHIANNFDIPKGWSEEQNMFE

YTQWTAFADM NDVYYIKTYDDQVT SFSF DFDVDSK

DILTIKFEPKLD APS LKK

Wild-type clbA caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaaca taaaatacaagctcggaatacgaatcacgctatacacattgctaacaggaatgagattatcta aatgaggattgatatattaattggacatactagtttttttcatcaaaccagtagagataacttcctt cactatctcaatgaggaagaaataaaacgctatgatcagtttcattttgtgagtgataaagaact ctatattttaagccgtatcctgctcaaaacagcactaaaaagatatcaacctgatgtctcattac aatcatggcaatttagtacgtgcaaatatggcaaaccatttatagtttttcctcagttggcaaaaa agattttttttaacctttcccatactatagatacagtagccgttgctattagttctcactgcgagctt ggtgtcgatattgaacaaataagagatttagacaactcttatctgaatatcagtcagcattttttta ctccacaggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttttttggaaaa tgtggacgctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggactggatt gtattgaatttcatttaacaaataaaaaactaacttcaaaatatagaggttcacctgtttatttctct caatggaaaatatgtaactcatttctcgcattagcctctccactcatcacccctaaaataactatt gagctatttcctatgcagtcccaactttatcaccacgactatcagctaattcattcgtcaaatgg gcagaattgaatcgccacggataatctagacacttctgagccgtcgataatattgattttcatat tccgtcggtggtgtaagtatcccgcataatcgtgccattcacatttag

clbA knock-out ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaagaca agtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatcttaacatatca ataaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagctcggaatacgaat cacgctatacacattgctaacaggaatgagattatctaaatgaggattgaTGTGTAGG

CTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAG

GAACTTCGGAATAGGAACTTCGGAATAGGAACTAAGGA

GGATATTCATATGtcgtcaaatgggcagaattgaatcgccacggataatctagaca cttctgagccgtcgataatattgattttcatattccgtcggtgg

ABCB11 bile salt GAATGATGAAAACCGAGGTTGGAAAAGGTTGTGAAACC exporter Homo TTTTAACTCTCCACAGTGGAGTCCATTATTTCCTCTGGC sapiens TTCCTCAAATTCATATTCACAGGGTCGTTGGCTGTGGGT

TGCAATTACCATGTCTGACTCAGTAATTCTTCGAAGTAT

AAAGAAATTTGGAGAGGAGAATGATGGTTTTGAGTCAG

ATAAATCATATAATAATGATAAGAAATCAAGGTTACAA

GATGAGAAGAAAGGTGATGGCGTTAGAGTTGGCTTCTT

TCAATTGTTTCGGTTTTCTTCATCAACTGACATTTGGCTG

ATGTTTGTGGGAAGTTTGTGTGCATTTCTCCATGGAATA

GCCCAGCCAGGCGTGCTACTCATTTTTGGCACAATGAC

AGATGTTTTTATTGACTACGACGTTGAGTTACAAGAACT

CCAGATTCCAGGAAAAGCATGTGTGAATAACACCATTG

TATGGACTAACAGTTCCCTCAACCAGAACATGACAAAT

GGAACACGTTGTGGGTTGCTGAACATCGAGAGCGAAAT

GATCAAATTTGCCAGTTACTATGCTGGAATTGCTGTCGC

AGTACTTATCACAGGATATATTCAAATATGCTTTTGGGT

CATTGCCGCAGCTCGTCAGATACAGAAAATGAGAAAAT

TTTACTTTAGGAGAATAATGAGAATGGAAATAGGGTGG

TTTGACTGCAATTCAGTGGGGGAGCTGAATACAAGATT

CTCTGATGATATTAATAAAATCAATGATGCCATAGCTG

ACCAAATGGCCCTTTTCATTCAGCGCATGACCTCGACCA

TCTGTGGTTTCCTGTTGGGATTTTTCAGGGGTTGGAAAC

TGACCTTGGTTATTATTTCTGTCAGCCCTCTCATTGGGA

TTGGAGCAGCCACCATTGGTCTGAGTGTGTCCAAGTTTA

CGGACTATGAGCTGAAGGCCTATGCCAAAGCAGGGGTG

GTGGCTGATGAAGTCATTTCATCAATGAGAACAGTGGC

TGCTTTTGGTGGTGAGAAAAGAGAGGTTGAAAGGTATG

AGAAAAATCTTGTGTTCGCCCAGCGTTGGGGAATTAGA

AAAGGAATAGTGATGGGATTCTTTACTGGATTCGTGTG GTGTCTCATCTTTTTGTGTTATGCACTGGCCTTCTGGTAC

GGCTCCACACTTGTCCTGGATGAAGGAGAATATACACC

AGGAACCCTTGTCCAGATTTTCCTCAGTGTCATAGTAGG

AGCTTTAAATCTTGGCAATGCCTCTCCTTGTTTGGAAGC

CTTTGCAACTGGACGTGCAGCAGCCACCAGCATTTTTGA

GACAATAGACAGGAAACCCATCATTGACTGCATGTCAG

AAGATGGTTACAAGTTGGATCGAATCAAGGGTGAAATT

GAATTCCATAATGTGACCTTCCATTATCCTTCCAGACCA

GAGGTGAAGATTCTAAATGACCTCAACATGGTCATTAA

ACCAGGGGAAATGACAGCTCTGGTAGGACCCAGTGGAG

CTGGAAAAAGTACAGCACTGCAACTCATTCAGCGATTC

TATGACCCCTGTGAAGGAATGGTGACCGTGGATGGCCA

TGACATTCGCTCTCTTAACATTCAGTGGCTTAGAGATCA

GATTGGGATAGTGGAGCAAGAGCCAGTTCTGTTCTCTA

CCACCATTGCAGAAAATATTCGCTATGGCAGAGAAGAT

GCAACAATGGAAGACATAGTCCAAGCTGCCAAGGAGG

CCAATGCCTACAACTTCATCATGGACCTGCCACAGCAA

TTTGACACCCTTGTTGGAGAAGGAGGAGGCCAGATGAG

TGGTGGCCAGAAACAAAGGGTAGCTATCGCCAGAGCCC

TCATCCGAAATCCCAAGATTCTGCTTTTGGACATGGCCA

CCTCAGCTCTGGACAATGAGAGTGAAGCCATGGTGCAA

GAAGTGCTGAGTAAGATTCAGCATGGGCACACAATCAT

TTCAGTTGCTCATCGCTTGTCTACGGTCAGAGCTGCAGA

TACCATCATTGGTTTTGAACATGGCACTGCAGTGGAAA

GAGGGACCCATGAAGAATTACTGGAAAGGAAAGGTGTT

TACTTCACTCTAGTGACTTTGCAAAGCCAGGGAAATCA

AGCTCTTAATGAAGAGGACATAAAGGATGCAACTGAAG

ATGACATGCTTGCGAGGACCTTTAGCAGAGGGAGCTAC

CAGGATAGTTTAAGGGCTTCCATCCGGCAACGCTCCAA

GTCTCAGCTTTCTTACCTGGTGCACGAACCTCCATTAGC

TGTTGTAGATCATAAGTCTACCTATGAAGAAGATAGAA

AGGACAAGGACATTCCTGTGCAGGAAGAAGTTGAACCT

GCCCCAGTTAGGAGGATTCTGAAATTCAGTGCTCCAGA

ATGGCCCTACATGCTGGTAGGGTCTGTGGGTGCAGCTG

TGAACGGGACAGTCACACCCTTGTATGCCTTTTTATTCA

GCCAGATTCTTGGGACTTTTTCAATTCCTGATAAAGAGG

AACAAAGGTCACAGATCAATGGTGTGTGCCTACTTTTTG

TAGCAATGGGCTGTGTATCTCTTTTCACCCAATTTCTAC

AGGGATATGCCTTTGCTAAATCTGGGGAGCTCCTAACA

AAAAGGCTACGTAAATTTGGTTTCAGGGCAATGCTGGG

GCAAGATATTGCCTGGTTTGATGACCTCAGAAATAGCC

CTGGAGCATTGACAACAAGACTTGCTACAGATGCTTCC

CAAGTTCAAGGGGCTGCCGGCTCTCAGATCGGGATGAT

AGTCAATTCCTTCACTAACGTCACTGTGGCCATGATCAT

TGCCTTCTCCTTTAGCTGGAAGCTGAGCCTGGTCATCTT

GTGCTTCTTCCCCTTCTTGGCTTTATCAGGAGCCACACA

GACCAGGATGTTGACAGGATTTGCCTCTCGAGATAAGC

AGGCCCTGGAGATGGTGGGACAGATTACAAATGAAGCC

CTCAGTAACATCCGCACTGTTGCTGGAATTGGAAAGGA

GAGGCGGTTCATTGAAGCACTTGAGACTGAGCTGGAGA AGCCCTTCAAGACAGCCATTCAGAAAGCCAATATTTAC

GGATTCTGCTTTGCCTTTGCCCAGTGCATCATGTTTATT

GCGAATTCTGCTTCCTACAGATATGGAGGTTACTTAATC

TCCAATGAGGGGCTCCATTTCAGCTATGTGTTCAGGGTG

ATCTCTGCAGTTGTACTGAGTGCAACAGCTCTTGGAAG

AGCCTTCTCTTACACCCCAAGTTATGCAAAAGCTAAAAT

ATCAGCTGCACGCTTTTTTCAACTGCTGGACCGACAACC

CCCAATCAGTGTATACAATACTGCAGGTGAAAAATGGG

ACAACTTCCAGGGGAAGATTGATTTTGTTGATTGTAAAT

TTACATATCCTTCTCGACCTGACTCGCAAGTTCTGAATG

GTCTCTCAGTGTCGATTAGTCCAGGGCAGACACTGGCG

TTTGTTGGGAGCAGTGGATGTGGCAAAAGCACTAGCAT

TCAGCTGTTGGAACGTTTCTATGATCCTGATCAAGGGAA

GGTGATGATAGATGGTCATGACAGCAAAAAAGTAAATG

TCCAGTTCCTCCGCTCAAACATTGGAATTGTTTCCCAGG

AACCAGTGTTGTTTGCCTGTAGCATAATGGACAATATCA

AGTATGGAGACAACACCAAAGAAATTCCCATGGAAAG

AGTCATAGCAGCTGCAAAACAGGCTCAGCTGCATGATT

TTGTCATGTCACTCCCAGAGAAATATGAAACTAACGTT

GGGTCCCAGGGGTCTCAACTCTCTAGAGGGGAGAAACA

ACGCATTGCTATTGCTCGGGCCATTGTACGAGATCCTAA

AATCTTGCTACTAGATGAAGCCACTTCTGCCTTAGACAC

AGAAAGTGAAAAGACGGTGCAGGTTGCTCTAGACAAA

GCCAGAGAGGGTCGGACCTGCATTGTCATTGCCCATCG

CTTGTCCACCATCCAGAACGCGGATATCATTGCTGTCAT

GGCACAGGGGGTGGTGATTGAAAAGGGGACCCATGAA

GAACTGATGGCCCAAAAAGGAGCCTACTACAAACTAGT

CACCACTGGATCCCCCATCAGTTGACCCAATGCAAGAA

TCTCAGACACACATGACGCACCAGTTACAGGGGTTGTT

TTTAAAGAAAAAAACAATCCCAGCAGGAGGGATTGCTG

GGATTGTTTTTTCTTTAAAGAAGAATGTTAATATTTTAC

TTTTACAGTCATTTTCCTACATCGGAATCCAAGCTAATT

TCTAATGGCCTTCCATAATAATTCTGCTTTAGATGTGTA

TACAGAAAATGAAAGAAACTAGGGTCCATATGAGGGA

AAACCCAATGTCAAGTGGCAGCTCAGCCACCACTCAGT

GCTTCTCTGTGCAGGAGCCAGTCCTGATTAATATGTGGG

AATTAGTGAGACATCAGGGAGTAAGTGACACTTTGAAC

TCCTCAAGGGCAGAGAACTGTCTTTCATTTTTGAACCCT

CGGTGTACACAGAGGCGGGTCTATAACAGGCAATCAAC

AAACGTTTCTTGAGCTAGACCAAGGTCAGATTTGAAAA

GAACAGAAGGACTGAAGACCAGCTGTGTTTCTTAACTA

AATTTGTCTTTCAAGTGAAACCAGCTTCCTTCATCTCTA

AGGCTAAGGATAGGGAAAGGGTGGATGCTCTCAGGCTG

AGGGAGGCAGAAAGGGAAAGTATTAGCATGAGCTTTCC

AGTTAGGGCTGTTGATTTATGCTTTAACTTCAGAGTGAG

TGTAGGGGTGGTGATGCT

ABCB11 bile salt MSDSVILRSIKKFGEENDGFESDKSYNNDKKSRLQDEKKG exporter protein DGVRVGFFQLFRFSSSTDIWLMFVGSLCAFLHGIAQPGVLL Homo sapiens IFGTMTDVFIDYDVELQELQIPGKACVNNTIVWTNSSLNQ NMTNGTRCGLLNIESEMIKFASYYAGIAVAVLITGYIQICF

WVIAAARQIQKMRKFYFRRIMRMEIGWFDCNSVGELNTR

FS DDINKIND AIADQM ALFIQRMTS TIC GFLLGFFRGWKLT

LVIIS VS PLIGIG A ATIGLS VS KFTD YELKA Y AKAG V V ADE

VISSMRTVAAFGGEKREVERYEKNLVFAQRWGIRKGIVM

GFFTGFVWCLIFLCYALAFWYGSTLVLDEGEYTPGTLVQI

FLS VIVG ALNLGN AS PCLE AF ATGR A A ATS IFETIDRKPIID

CMSEDGYKLDRIKGEIEFHNVTFHYPSRPEVKILNDLNMVI

KPGEMTALVGPSGAGKSTALQLIQRFYDPCEGMVTVDGH

DIRSLNIQWLRDQIGIVEQEPVLFSTTIAENIRYGREDATME

DIVQAAKEANAYNFIMDLPQQFDTLVGEGGGQMSGGQK

QRVAIARALIRNPKILLLDMATSALDNESEAMVQEVLSKIQ

HGHTIIS V AHRLS T VR A ADTIIGFEHGT A VERGTHEELLER

KGVYFTLVTLQSQGNQALNEEDIKDATEDDMLARTFSRG

SYQDSLRASIRQRSKSQLSYLVHEPPLAVVDHKSTYEEDR

KDKDIPVQEEVEPAPVRRILKFSAPEWPYMLVGSVGAAVN

GTVTPLYAFLFSQILGTFSIPDKEEQRSQINGVCLLFVAMG

CVSLFTQFLQGYAFAKSGELLTKRLRKFGFRAMLGQDIA

WFDDLRNS PG ALTTRLATD AS Q VQG A AGS QIGMIVNS FTN

VTVAMIIAFSFSWKLSLVILCFFPFLALSGATQTRMLTGFA

SRDKQALEMVGQITNEALSNIRTVAGIGKERRFIEALETEL

EKPFKTAIQKANIYGFCFAFAQCIMFIANSASYRYGGYLIS

NEGLHFS Y VFRVIS AWLS AT ALGRAFS YTPS Y AKAKIS A A

RFFQLLDRQPPISVYNTAGEKWDNFQGKIDFVDCKFTYPS

RPDS Q VLNGLS VS IS PGQTLAF VGS S GCGKS TS IQLLERF Y

DPDQGKVMID GHDS KKVN VQFLRS NIGIVS QEP VLFAC S I

MDNIKYGDNTKEIPMERVIAAAKQAQLHDFVMSLPEKYE

TNVGSQGSQLSRGEKQRIAIARAIVRDPKILLLDEATSALD

TESEKTVQVALDKAREGRTCrVIAHRLSTIQNADIIAVMAQ

GVVIEKGTHEELMAQKGAYYKLVTTGSPIS

Streptococcus

thermophilus Msba

MEGRTVFVIAHRLSTIVNSDVILVMDHGRIIKRGDHDTLM

subfamily ABC

EQGGTYYRLYTGS LEID

transporter ATP- binding protein

STH8232_1633

Nostoc spp. atgtgggggaaacaaagacaaagaatcgccattgcacgagggggttttaagaatttgcagg Asll293 ABC ttttgattttagataaagcaacctcggcattggataataaaacagaagcagctattgagcgatc transporter gene gctggtgttgactgttgaccaatga

Nostoc spp.

MWGKQRQRIAIARGGF NLQVLILDKATSALDNKTEAAI

Asll293 ABC

ERSLVLTVDQ

transporter protein

Neisseria ATGAATATCCTCAGTAAAATCAGCAGCTTTATCGGAA meningitides AAACATTTTCCCTCTGGGCCGCGCTCTTTGCCGCCGCC (MC58) ASBTNM GCTTTTTTCGCGCCCGACACCTTCAAATGGGCGGGGCC bile acid sodium TTATATTCCTTGGCTGTTGGGCATTATTATGTTCGGTAT symporter GGGTTTGACGCTCAAACCTTCCGACTTCGATATTTTGT (NMB0705) TCAAACATCCCAAAGTCGTCATCATCGGCGTAATCGC

ACAAT CGCCATTATGCCGGCAACCGCCTGGCTGCTGT

CCAAACTGTTGAACCTGCCTGCCGAAATCGCGGTCGG CGTGATTTTGGTCGGCTGCTGCCCGGGCGGTACGGCTT

CCAATGTGATGACCTATCTGGCGCGTGGCAATGTGGC

TTTGTCGGTTGCCGTTACGTCTGTTTCCACCCTGATTTC

CCCATTGCTGACTCCCGCCATCTTCCTGATGCTTGCCG

GCGAAATGCTGGAAATCCAAGCGGCCGGTATGTTGAT

GTCCATCGTCAAAATGGTTTTGCTCCCCATTGTTTTGG

GTTTGATTGTCCATAAGGTTTTGGGCAGTAAAACCGA

AAAGCTGACCGATGCGCTGCCGCTGGTTTCCGTTGCCG

CCATCGTGCTGATTATCGGCGCGGTTGTTGGGGCAAG

CAAAGGCAAGATTATGGAAAGCGGCCTGCTGATTTTT

GCGGTTGTCGTACTCCACAACGGCATCGGCTACCTGCT

CGGCTTCTTTGCCGCCAAATGGACCGGCCTGCCTTATG

ATGCACAAAAAACGCTGACCATCGAAGTCGGTATGCA

AAACTCGGGCCTGGCCGCCGCGCTTGCCGCCGCACAC

TTTGCCGCCGCGCCGGTCGTTGCCGTTCCGGGCGCATT

GTTCAGCGTGTGGCACAATATCTCCGGCTCGCTGCTGG

CAACTTATTGGGCGGCCAAAGCCGGTAAACATAAAAA

ACCCTAA

Neisseria MNILS KIS S FIGKTFS LW A ALFA A A AFFAPDTFKW AGP YI meningitides PWLLGIIMFGMGLTLKPSDFDILFKHPKVVIIGVIAQFAIM (MC58) ASBTNM PATAWLLSKLLNLPAEIAVGVILVGCCPGGTASNVMTYL bile acid sodium ARGN V ALS V A VTS VS TLIS PLLTP AIFLMLAGEMLEIQ A A symporter protein GMLMS IVKM VLLPIVLGLIVHKVLGS KTEKLTD ALPLVS

VA AIVLIIG A V VG AS KGKIMES GLLIF A V V VLHNGIG YLL GFFAAKWTGLPYDAQKTLTIEVGMQNSGLAAALAAAHF AA AP V V A VPG ALFS VWHNIS GS LLAT YW A AKAGKHKK PGSENLYFQ

Clostridium AAAAGATATTAAGCATTAAGAAAATGCACAAAAAATCA scindens GCGTGTGAGAGGGAGGGCAAGGAGTTGAAGCGTGACTT

VPI 12708 bile TTTTAACAAGTTTAATTTGGGGACATCGAACTTTGTCAC acid-inducible GCCGGGAAAACAGTTGGAATACGTTTCGGAATGCAAGC operon (GenBank CAGATTCTACTGCGGTCATTTGCTTAGATAAAGAACAG

U57489.2) AACTGTTCCGTTATTACTTGGCATCAGCTGCACGTCTAT

TCCAGCCAGCTGGCATGGTACCTTATAGAAAATGAGAT

TGGCCCGGGGTCGATCGTACTTACAATGTTTCCGAACA

GCATCGAGCACATTATTGCGGTATTTGCAATCTGGAAG

GCGGGCGCCTGCTATATGCCCATGTCCTATAAGGCGGC

GGAATCCGAGATCAGGGAGGCCTGCGATACCATCCACC

CGAATGCGGCTTTTGCGGAATGCAAGATTCCAGGATTA

AAATTCTGCCTTAGCGCAGACGAGATATATGAGGCGAT

GGAAGGAAGATCCAAGGAGATGCCTTCGGACCGTCTGG

CCAATCCGAACATGATATCCTTATCAGGCGGAACCAGC

GGAAAGATGAAGTTCATCCGTCAGAACCTTCCATGCGG

GCTGGACGATGAGACGATCAGAAGCTGGTCTTTGATGT

CTGGAATGGGATTTGAGCAGCGCCAGCTGCTGGTAGGC

CCGCTGTTTCATGGCGCGCCTCACTCCGCGGCGTTTAAT

GGACTGTTCATGGGCAACACCCTGGTACTGACCAGGAA

CCTTTGCCCGGGAAATATCCTGAACATGATTAAGAAAT

ATAAGATTGAATTTATACAGATGGTGCCGACCCTGATG

AACCGGCTTGCCAAACTGGAGGGAGTCGGAAAAGAAG ACTTTGCATCCCTGAAGGCGCTGTGCCATACAGGGGGC

GTCTGTTCTCCCTGGCTTAAGCAGATCTGGATCGACCTG

CTGGGGCCTGAAAAGATCTATGAGATGTATTCCATGAC

GGAATGCATCGGCCTTACCTGCATCCGGGGAGACGAGT

GGGTGAAGCATCCGGGAAGCATCGGACGGCCAGTGGG

CGATAGCAAGGTGTCTATCCGGGATGAGAATGGCAAGG

AAGTTGCGCCTTTTGAGATTGGCGAGATCTATATGACA

GCGCCGGCCTCCTATCTGGTTACCGAGTACATCAATTGG

GAACCGCTGGAAGTGAAAGAGGGAGGCTTCCGAAGCG

TAGGGGATATCGGCTACGTGGATGAGCAGGGCTATCTG

TACTTTTCTGACCGGCGCAGCGACATGCTGGTATCAGGC

GGAGAAAACGTGTTCGCCACCGAAGTCGAGACGGCGCT

TTTGAGATATAAGGATATCCTGGACGCTGTAGTGGTAG

GGATACCGGATGAAGATCTGGGGCGAAGGCTCCATGCG

GTCATTGAGACAGGGAAAGAGATACCGGCAGAGGAAC

TGAAAACATTCCTGAGAAAGTATCTGACTCCATATAAG

ATACCAAAGACGTTCGAGTTCGTAAGGAGCATACGAAG

GGGAGACAATGGAAAGGCCGACAGGAAGCGGATCCTG

GAAGATTGTATTGCCCGCGGGGGATGATTCTATAAATG

CAAAGAAAACAAATTATATAAAGGAGGAGTAACAAAA

TGAGTTACGAAGCACTTTTTTCACCATTCAAGGTCAGAG

GACTGGAACTTAAAAACCGTATCGTCCTGCCTGGAATG

AACACCAAGATGGCAAAGAACAAGCACGACATAGGCG

AGGATATGATAGCCTACCATGTTGCCAGGGCAAAAGCG

GGATGCGCGTTAAATATATTTGAATGCGTAGCATTATGT

CCGGCGCCTCACGCTTATATGTATATGGGGCTTTATACG

GACCATCATGTAGAACAGCTTAAGAAATTGACGGATGC

AGTCCATGAAGCAGGCGGCAAGATGGGCATCCAGCTGT

GGCATGGAGGATTCAGCCCGCAGATGTTCTTTGACGAG

ACCAACACCCTGGAAACTCCGGACACTCTTACGGTAGA

GAGGATTCATGAGATCGTAGAAGAATTCGGACGCGGCG

CAAGGATGGCTGTTCAGGCTGGATTTGACGCAGTAGAA

TTCCATGCGGCTCACAGTTATCTGCCTCACGAGTTCTTA

AGCCCTGGAATGAACAAACGTACGGATGAGTACGGCGG

AAGTTTTGAGAACCGCTGCAGATTCTGTTATGAAGTCGT

TCAGGCAATCCGTTCCAATATCCCGGATGACATGCCATT

CTTTATGCGTGCAGACTGCATCGACGAATTAATGGAAC

AGACCATGACAGAGGAAGAGATCGTTACATTTATCAAT

AAGTGCGCAGAACTTGGCGTGGATGTGGCAGACCTTTC

CCGTGGAAACGCGACTTCATTCGCAACCGTATATGAAG

TTCCGCCATTCAACCTGGCTCATGGCTTCAACATAGAGA

ATATTTACAACATCAAAAAGCAGATCAATATCCCGGTT

ATGGGAGTTGGCCGTATCAATACAGGAGAGATGGCAAA

CAAGGTCATTGAAGAAGGCAAGTTTGACCTGGTAGGCA

TCGGACGCGCCCAGCTTGCAGATCCAAACTGGATCACC

AAAGTAAGAGAAGGCAAAGAAGACCTGATCCGCCACT

GTATCGGATGTGACCAGGGATGCTATGACGCAGTCATC

AATCCAAAGATGAAGCATATCACCTGCACCCACAATCC

AGGATTGTGCTTAGAGTATCAGGGAATGCCAAAGACAG

ACGCTCCTAAGAAAGTCATGATCGTAGGAGGCGGAATG GCAGGCATGATCGCTGCGGAAGTATTAAAGACCAGAGG

CCATAACCCGGTAATCTTCGAGGCATCCGACAAGCTTG

CAGGACAGTTCAGGCTGGCAGGCGTAGCGCCGATGAAG

CAGGATTGGGCAGATGTTGCAGAATGGGAAGCAAAAG

AAGTAGAGCGCCTTGGAATCGAAGTACGTCTGAATACC

GAAGTGACTGCAGAGACCATCAAGGAATTCAATCCGGA

TAATGTCATCATCGCAGTAGGCTCTACCTATGCGCTGCC

TGAGATTCCGGGAATCGACAGCCCAAGCGTATACTCCC

AGTATCAGGTACTGAAAGGGGAAGTAAATCCGACAGGC

CGTGTAGCCGTTATCGGATGCGGACTGGTTGGTACGGA

AGTCGCAGAACTTCTGGCATCCAGAGGCGCACAGGTAA

TCGCGATCGAGAGGAAGGGCGTAGGTACCGGCCTTAGC

ATGCTTCGCAGAATGTTCATGAACCCGGAATTCAAATA

TTACAAGATCGCCAAGATGTCCGGAACAAATGTCACCG

CTTTAGAGCAGGGCAAGGTTCACTACATCATGACAGAC

AAGAAGACCAAAGAAGTGACGCAGGGAGTCCTGGAAT

GCGACGCTACCGTTATCTGTACAGGAATTACCGCACGT

CCAAGCGATGGGCTTAAGGCAAGATGCGAAGAACTTGG

AATCCCGGTTGAGGTGATCGGAGACGCTGCTGGCGCAA

GAGACTGCACGATCGCGACACGCGAAGGCTATGACGCA

GGAATGGCAATCTAGAAAATCAGAACTTATCAATCTTA

CATATAGAAAGGATGATACATATGACATTAGAAGAGAG

AGTTGAAGCATTAGAAAAAGAATTGC

AGGAGATGAAGGATATTGAGGCAATCAAGGAACTGAA

AGGAAAGTATTTCCGCTGCCTGGACGGAAAGATGTGGG

ATGAGCTGGAGACCACCCTGTCACCAAATATCGTAACC

TCTTATTCCAACGGGAAACTGGTATTCCATAGCCCGAA

GGAAGTTACCGATTACTTAAAGAGCTCGATGCCAAAAG

AAGAGATCAGCATGCATATGGGCCACACGCCGGAGATC

ACCATTGACAGCGAGACTACGGCTACGGGCAGATGGTA

TCTGGAAGATAGACTGATCTTTACGGACGGTAAGTACA

AAGACGTAGGAATCAATGGCGGCGCGTTCTATACAGAC

AAATATGAGAAGATAGACGGCCAGTGGTACATCCTTGA

AACCGGCTATGTACGAATCTATGAAGAACATTTCATGC

GTGATCCAAAGATCCATATCACGATGAACATGCACAAA

TAAGAATATTGTAAAAGAAAGGCAGGAGTAAGAGTAT

GAATCTCGTACAAGACAAAGTTACGATCATCACAGGCG

GCACAAGAGGTATTGGATTCGCCGCTGCCAAAATATTT

ATCGACAATGGCGCAAAAGTATCCATCTTCGGAGAGAC

GCAGGAAGAAGTAGATACAGCGCTTGCACAGTTAAAAG

AACTTTATCCGGAAGAAGAGGTTCTGGGATTCGCGCCG

GATCTTACATCCAGAGACGCAGTTATGGCAGCGGTAGG

CCAGGTAGCACAGAAATATGGCAGACTGGATGTCATGA

TCAACAATGCAGGAATTACCAGCAACAACGTATTCTCC

AGAGTGTCTGAAGAAGAGTTCAAGCATATTATGGACAT

CAACGTAACAGGCGTATTCAACGGCGCATGGTGCGCAT

ACCAGTGCATGAAGGATGCCAAAAAGGGCGTTATCATC

AACACGGCATCCGTTACAGGCATCTTCGGATCACTCTCA

GGCGTAGGATATCCGGCCAGCAAGGCAAGCGTGATCGG

ACTCACCCATGGACTTGGAAGAGAGATCATCCGCAAGA ATATCCGTGTAGTAGGAGTGGCTCCTGGAGTTGTGAAC

ACGGATATGACCAATGGCAATCCTCCGGAGATCATGGA

AGGATATCTGAAGGCGCTTCCGATGAAGAGAATGCTTG

AGCCGGAAGAGATCGCTAATGTATACCTGTTCCTGGCA

TCTGACTTGGCAAGCGGCATTACGGCTACTACGGTCAG

CGTAGACGGGGCTTACAGACCATAATTTTAATTTTTACT

AAGTAGAATATGTGATATAGAAAAGGAGATATAAAAA

CATGGCTGGAATAAAAGATTTTCCAAAATTCGGAGCTC

TTGCAGGGCTTAAGATACTTGACAGCGGATCTAACATC

GCCGGACCTTTAGGCGGAGGCCTTCTGGCAGAATGCGG

AGCAACGGTCATCCATTTTGAAGGACCAAAGAAACCTG

ATAACCAGAGAGGATGGTACGGCTATCCACAGAATCAC

CGTAATCAGCTGTCTATGGTAGCAGACATCAAATCTGA

AGAAGGAAGAAAGATCTTCCTTGATCTGATCAAATGGG

CAGATATCTGGGTAGAGTCATCCAAAGGCGGACAGTAT

GACAGGCTGGGACTTTCCGATGAAGTCATCTGGGAAGT

AAATCCTAAGATTGCCATCGTGCACGTATCCGGATATG

GACAGACAGGAGACCCGTCTTACGTTACACGTGCATCC

TATGACGCAGTAGGCCAGGCATTCAGCGGCTATATGTC

ACTGAACGGAACAACGGAAGCGCTGAAGATCAATCCTT

ATCTGAGCGATTTCGTATGCGGACTTACCACATGCTGGG

CTATGCTTGCCTGCTATGTAAGCACCATTCTTACCGGAA

AAGGCGAATCTGTTGACGTTGCACAGTACGAAGCGCTG

GCACGTATCATGGACGGACGTATGATCCAGTACGCTAC

AGACGGCGTGAAGATGCCAAGAACCGGCAATAAGGAT

GCGCAGGCTGCCCTGTTCAGCTTCTACACCTGTAAAGAC

GGACGTACGATCTTTATCGGAATGACTGGCGCGGAAGT

ATGTAAGAGAGGCTTCCCGATCATCGGACTTCCGGTAC

CTGGAACCGGAGACCCGGACTTCCCGGAAGGCTTCACA

GGCTGGATGATCTATACTCCTGTAGGACAGAGAATGGA

AAAGGCTATGGAGAAGTATGTATCTGAGCATACGATGG

AAGAAGTAGAGGCTGAGATGCAGGCACACCAGATTCCA

TGCCAGAGAGTATACGAGCTGGAAGACTGCCTGAACGA

TCCTCACTGGAAAGCACGTGGAACTATTACGGAGTGGG

ATGACCCGATGATGGGACATATCACAGGCCTTGGACTG

ATCAACAAGTTCAAGAGAAATCCTTCCGAAATCTGGAG

AGGCGCTCCGCTGTTCGGTATGGATAACCGCGATATCCT

GAAAGACCTGGGATATGACGATGCAAAGATCGATGAAC

TCTATGAGCAGGGCATCGTCAATGAATTCGACCTTGAC

ACTACTATCAAACGCTATAGACTGGATGAAGTAATTCC

ACATATGAGAAAGAAAGAGGAGTAAGAGTATGAGCAC

CGTAGCCAATCCAAATTATAAGAAAGGTTTTGTCCCCTT

TGCAATTGCAGCACTCCTGGTGAGCCTGATCGGCGGTTT

TACCGCCGTTCTCGGCCCGGCCTTCGTGGCGGACCAGG

GGATTGACTATAATAATACCACATGGATTTCCCTGGCGC

TGGCGATGTCTTCCGCCGCATGCGCTCCAATCCTTGGAA

AACTGGGAGACGTGCTAGGACGCAGGACGACGCTGCTT

CTGGGTATTGTGATCTTTGCGGCCGGCAATGTGCTGACA

GCCGTAGCCACGTCCCTGATATTCATGCTGGCAGCCCGT

TTTATCGTAGGTATCGGAACAGCAGCGATCTCACCGAT CGTTATGGCCTATATCGTAACCGAGTATCCGCAGGAGG

AGACAGGAAAGGCCTTTGGCCTGTATATGCTGATCTCC

AGCGGCGCCGTCGTGGTAGGACCTACCTGTGGCGGCCT

GATCATGAATGCGGCTGGCTGGAGAGTCATGATGTGGG

TATGCGTCGCTCTGTGCGTCGTTGTATTCCTGATCTGCA

CATTCTCCATCAAGAAGACTGCATTTGAGAAGAAGAGC

ATGGCAGGATTTGACAAGCCGGGCGCAGCCCTGGTAGT

CGTATTCTTCAGTTTGTTCCTGTGCATCCCATCCTTCGGA

CAGAATATCGGATGGTCTTCCACAGCATTTATCGCAGC

AGCGGCAGTAGCGCTGGTAGCACTTTTCATCCTGGTAAT

GGTAGAAAAGAAAGCGAAGAGTCCGATCATGAACGGC

AAGTTTATGGCACGCAAGGAATTCGTGCTTCCAGTATTG

ATCCTGTTCCTTACACAGGGACTTATGATGGCAAATATG

ACCAATGTCATCGTGTTCGTGCGCTATACGCAGCCGGA

CAATGTCATTATATCAAGTTTTGCGATCTCCATCATGTA

CATAGGAATGTCCTTAGGCTCCGTTATCATTGGACCTGT

TGCAGATAAGAAAGAGCCAAAGACGGTTCTGACATTCT

CTCTGGTACTGACAGCCATCGGCTGTGCGCTGATGTATC

TGTTCAAGGCAGATTCCTCCGTCGCTATCTTTGCGGCAT

CCTTGGGAATCCTTGGATTTGGCCTTGGAGGAAATGCA

ACCATCTTCATGAAGGTAGCGCTTTCCGGCCTGTCCAGC

GAAGTAGCTGGCTCTGGTACTGGAACCTATGGCCTGTTC

AGAGATATCTCGGCACCATTCGGCGTGGCAGTGTTCGT

GCCTATGTTTGCCAACGGCGTAACAGCGAATATTGCGA

AATACGCGTCAGGCGGCATGGAAGAAGGCGCCGCTACG

GTAAAAGCAGCCATCTCATCCATCCAGACGCTGACACT

GGTTGAACTTGGATGTATCGTTGTGGGAATCATCCTTGT

GAGAATGCTGCCAAGAATCTATCAGAAGAAAGAGGCAT

AAATAAGTTAAGAAAAGAGGTAATTATAAATGGATATG

AAACATTCCAGATTATTTTCGCCGCTTCAGATCGGATCC

CTGACACTGTCTAACCGTGTCGGCATGGCTCCCATGAGC

ATGGACTATGAAGCAGCAGACGGAACTGTGCCCAAGAG

GCTGGCGGACGTATTTGTCCGCCGCGCCGAGGGAGGCA

CAGGCTACGTCATGATCGACGCGGTGACGATAGACAGC

AAGTATCCTTATATGGGAAATACAACGGCCCTTGACCG

TGATGAACTGGTTCCCCAGTTTAAGGAATTTGCTGACAG

AGTAAAAGAAGCAGGCAGCACGCTGGTGCCGCAGATC

ATTCATCCGGGTCCGGAATCCGTATGCGGCTACCGGCA

TATCGCTCCGCTTGGACCTTCTGCCAACACCAATGCAAA

CTGCCACGTGAGCAGATCGATCAGCATAGATGAGATCC

ATGACATCATTAAGCAGTTCGGCCAGGCGGCACGCCGC

GCCGAAGAAGCAGGATGCGGGGCAATCTCCCTGCACTG

CGCGCATGCGTATATGCTGCCAGGATCCTTCCTGTCACC

GCTTCGCAACAAGCGCATGGATGAATATGGCGGAAGCC

TTGACAACCGTGCCCGTTTCGTGATCGAGATGATTGAG

GAGGCCCGCAGGAATGTGAGTCCTGATTTCCCGATCTTC

CTTCGTATCTCCGGAGACGAGAGAATGGTAGGAGGCAA

CAGCCTTGAAGATATGCTCTACCTGGCACCGAAGTTCG

AGGCTGCCGGCGTAAGCATGCTGGAAGTATCCGGCGGA

ACCCAGTATGAAGGCCTGGAACATATCATTCCTTGCCA GAATAAGAGCAGGGGCGTCAATGTATATGAAGCTTCTG

AGATCAAGAAAGTAGTGGGCATCCCGGTATACGCAGTA

GGAAAGATCAACGATATACGCTATGCGGCAGAGATCGT

AGAACGCGGCCTGGTAGACGGCGTGGCTATGGGACGTC

CGCTTCTGGCAGATCCGGACCTTTGCAAGAAGGCAGTG

GAAGGCCAGTTTGACGAGATCACTCCATGCGCAAGCTG

CGGCGGAAGCTGCATCAGCCGTTCTGAGGCAGCGCCTG

AGTGCCATTGCCATATTAATCCAAGGCTTGGCCGGGAG

TATGAATTCCCGGATGTGCCTGCCGAGAAGTCCAAGAA

GGTACTGGTTATCGGCGCAGGCCCTGGAGGAATGATGG

CTGCCGTGACAGCTGCGGAACGCGGCCATGATGTTACG

GTATGGGAGGCTGACGACAAGATCGGCGGCCAGCTGAA

CCTGGCAGTAGTGGCTCCTGGCAAGCAGGAGATGACCC

AGTGGATGGTACATCTGAACTATCGCGCGAAGAAAGCA

GGCGTGAAGTTTGAATTCAATAAAGAAGCGACGGCAGA

AGATGTCAAGGCGCTGGCGCCGGAAGCAGTGATCGTTG

CTACAGGCGCGAAGCCGCTGGTTCCTCCGATTAAAGGA

ACACAGGATTATCCGGTGCTTACTGCCCATGATTTCCTT

CGCGGCAAGTTCGTGATTCCGAAGGGACGCGTCTGCGT

GCTGGGAGGAGGCGCGGTTGCCTGCGAGACTGCCGAGA

CAGCCCTGGAGAATGCACGTCCGAATTCTTATACCAGA

GGATACGATGCAAGCATCGGAGATATCGATGTCACGCT

TGTGGAGATGCTTCCGCAGCTCCTTACCGGCGTATGCGC

GCCGAACCGCGAGCCTTTGATCCGCAAGTTAAAGAGCA

AGGGCGTACACATCAACGTCAATACCAAGATCATGGAA

GTAACAGACCATGAAGTAAAGGTTCAGAGACAGGATG

GAACGCAGGAATGGCTGGAAGGATTTGACTATGTCCTC

TTTGGCCTTGGTTCCAGAAATTACGATCCGCTTTCAGAG

ACCCTCAAGGAATTCGTTCCGGAAGTACATGTCATCGG

CGATGCCGTAAGGGCGCGCCAGGCAAGCTACGCAATGT

GGGAAGGATTTGAGAAGGCATACAGCCTGTAAAAGCG

GTTTGAGTAAAAGGAGGCTTAAGAAATGGCAGTGAAGG

CAATCTCAGGCTGCGACAAGGATCAGGAACTGATCA

Clostridium ATGCACAAAAAATCAGCGTGTGAGAGGGAGGGCAAGG scindens AGTTGAAGCGTGACTTTTTTAACAAGTTTAATTTGGGGA

VPI 12708 bile CATCGAACTTTGTCACGCCGGGAAAACAGTTGGAATAC acid-coenzyme A G I N CGGAATGCAAGCCAGATTCTACTGCGGTCATTTG ligase (BaiB) CTTAGATAAAGAACAGAACTGTTCCGTTATTACTTGGC

ATCAGCTGCACGTCTATTCCAGCCAGCTGGCATGGTAC

CTTATAGAAAATGAGATTGGCCCGGGGTCGATCGTACT

TACAATGTTTCCGAACAGCATCGAGCACATTATTGCGG

TATTTGC AATCTGGA AG G CG G G CG CCTGCT AT ATG CC

CATGTCCTATAAGGCGGCGGAATCCGAGATCAGGGAGG

CCTGCGATACCATCCACCCGAATGCGGCTTTTGCGGAA

TGCAAGATTCCAGGATTAAAATTCTGCCTTAGCGCAGA

CGAGATATATGAGGCGATGGAAGGAAGATCCAAGGAG

ATGCCTTCGGACCGTCTGGCCAATCCGAACATGATATC

CTTATCAGGCGGAACCAGCGGAAAGATGAAGTTCATCC

GTCAGAACCTTCCATGCGGGCTGGACGATGAGACGATC

AGAAGCTGGTCTTTGATGTCTGGAATGGGATTTGAGCA GCGCC AGCTGCTGGTAGGCCCGCTGTTTC ATG G CG CG C

CTC ACTCCG CG G CGTTT A ATGGACTGTTC ATGGGC AAC

ACCCTGGT ACTGACC AGGAACCTTTG CCCG G G AAATAT

CCTGAACATGATTAAGAAATATAAGATTGAATTTATAC

AGATGGTGCCGACCCTGATGAACCGGCTTGCCAAACT

GGAGGGAGTCGGAAAAGAAGACTTTGCATCCCTGAAG

GCGCTGTGCCATACAGGGGGCGTCTGTTCTCCCTGGC

TTAAGCAGATCTGGATCGACCTGCTGGGGCCTGAAAAG

ATCTATGAGATGTATTCCATGACGGAATGCATCGGCCT

TACCTGCATCCGGGGAGACGAGTGGGTGAAGCATCCGG

GAAGCATCGGACGGCCAGTGGGCGATAGCAAGGTGTC

TATCCGGGATGAGAATGGCAAGGAAGTTGCGCCTTTTG

AGATTGGCGAGATCTATATGACAGCGCCGGCCTCCTAT

CTGGTTACCGAGTACATCAATTGGGAACCGCTGGAAGT

GAAAGAGGGAGGCTTCCGAAGCGTAGGGGATATCGGCT

ACGTGGATGAGCAGGGCTATCTGTACTTTTCTGACCGG

CG C AG CG AC ATG CTGGTATC AGGCGGAG AAAACGTGT

TCGCCACCGAAGTCGAGACGGCGCTTTTGAGATATAAG

GATATCCTGGACGCTGTAGTGGTAGGGATACCGGATGA

AGATCTGGGGCGAAGGCTCCATGCGGTCATTGAGACA

GGGAAAGAGATACCGGCAGAGGAACTGAAAACATTCC

TGAGAAAGTATCTGACTCCATATAAGATACCAAAGACG

TTCGAGTTCGTAAGGAGCATACGAAGGGGAGACAATG

GAAAGGCCGACAGGAAGCGGATCCTGGAAGATTGTAT

TGCCCGCGGGGGATGA

96. Clostridium MHKKSACEREGKELKRDFFNKFNLGTSNFVTPGKQLEYV scindens SECKPDSTAVICLDKEQNCSVrrWHQLHVYSSQLAWYLIE

VPI 12708 bile NEIGPGSIVLTMFPNSIEHIIAVFAIWKAGACYMPMSYKAA acid-coenzyme A ESEIREACDTIHPNAAFAECKIPGLKFCLSADEIYEAMEGRS ligase (BaiB) KEMPS DRLANPNMIS LS GGTS GKMKFIRQNLPC GLDDETI protein RSWSLMSGMGFEQRQLLVGPLFHGAPHSAAFNGLFMGNT

LVLTRNLCPGNILNMIKKYKIEFIQMVPTLMNRLAKLEGV

GKEDFASLKALCHTGGVCSPWLKQIWIDLLGPEKIYEMYS

MTECIGLTCIRGDEWVKHPGS IGRPVGDS KVS IRDENGKE

VAPFEIGEIYMTAPASYLVTEYINWEPLEVKEGGFRSVGDI

GYVDEQGYLYFSDRRSDMLVSGGENVFATEVETALLRYK

DILDAVVVGIPDEDLGRRLHAVIETGKEIPAEELKTFLRKY

LTPYKIPKTFEFVRSIRRGDNGKADRKRILEDCIARGG

97. Clostridium ATGAGTTACGAAGCACTTTTTTCACCATTCAAGGTCAGA scindens GGACTGGAACTTAAAAACCGTATCGTCCTGCCTGGAAT

VPI 12708 BaiCD GAAC ACC AAGATGGC AAAGAAC AAGC ACGAC AT AG G C

G AG G AT ATG AT AG CCT ACC ATGTTG CC AGGGC AAAAG

CGGGATGCGCGTTAAATATATTTGAATGCGTAGCATTA

TGTCCG G CG CCTCACG CTT AT ATGT ATATGGGGCTTT A

TACGGACCATCATGTAGAACAGCTTAAGAAATTGACGG

ATGCAGTCCATGAAGCAGGCGGCAAGATGGGCATCCA

GCTGTGGCATGGAGGATTCAGCCCGCAGATGTTCTTTG

ACGAGACCAACACCCTGGAAACTCCGGACACTCTTACG

GTAGAGAGGATTCATGAGATCGTAGAAGAATTCGGAC

GCGGCGCAAGGATGGCTGTTCAGGCTGGATTTGACGCA GTAGAATTCCATGCGGCTCACAGTTATCTGCCTCACGA

GTTCTTAAGCCCTGGAATGAACAAACGTACGGATGAGT

ACGGCGGAAGTTTTGAGAACCGCTGCAGATTCTGTTAT

GAAGTCGTTCAGGCAATCCGTTCCAATATCCCGGATGA

CATGCCATTCTTTATGCGTGCAGACTGCATCGACGAAT

TAATGGAACAGACCATGACAGAGGAAGAGATCGTTAC

ATTT ATC AAT AAGTG CG C AG AACTTGGCGTGGATGTGG

CAGACCTTTCCCGTGGAAACGCGACTTCATTCGCAACC

GTATATGAAGTTCCGCCATTCAACCTGGCTCATGGCTTC

AACATAGAGAATATTTACAACATCAAAAAGCAGATCAA

TATCCCGGTTATGGGAGTTGGCCGTATCAATACAGGAG

AGATGGCAAACAAGGTCATTGAAGAAGGCAAGTTTGAC

CTGGTAGGCATCGGACGCGCCCAGCTTGCAGATCCAAA

CTGGATCACCAAAGTAAGAGAAGGCAAAGAAGACCTGA

TCCGCCACTGTATCGGATGTGACCAGGGATGCTATGAC

GCAGTCATCAATCCAAAGATGAAGCATATCACCTGCAC

CC AC AATCC AGGATTGTGCTTAGAGT ATC AG G G AATG C

CAAAGACAGACGCTCCTAAGAAAGTCATGATCGTAGGA

GGCGGAATGGCAGGCATGATCGCTGCGGAAGTATTAAA

GACCAGAGGCCATAACCCGGTAATCTTCGAGGCATCCG

ACAAGCTTGCAGGACAGTTCAGGCTGGCAGGCGTAGC

GCCGATGAAGCAGGATTGGGCAGATGTTGCAGAATGG

GAAGCAAAAGAAGTAGAGCGCCTTGGAATCGAAGTAC

GTCTGAATACCGAAGTGACTGCAGAGACCATCAAGGAA

TTCAATCCGGATAATGTCATCATCGCAGTAGGCTCTAC

CTATGCGCTGCCTGAGATTCCGGGAATCGACAGCCCAA

GCGTATACTCCC AGT ATC AG GT ACTG A AAG G G GAAGTA

AATCCGACAGGCCGTGTAGCCGTTATCGGATGCGGACT

GGTTGGTACGGAAGTCGCAGAACTTCTGGCATCCAGA

GGCGCACAGGTAATCGCGATCGAGAGGAAGGGCGTAG

GT ACCG G CCTT AG C ATG CTTCG C AGAATGTTC ATGAAC

CCGGAATTCAAATATTACAAGATCGCCAAGATGTCCGG

AACAAATGTCACCGCTTTAGAGCAGGGCAAGGTTCACT

ACATCATGACAGACAAGAAGACCAAAGAAGTGACGCA

GGGAGTCCTGGAATGCGACGCTACCGTTATCTGTACAG

GAATTACCGC ACGTCC AAG CG ATG G G CTT AAG G C A AG

ATGCGAAGAACTTGGAATCCCGGTTGAGGTGATCGGAG

ACGCTGCTGGCGCAAGAGACTGCACGATCGCGACACG

CGAAGGCTATGACGCAGGAATGGCAATCTAG

Clostridium MSYEALFSPFKVRGLELKNRIVLPGMNTKMAKNKHDIGE scindens DMIAYHVARAKAGCALNIFECVALCPAPHAYMYMGLYT

VPI 12708 BaiCD DHHVEQLKKLTDAVHEAGGKMGIQLWHGGFSPQMFFDE protein TNTLETPDTLTVERIHEIVEEFGRGARMAVQAGFDAVEFH

AAHS YLPHEFLS PGMNKRTDE YGGS FENRCRFC YE V VQ AI

RSNIPDDMPFFMRADCIDELMEQTMTEEEIVTFINKCAELG

VDVADLSRGNATSFATVYEVPPFNLAHGFNIENIYNIKKQI

NIPVMGVGRINTGEMANKVIEEGKFDLVGIGRAQLADPN

WITKVREGKEDLIRHCIGCDQGCYDAVINPKMKHITCTHN

PGLCLEYQGMPKTDAPKKVMIVGGGMAGMIAAEVLKTR

GHNPVIFEASDKLAGQFRLAGVAPMKQDWADVAEWEAK EVERLGIEVRLNTEVTAETIKEFNPDNVIIAVGSTYALPEIP

GIDSPSVYSQYQVLKGEVNPTGRVAVIGCGLVGTEVAELL

ASRGAQVIAIERKGVGTGLSMLRRMFMNPEFKYYKIAKM

SGTNVTALEQGKVHYIMTDKKTKEVTQGVLECDATVICT

GITARPSDGLKARCEELGIPVEVIGDAAGARDCTIATREGY

DAGMAI

99. Clostridium ATGACATTAGAAGAGAGAGTTGAAGCATTAGAAAAAG scindens AATTGCAGGAGATGAAGGATATTGAGGCAATCAAGGA

VPI 12708 bile acid ACTGAAAGGAAAGTATTTCCGCTGCCTGGACGGAAAGA 7-a dehydratase TGTGGGATGAGCTGGAGACCACCCTGTCACCAAATATC (BaiE) GTAACCTCTTATTCCAACGGGAAACTGGTATTCCATAGC

CCGAAGGAAGTTACCGATTACTTAAAGAGCTCGATGCC

AAAAGAAGAGATCAGCATGCATATGGGCCACACGCCG

GAGATCACCATTGACAGCGAGACTACGGCTACGGGCAG

ATGGTATCTGGAAGATAGACTGATCTTTACGGACGGTA

AGTACAAAGACGTAGGAATCAATGGCGGCGCGTTCTAT

ACAGACAAATATGAGAAGATAGACGGCCAGTGGTACAT

CCTTGAAACCGGCTATGTACGAATCTATGAAGAACATT

TCATGCGTGATCCAAAGATCCATATCACGATGAACATG

CACAAATAA

100 Clostridium MTLEERVEALEKELQEMKDIEAIKELKGKYFRCLDGKMW scindens DELETTLS PNIVTS YS NGKLVFHS PKE VTD YLKS S MPKEEIS

VPI 12708 bile acid MHMGHTPEITIDSETTATGRWYLEDRLIFTDGKYKDVGIN 7-a dehydratase GGAFYTDKYEKIDGQWYILETGYVRIYEEHFMRDPKIHIT (BaiE) protein MNMHK

101 Clostridium ATGAATCTCGTACAAGACAAAGTTACGATCATCACAGG scindens CGGCACAAGAGGTATTGGATTCGCCGCTGCCAAAATAT

VPI 12708 3-alpha TTATCGACAATGGCGCAAAAGTATCCATCTTCGGAGAG hydroxysteroid ACGCAGGAAGAAGTAGATACAGCGCTTGCACAGTTAAA dehydrogenase AGAACTTTATCCGGAAGAAGAGGTTCTGGGATTCGCGC (BaiA2) CGGATCTTACATCCAGAGACGCAGTTATGGCAGCGGTA

GGCCAGGTAGCACAGAAATATGGCAGACTGGATGTCAT

GATCAACAATGCAGGAATTACCAGCAACAACGTATTCT

CCAGAGTGTCTGAAGAAGAGTTCAAGCATATTATGGAC

ATCAACGTAACAGGCGTATTCAACGGCGCATGGTGCGC

ATACCAGTGCATGAAGGATGCCAAAAAGGGCGTTATCA

TCAACACGGCATCCGTTACAGGCATCTTCGGATCACTCT

CAGGCGTAGGATATCCGGCCAGCAAGGCAAGCGTGATC

GGACTCACCCATGGACTTGGAAGAGAGATCATCCGCAA

GAATATCCGTGTAGTAGGAGTGGCTCCTGGAGTTGTGA

ACACGGATATGACCAATGGCAATCCTCCGGAGATCATG

GAAGGATATCTGAAGGCGCTTCCGATGAAGAGAATGCT

TGAGCCGGAAGAGATCGCTAATGTATACCTGTTCCTGG

CATCTGACTTGGCAAGCGGCATTACGGCTACTACGGTC

AGCGTAGACGGGGCTTACAGACCATAA

102 Clostridium MNLVQDKVTIITGGTRGIGFAAAKIFIDNGAKVSIFGETQE scindens EVDTALAQLKELYPEEEVLGFAPDLTSRDAVMAAVGQVA

VPI 12708 3-alpha QKYGRLDVMINNAGITSNNVFSRVSEEEFKHIMDINVTGV hydroxysteroid FNGAWCAYQCMKDAKKGVIINTASVTGIFGSLSGVGYPA dehydrogenase S KAS VIGLTHGLGREIIRKNIR V VG V APG V VNTDMTNGNP (BaiA2) protein PEIMEGYLKALPMKRMLEPEEIANVYLFLASDLASGITATT VSVDGAYRP

103 Clostridium ATGGCTGGAATAAAAGATTTTCCAAAATTCGGAGCTCT scindens TGCAGGGCTTAAGATACTTGACAGCGGATCTAACATCG

VPI 12708 BaiF CCGGACCTTTAGGCGGAGGCCTTCTGGCAGAATGCGGA

GCAACGGTCATCCATTTTGAAGGACCAAAGAAACCTGA

TAACCAGAGAGGATGGTACGGCTATCCACAGAATCACC

GTAATCAGCTGTCTATGGTAGCAGACATCAAATCTGAA

GAAGGAAGAAAGATCTTCCTTGATCTGATCAAATGGGC

AGATATCTGGGTAGAGTCATCCAAAGGCGGACAGTATG

ACAGGCTGGGACTTTCCGATGAAGTCATCTGGGAAGTA

AATCCTAAGATTGCCATCGTGCACGTATCCGGATATGG

ACAGACAGGAGACCCGTCTTACGTTACACGTGCATCCT

ATGACGCAGTAGGCCAGGCATTCAGCGGCTATATGTCA

CTGAACGGAACAACGGAAGCGCTGAAGATCAATCCTTA

TCTGAGCGATTTCGTATGCGGACTTACCACATGCTGGGC

TATGCTTGCCTGCTATGTAAGCACCATTCTTACCGGAAA

AGGCGAATCTGTTGACGTTGCACAGTACGAAGCGCTGG

CACGTATCATGGACGGACGTATGATCCAGTACGCTACA

GACGGCGTGAAGATGCCAAGAACCGGCAATAAGGATG

CGCAGGCTGCCCTGTTCAGCTTCTACACCTGTAAAGACG

GAC GT AC G ATCTTT ATC GG A ATG ACTGGC GC GG A AGT A

TGTAAGAGAGGCTTCCCGATCATCGGACTTCCGGTACCT

GGAACCGGAGACCCGGACTTCCCGGAAGGCTTCACAGG

CTGGATGATCTATACTCCTGTAGGACAGAGAATGGAAA

AGGCTATGGAGAAGTATGTATCTGAGCATACGATGGAA

GAAGTAGAGGCTGAGATGCAGGCACACCAGATTCCATG

CCAGAGAGTATACGAGCTGGAAGACTGCCTGAACGATC

CTCACTGGAAAGCACGTGGAACTATTACGGAGTGGGAT

GACCCGATGATGGGACATATCACAGGCCTTGGACTGAT

CAACAAGTTCAAGAGAAATCCTTCCGAAATCTGGAGAG

GCGCTCCGCTGTTCGGTATGGATAACCGCGATATCCTGA

AAGACCTGGGATATGACGATGCAAAGATCGATGAACTC

TATGAGCAGGGCATCGTCAATGAATTCGACCTTGACAC

TACTATCAAACGCTATAGACTGGATGAAGTAATTCCAC

ATATGAGAAAGAAAGAGGAGTAA

104 Clostridium MAGIKDFPKFGALAGLKILDSGSNIAGPLGGGLLAECGAT scindens VIHFEGPKKPDNQRGWYGYPQNHRNQLSMVADIKSEEGR

VPI 12708 BaiF KIFLDLIKWADIWVESSKGGQYDRLGLSDEVIWEVNPKIAI protein VH VS G YGQTGDPS Y VTRAS YD A VGQ AFS GYMS LNGTTE A

LKINP YLS DFVC GLTTC W AMLAC Y VS TILTGKGES VD V AQ

YEALARIMDGRMIQYATDGVKMPRTGNKDAQAALFSFYT

CKDGRTIFIGMTGAEVCKRGFPIIGLPVPGTGDPDFPEGFTG

WMIYTPVGQRMEKAMEKYVSEHTMEEVEAEMQAHQIPC

QRVYELEDCLNDPHWKARGTITEWDDPMMGHITGLGLIN

KFKRNPSEIWRGAPLFGMDNRDILKDLGYDDAKIDELYEQ

GIVNEFDLDTTIKRYRLDEVIPHMRKKEE

105 Clostridium ATGAGCACCGTAGCCAATCCAAATTATAAGAAAGGTTT scindens TGTCCCCTTTGCAATTGCAGCACTCCTGGTGAGCCTGAT

VPI 12708 bile acid CGGCGGTTTTACCGCCGTTCTCGGCCCGGCCTTCGTGGC transporter (BaiG) GGACCAGGGGATTGACTATAATAATACCACATGGATTT

CCCTGGCGCTGGCGATGTCTTCCGCCGCATGCGCTCCAA

TCCTTGGAAAACTGGGAGACGTGCTAGGACGCAGGACG

ACGCTGCTTCTGGGTATTGTGATCTTTGCGGCCGGCAAT

GTGCTGACAGCCGTAGCCACGTCCCTGATATTCATGCTG

GCAGCCCGTTTTATCGTAGGTATCGGAACAGCAGCGAT

CTCACCGATCGTTATGGCCTATATCGTAACCGAGTATCC

GCAGGAGGAGACAGGAAAGGCCTTTGGCCTGTATATGC

TGATCTCCAGCGGCGCCGTCGTGGTAGGACCTACCTGT

GGCGGCCTGATCATGAATGCGGCTGGCTGGAGAGTCAT

GATGTGGGTATGCGTCGCTCTGTGCGTCGTTGTATTCCT

GATCTGCACATTCTCCATCAAGAAGACTGCATTTGAGA

AGAAGAGCATGGCAGGATTTGACAAGCCGGGCGCAGC

CCTGGTAGTCGTATTCTTCAGTTTGTTCCTGTGCATCCC

ATCCTTCGGACAGAATATCGGATGGTCTTCCACAGCATT

TATCGCAGCAGCGGCAGTAGCGCTGGTAGCACTTTTCA

TCCTGGTAATGGTAGAAAAGAAAGCGAAGAGTCCGATC

ATGAACGGCAAGTTTATGGCACGCAAGGAATTCGTGCT

TCCAGTATTGATCCTGTTCCTTACACAGGGACTTATGAT

GGCAAATATGACCAATGTCATCGTGTTCGTGCGCTATAC

GCAGCCGGACAATGTCATTATATCAAGTTTTGCGATCTC

CATCATGTACATAGGAATGTCCTTAGGCTCCGTTATCAT

TGGACCTGTTGCAGATAAGAAAGAGCCAAAGACGGTTC

TGACATTCTCTCTGGTACTGACAGCCATCGGCTGTGCGC

TGATGTATCTGTTCAAGGCAGATTCCTCCGTCGCTATCT

TTGCGGCATCCTTGGGAATCCTTGGATTTGGCCTTGGAG

GAAATGCAACCATCTTCATGAAGGTAGCGCTTTCCGGC

CTGTCCAGCGAAGTAGCTGGCTCTGGTACTGGAACCTA

TGGCCTGTTCAGAGATATCTCGGCACCATTCGGCGTGGC

AGTGTTCGTGCCTATGTTTGCCAACGGCGTAACAGCGA

ATATTGCGAAATACGCGTCAGGCGGCATGGAAGAAGGC

GCCGCTACGGTAAAAGCAGCCATCTCATCCATCCAGAC

GCTGACACTGGTTGAACTTGGATGTATCGTTGTGGGAAT

CATCCTTGTGAGAATGCTGCCAAGAATCTATCAGAAGA

AAGAGGCATAA

106 Clostridium MS T V ANPN YKKGFVPF AIA ALLVS LIGGFT A VLGP AF V AD scindens QGIDYNNTTWISLALAMSSAACAPILGKLGDVLGRRTTLL

VPI 12708 bile acid LGIVIF A AGN VLT A V ATS LIFMLA ARFIVGIGT A AIS PIVM A transporter (BaiG) YIVTEYPQEETGKAFGLYMLISSGAVVVGPTCGGLIMNAA protein GWRVMMWVCVALCVVVFLICTFSIKKTAFEKKSMAGFD

KPG A ALV V VFFS LFLCIPS FGQNIGWS S TAFIA A AAV ALVA LFILVMVEKKAKSPIMNGKFMARKEFVLPVLILFLTQGLM M ANMTN VIVFVR YTQPDN VIIS S FAIS IM YIGMS LGS VIIGP VADKKEPKTVLTFSLVLTAIGCALMYLFKADSSVAIFAAS LGILGFGLGGNATIFMKVALS GLS SE VAGS GTGT YGLFRDI SAPFGVAVFVPMFANGVTANIAKYASGGMEEGAATVKA AIS S IQTLTLVELGCIV VGIILVRMLPRIYQKKEA

107 Clostridium AAGAGGTAATTATAAATGGATATGAAACATTCCAGATT scindens ATTTTCGCCGCTTCAGATCGGATCCCTGACACTGTCTAA VPI 12708 CCGTGTCGGCATGGCTCCCATGAGCATGGACTATGAAG NADH: Flavin CAGCAGACGGAACTGTGCCCAAGAGGCTGGCGGACGTA oxidoreductase TTTGTCCGCCGCGCCGAGGGAGGCACAGGCTACGTCAT (BaiH) GATCGACGCGGTGACGATAGACAGCAAGTATCCTTATA

TGGGAAATACAACGGCCCTTGACCGTGATGAACTGGTT CCCCAGTTTAAGGAATTTGCTGACAGAGTAAAAGAAGC AGGCAGCACGCTGGTGCCGCAGATCATTCATCCGGGTC CGGAATCCGTATGCGGCTACCGGCATATCGCTCCGCTTG GACCTTCTGCCAACACCAATGCAAACTGCCACGTGAGC AGATCGATCAGCATAGATGAGATCCATGACATCATTAA GCAGTTCGGCCAGGCGGCACGCCGCGCCGAAGAAGCA GGATGCGGGGCAATCTCCCTGCACTGCGCGCATGCGTA TATGCTGCCAGGATCCTTCCTGTCACCGCTTCGCAACAA GCGCATGGATGAATATGGCGGAAGCCTTGACAACCGTG CCCGTTTCGTGATCGAGATGATTGAGGAGGCCCGCAGG AATGTGAGTCCTGATTTCCCGATCTTCCTTCGTATCTCC GGAGACGAGAGAATGGTAGGAGGCAACAGCCTTGAAG ATATGCTCTACCTGGCACCGAAGTTCGAGGCTGCCGGC GTAAGCATGCTGGAAGTATCCGGCGGAACCCAGTATGA AGGCCTGGAACATATCATTCCTTGCCAGAATAAGAGCA GGGGCGTCAATGTATATGAAGCTTCTGAGATCAAGAAA GTAGTGGGCATCCCGGTATACGCAGTAGGAAAGATCAA CGATATACGCTATGCGGCAGAGATCGTAGAACGCGGCC TGGTAGACGGCGTGGCTATGGGACGTCCGCTTCTGGCA GATCCGGACCTTTGCAAGAAGGCAGTGGAAGGCCAGTT TGACGAGATCACTCCATGCGCAAGCTGCGGCGGAAGCT GCATCAGCCGTTCTGAGGCAGCGCCTGAGTGCCATTGC CATATTAATCCAAGGCTTGGCCGGGAGTATGAATTCCC GGATGTGCCTGCCGAGAAGTCCAAGAAGGTACTGGTTA TCGGCGCAGGCCCTGGAGGAATGATGGCTGCCGTGACA GCTGCGGAACGCGGCCATGATGTTACGGTATGGGAGGC TGACGACAAGATCGGCGGCCAGCTGAACCTGGCAGTAG TGGCTCCTGGCAAGCAGGAGATGACCCAGTGGATGGTA CATCTGAACTATCGCGCGAAGAAAGCAGGCGTGAAGTT TGAATTCAATAAAGAAGCGACGGCAGAAGATGTCAAG GCGCTGGCGCCGGAAGCAGTGATCGTTGCTACAGGCGC GAAGCCGCTGGTTCCTCCGATTAAAGGAACACAGGATT ATCCGGTGCTTACTGCCCATGATTTCCTTCGCGGCAAGT TCGTGATTCCGAAGGGACGCGTCTGCGTGCTGGGAGGA GGCGCGGTTGCCTGCGAGACTGCCGAGACAGCCCTGGA GAATGCACGTCCGAATTCTTATACCAGAGGATACGATG CAAGCATCGGAGATATCGATGTCACGCTTGTGGAGATG CTTCCGCAGCTCCTTACCGGCGTATGCGCGCCGAACCGC GAGCCTTTGATCCGCAAGTTAAAGAGCAAGGGCGTACA CATCAACGTCAATACCAAGATCATGGAAGTAACAGACC ATGAAGTAAAGGTTCAGAGACAGGATGGAACGCAGGA ATGGCTGGAAGGATTTGACTATGTCCTCTTTGGCCTTGG TTCCAGAAATTACGATCCGCTTTCAGAGACCCTCAAGG AATTCGTTCCGGAAGTACATGTCATCGGCGATGCCGTA AGGGCGCGCCAGGCAAGCTACGCAATGTGGGAAGGATT TGAGAAGGCATACAGCCTGTAA 108 Clostridium MDMKHS RLFS PLQIGS LTLS NR VGM APMS MD YE A ADGT V scindens PKRLADVFVRRAEGGTGYVMIDAVTIDSKYPYMGNTTAL VPI 12708 DRDELVPQFKEFADRVKEAGSTLVPQIIHPGPESVCGYRHI NADH: Flavin APLGPS ANTN ANCH VS RS IS IDEIHDIIKQFGQ A ARRAEE AG oxidoreductase CGAISLHCAHAYMLPGSFLSPLRNKRMDEYGGSLDNRAR (BaiH) protein FVIEMIEE ARRN VS PDFPIFLRIS GDERM VGGNS LEDMLYL

APKFEAAGVSMLEVSGGTQYEGLEHIIPCQNKSRGVNVYE

ASEIKKVVGIPVYAVGKINDIRYAAEIVERGLVDGVAMGR

PLLADPDLC KKA VEGQFDEITPC AS CGGS CIS RS E A APECH

CHINPRLGREYEFPDVPAEKSKKVLVIGAGPGGMMAAVT

AAERGHDVTVWEADDKIGGQLNLAVVAPGKQEMTQWM

VHLNYRAKKAGVKFEFNKEATAEDVKALAPEAVIVATGA

KPLVPPIKGTQDYPVLTAHDFLRGKFVIPKGRVCVLGGGA

VACETAETALENARPNSYTRGYDASIGDIDVTLVEMLPQL

LTGVCAPNREPLIRKLKSKGVHINVNTKIMEVTDHEVKVQ

RQDGTQEWLEGFDYVLFGLGSRNYDPLSETLKEFVPEVH

VIGD A VRARQ AS Y AMWEGFEKA YS L

109 Clostridium

AGGAGGCTTAAGAAATGGCAGTGAAGGCAATCTCAGGC

scindens

TGCGACAAGGATCAGGAACTGATCA

VPI 12708 Bail

110 Clostridium

scindens

MA VKAIS GCDKDQELI

VPI 12708 Bail

protein

111 Clostridium GCAAATTGATTTTGATTGGTATTTCTTTCATTCAAAATA hiranonis TO-931 TCTCCTTTCCTTTATTTAGCTGTATTAAAATTTATAAAAA bile acid-inducible ATTTTCATTGTTAATAAAAAAATATTCTTTGTTAGTATT operon (GenBank ATAGCATAATTTATAAAAATAATGATAATGTTTTAATAT AF210152.2) TGAAATAATAAATATGTAAAAAGGTTGGAAATTTATTT

AAAAATGACCAGAGATAAAAAGCTCAGGTCATTTTTTT

TATTATTACAAGTAATTTGAAAAAAATATATGAAATGA

ATGGAGAAAATATAACTGAGATACATTTGATAATGAAA

AAAACATTTATCGAAATTGTAAATAGACTCATTGTTATA

ATTAATAAATATTTATTATGGCATAGTTGTTAAAATTAT

ACCCTAAAGAAACGTTTCCTCAAAAAGTGGGTTATAAA

ATAAATGTTTTTTGACGAAAGATGTGATTTTATTTGTAC

CCCTTTTGTATAAAGATTAAACAGTATTTTTGTATAAAT

ATATTGTATACAGTATAGAGAATGTCGATGTAAAAAAG

TATATAAAAGTAAATAATAATCAAAAAAACTAGTTTTA

ATTATTAAAAATGATAAAAAATATTAATAAAATAAAGA

GTCAAAAATACTTGTTAGTTAAATCACAGATTTTGTCTA

AGTATAGATTAGGTTTTGTATTTGAAAAGGTCATCTATA

GTGTTGTAAGAAAGCGAGTTATTAGCACATATTGTATCT

CAAAAAAATGTTAAGATAATATCAAGATAGGGCGATAA

AGAAAAAAGCAAATTGAAAAAGAAAAAAGTAACTATA

AGTTTTTACAATAAATCAAAAGAGAATTGATTTTAAAA

GAGGGAGGCAAAATACCGATATGAATGATGTGAAATGT

AAATATTTTAATAAATTTAATACAGGAATGTCAGATTTT

GTTACTCCAGGAAAACAGTTAGAATATGTAGCAAAATG

CAAGCCAGATGAAAAAGCTATCATATATATAGATAAAG AAGACAATGTGAGAGATATCACTTGGAAGGAACTTCAC

ATAGCTTCAAATAAACTAGCTTGGCATTTAATGAAAAA

GGGATTTGGAAAAGGTCAGGTAGCAATGGTATCTTTCC

CAAATGGTATAGAACATATATTAGCAACATTAGCTGTTT

GGAAAACAGGAGGTTGCTACATGCCAGTTTCTTGTAAG

ATAACAGATACAGAGCTTGGTGATATATGCAGAATAAT

AAAACCAACAGTTTCTTTTACAGATAAAGAAATGCCTT

GTAGAACAGAAAGTATAAAAATAGGATCAGTATTCGAT

GTTTGTAAAGACGAATCAGAAGAAATGCCAGAAGATAT

AGCTGCAAATCCAAATATGATTTCTCCATCTGGAGGAA

CAACAGGAGAGCCTAAGTTCATAAAACAGAATGTGGCA

AGTGGCTTATCTGATGAAATTATAAAAAGCTGGTTTGA

AATGTCAGGTATGGAATTTGAACAAAGACAATTATTAG

TAGGACCACTTTTCCATGGTGCTCCTCATACAGCAGCAT

TTAATGGATTATTTGTAGGAAATACATTGATAATACCTA

GAAATTTAAGACCTGAAAGTATAGTTAGATATATAAAA

GAATACAAAATAGAATTTATACAGATGATCCCAACATT

AATGAATAGAATAATAAAATTAGCTGATGTTGATAAAG

AAGATTTTAAATCAATAAAAGCACTACACCATACTGGT

GGATATTGTTCTCCATATTTAAAAGAAAAGTGGATCGA

TATAATAGGAGCTGAAAAAGTTCACGAAATGTACTCTA

TGACAGAGGCAATCGGTATCACTTGTATAAGAGGAGAT

GAATGGCTTAAACACTATGGAAGCGTAGGACTTCCACT

AGGAGGAAGCAGAATATCAATAAGAGATGAAGAAGGA

AATGAATTAGGACCACATGAGGTTGGAGAAATTCATAT

GACTTCACCAAGTGCTTGTTGCATGACAGAATACATAA

ACCATAAACCACTTGAAACTAAAGATGGTGGATTTAGA

AGTGTTGGTGATTTCGGTTATGTAGATGAAGATGGATA

CCTTTACTTCTCAGATAGAAGAAGCGACATGCTTGTTAT

AGGTGGAGAAAACGTATTTGCGACTGAAGTTGAACCAG

TACTACCAGCTTATGAAAAAGTAGTTGATGCTGTGGTA

GTTGGAATACCTGATGAAGAGTGGGGAAGAAGATTACA

CGCAATAGTACAGAAGAAAGAAGAAGTTTCAGCAGAA

GAATTAATCGAGTACTTAGGAAAACACTTATTACCATA

TAAAGTTCCAAAGAGCTTTACATTTGTTCCTTGCATACC

AAGAGGTGACAATGGAAAGGTAAACAGAGATAAGATG

CTAAAAGGCTTAATAGAAAAAAATCTAGTTAATAAAGT

TTGCTAGGATATAAATTCAGTTAACTATCTGCACCAAGT

GCAGTGGAAAATAAATCAAAATTAATAAAATAAATTAA

TAAGGTAAATTTAGGAGGTCTAAAATGAGTTACGACGC

ACTTTTTTCACCATTTAAAATCAGAGGATTAGAACTTAA

AAACAGAATAGTTCTACCAGGTATGAATACAAAAATGG

CAAAAAATAAACATGATTTAAGCGATGATATGATAGCT

TACCATGTTGCAAGAGCAAAAGCAGGTTGTGCATTAAA

TATATTTGAATGTGTTGCGCTATGTCCAGCACCTCATGC

ATATATGTACATGGGATTATACAATGACAATCATGTAG

CTCAGTTAAAAAAATTAACAGATGCTGTTCACGAAGTT

GGCGGTAAAATGGCTGTTCAGTTATGGCATGGTGGTTTC

AGCCCACAGATGTTCTTTGATAAAACAAATACATTAGA

AACACCAGATACTATAACAGTTGAACGTATTCATGAAA TAGTTAAAGAGTTTGGAGAAGGTGCAAGAAGAGCTGTT

GAAGCTGGATTCGATGCAGTTGAATTCCATGCAGCACA

CAGTTACTTACCTCACGAATTCCTAAGTCCAGGAATGA

ACAAAAGAACTGACGAATATGGTGGAAACTTCGAAAAT

CGTTGCAGATTCTGCTTCGAAGTAGTTGAAGCTATACGT

GCAAATATACCAGAAGATATGCCATTCTTCATGAGAGT

TGACTGCATAGATGAGTTAATGGATGAAGTAATGACAG

AAGAAGAAATAGTAGAATTCATAAATAGATGTGCTGAT

CTAGGAGTAGACGTAGCTGACTTATCAAGAGGTAATGC

TCAGTCATTCGCAACAGTTTACGAAGTTCCTCCTTTCAA

CTTACAGCACGGTTTCAATATAGAAAACATATACAACA

TCAAAAAACAGATAAAAATACCAGTAATGGGTGTTGGA

CGTATAAACACAGGAGAAATGGCTAACCAGGTAATAGC

AGATGGAAAATTTGACTTAGTTGGTATAGGTCGTGCTC

AGTTAGCAGATCAGGATTGGGTTGCTAAAGTTAGAGAA

GGTAAAGAAGATTTAATACGTCATTGTATAGGATGTGA

CCAGGGATGCTACGATGCAGTTATAAACCCTCAGATGA

CTCATATAACTTGTACAAGAAACCCTCACTTATGCTTAG

AATACAAAGGTATGCCAAAAACTGATGAACCTAAAAAA

GTTATGATAATCGGTGGTGGTATGGCTGGTATATTAGCA

GCTGAAGTACTTAAAAAACGTGGACATGAACCAGTTAT

ATTCGAAGCTTCTGATCACTTAGCAGGACAGTTCGTATT

AGCAGGTAAAGCTCCAATGAAAGAAGACTGGGCAGCT

GCAGCTAAATGGGAAGCTGAAGAAGTAGCTCGTTTAGG

AATAGAAGTTAGATACAATACAAAAGTTACTCCAGAAT

TAATAGAAGAATTCGCTCCAGACCACGTTGTTATAGCT

ATAGGATCTGATTACGTAGCTCCAGCTATACCAGGTAT

AGATAGTGACAAAGTTTACACTCAGTATCAGGTATTAA

AAGGTGAAGTAGAACCAAAAGGACATGTAGCAGTAGTT

GGTTGTGGATTAGTTGGTACAGAAGTTGCTCAGTACTTA

GCAGCTAGAGGAGCTCAGGTAACAGCTATAGAAAGAA

AAGGTGTTGGTACAGGTCTAAGCATGCTTAGAAGAATG

TTCATGAACCCAGAATTCAAATACTACAAAATAAACAA

AATGTCTGGAACTAACATAGTTGGTATAGAACCAGGAA

AACTTCACTACATAATGACTAACAAGAAAACTCAGGAA

GTTACTGAAGGTGTGTTAGAATGTGATGCAGCAGTAAT

CTGTACAGGTATAACTGCTAGACCAAGTGAAGATTTAC

AGGAAAAATGTAAAGAATTAGGTGTTCCATTCAACGTA

ATAGGTGACGCAGCTGGTGCTAGAGATGCTAGAATAGC

TACTCAGGAAGGTTACGAAGTAGGTATGAGTATATAAT

TTAAAAATTATATAATTATATAAATTAAAAGTTATTAAA

TTACAAGAAAGAGGCGAATAAAATGACTTTAGAAGCAA

GAATAGAAGCATTAGAAAAAGAAATACAGAGATTAAA

CGATATAGAAGCTATAAAACAGTTAAAAGCTAAATATT

TCCGTTGCCTAGATGGAAAATTATGGGATGAATTAGAA

ACTACTCTTTCTCCTAACATAGAAACTTCTTACTCTGAT

GGAAAATTAGTATTCCACAGCCCAAAAGAAGTAACTGA

ATATTTAGCAGCAGCAATGCCTAAAGAAGAAATAAGTA

TGCACATGGGACATACTCCAGAAATAACTATAGACAGC

GAAAATACTGCTACAGGAAGATGGTACTTAGAAGATAA CCTAATATTCACAGACGGAAAATACAAAAACGTTGGAA

TAAACGGTGGAGCATTCTACACAGATAAATATGAAAAA

ATAGACGGACAGTGGTACATAAAAGAAACTGGATATGT

TCGTATATTTGAAGAACATTTCATGAGAGATCCAAAAA

TACATATAACTAGCAACATGCATAAAGAAAAATAATAA

CTGATTGCTAATAAACAAGATATAAACAGGGGGCTGGT

AAACAGCCAGCCCTCTGAAAAATAAACTAAAAAACTAT

AATCTTTTAAAATCTTAATTAAAGTAGAAGGAGATAAG

ACAATGAACTTAGTACAGGACAAAATAGTTATAATAAC

AGGTGGAACAAGTGGTATAGGTCTTTGCGCAGCAAAAA

TATTCATGGATAACGGTGCAACAGTTTCTATATTCGGAA

AAACTCAGGAAGAAGTAGATGCTGCTAAAGCAGAATTA

AAAGAAACTCACCCAGATAAAGAAGTATTAGGATTTGC

TCCAGATTTAACTAATAGAGATGAAGTTATGGCTGCAG

TTGGTGCAGTAGCTGAAAAATACGGAAGATTAGACGTT

ATGATAAACAATGCTGGTGTTACTAGCTCAAACGTATTC

TCAAGAGTTAGCCCAGAAGAATTCACATATTTAATGGA

TATAAACGTTACAGGTGTATTCCATGGTGCTTGGGCTGC

TTACCACTGCCTGAAAGGTGAAAAGAAGATTATAATAA

ATACTGCTTCAGTAACAGGAATACACGGATCATTATCA

GGAGTTGGATACCCAACAAGTAAATCAGCTGTTGTAGG

ATTCACTCAGGCTCTTGGTAGAGAAATAATACGTAAAA

ACATAAGAGTTGTTGGTGTTGCACCAGGTGTTGTTAACA

CTCCAATGGTTGGTAATATACCAGATGAAATATTAGAT

GGATACCTAAGCTCATTCCCAATGAAGAGAATGTTAGA

ACCAGAAGAAATAGCTAACACTTACTTATTCTTAGCTTC

TGACTTAGCTAGTGGTATAACAGCTACAACTGTAAGCG

TTGACGGTGCTTATAGACCATCATAAGATTTACTTTAAT

TTAAAACTGTAATTAGATAGATAATACGACGATTAATA

TAAAAAATGTTCTTTAAAAGAAAAGGAGAAATAAAATG

GCTGGATTAAAAGATTTTCCTAAATTTGGTGCACTTTCT

GGATTAAAAATATTAGATAGTGGATCTAACATAGCTGG

ACCTCTAGGTGGTGGACTTTTAGCAGAATGTGGTGCTAC

AGTTATACACTTCGAAGGACCTAAAAAACCTGACAACC

AGAGAGGTTGGTATGGATACCCTCAGAACCACAGAAAC

CAGTTATCAATGGTTGCTGATATAAAATCTGAAGAAGG

TAGAAAAATATTCTTAGACTTAATAAAATGGGCTGACA

TATGGGTTGAATCATCAAAAGGTGGACAGTACGACAGA

CTAAGTCTTTCTGATGAAGTTATATGGTCAGTAAACCCT

AAAATAGCTATAGTTCACGTTTCTGGATACGGACAGGT

TGGAGATCCATCATACGTAACAAAAGCTTCTTATGATG

CTGTTGGACAGGCATTCAGTGGATACATGTCATTAAAT

GGTGTTAATGAAGCATTAAAAATAAATCCTTACCTAAG

TGACTTCGTATGTGTTCTTACTACTTGCTGGGCAATGTT

AGCATGCTACGTAAGTACTCAGTTAACTGGAAAAGGAG

AATCTGTAGACGTTGCTCAGTACGAAGCATTAGCTCGT

ATAATGGACGGACGTATGATACAGTACGCTACTGATGG

TGTAAGTGTTCCAAAAACTGGTAACAAAGATGCTCAGG

CAGCTCTATTCAGCTTCTATACTTGTAAAGATGGAAGAA

CTATATTCATAGGTATGACTGGTGCTGAAGTATGTAAG AGAGGATTCCCTGTAATAGGGCTTCCAGTTCCTGGTACA

GGTGACCCTGACTTCCCAGAAGGATTCACAGGATGGAT

GATAAATACTCCAGTTGGACAGAGAATGGAAAAAGCTA

TGGAAGCATTCGTTGCTGAAAGAACTATGCCAGAAGTT

GAAAAAGCTATGATAGATGCTCAGATACCATGCCAGAG

AGTTTATGATCTTGAAGACTGCTTAAACGACCCTCACTG

GAATGCTCGTGGAACTATAATGGAATGGGATGACCCAA

TGATGGGACACATAAAAGGTCTTGGATTAATAAACAAA

TTCAAAAACAACCCTTCTGAAATATGGAGAGGTGCTCC

ATTATTCGGTATGGACAACAGAGACATAATTAGAGACC

TTGGATATTCTGAGGAGGAAGTTAACGATTTATACGCT

AAAGGTATTGTAAACGAATTCGACCTTGAAACAACTAT

AAAACGTTACAAACTTGATCAGGTTATACCTCACATGG

CTAAAAAAGATAAATAAGAAACGTATTAAATAATAAAA

TATAAATGTCGAGCCTGCCAGAATGAGAATTTTGACAG

GCTTGATATTATAACGAAATGTTATAAAAAAAACAAAA

TAAAAATTGCTTAAATTTTATACAAGGAGAATTGAAAT

GACAGCAACAAACGCAAACTATAAAAAAGGCTTTATCC

CATTTGCTATAGCAGCGTTACTAGTAGGTCTTATAGGTG

GTTTCACAGCCGTTCTAGCACCTGCATTCGTAGCAGATA

TGGGTCTTAACGATAACAATACTACATGGATAGCACTA

GCGCTTGCAATGTCTACAGCTGCATGTGCTCCAATACTT

GGTAAATTAGGTGACGTACTTGGACGTCGTAAAACTTT

ATTATTAGGAATCATAGTATTCACAATAGGTAACGTATT

AACAGCAATAGCATCTTCATTAATATTCATGCTAGGTGC

AAGATTTATAGTTGGGGTTGGTACAGCGGCTATAGCTC

CAGTTATAATGGCTTACATAGTTACAGAATATCCACCA

GAAGAAACTGGTAAGGGATTCGCTCTTTATATGTTAAT

ATCAAGTGCTGCAGTTGTTGTTGGTCCAACTTGTGGTGG

ATTAATAATGCAGGCATTTGGATGGAGAATGATGATGT

GGGTTTGTGTTGCCCTTTGTGTAGTAACATTCTTCATAT

GTTCAGTAATGATTAAGAAAACAGACTTTGAAAAGAAA

AGTCTTGATAACTTCGATAAAAAAGGTGCAGTATGCGT

ACTAATATTCTTCAGTTTAGTATTATGTATACCATCATTT

GGACAGAATATAGGTTGGACATCAGCGCCATTCCTAGG

TGTTACAGCAGTAGCTTTAGTAACATTATTCTTATTAAT

AAAAGCTGAAAGCAGTGCAGAAAACCCAATATTAAGTG

GTAAATTTATGAAACGTAAAGAATTCATATTACCAGTA

TTAATATTATTCCTTACTCAGGGATTAATGCAGGCTAAC

ATGACTAACGTAATATTATTCGTTAGAGCTACTCAGCCA

GAAAATACAATAATATCAAGTTTCGCAATATCAATCCTT

TACATAGGTATGTCTTTAGGTTCAGTATTCATAGGACCT

ATGGCAGATAAAAAAGAACCAAAAACTGTACTTACAGG

ATCACTTCTATTCACTGGTATAGGTTGTGCAATGATGTA

CTTCTTCACAGAAACTGCACCATTCGCAATGTTAGCTGG

ATCTCTAGGAATGTTAGGTATAGGACTTGGAGGAAATG

CTACAATACTAATGAAAGTTTCATTATCTGGATTATCTC

AGGCAGAAGCTGGATCAGGAACAGGAACATACGGATT

ATTCAGAGATATATCAGCTCCATTTGGTGTTGCGGTATT

CGTACCACTATTTGCAAACACAGTTACAACAAGAATGG CTGGAGTAATGGCTAACGGAACTGCAGAAGCTGCTGCT

AAATCATTAGCATCTGTTTCTTCTATACATACATTAGCA

TTAGTTGAAGTATGCTGTGTAATATTAGCAATAGTTGCA

GTTAGAATGCTACCAAAAATACACAATAAATAATTTAA

AAATAATAACAGAGTTGAAAAAACACTCAATTAAAAGA

GGGGCCTTGAGCCCCTTTTTTAGTGTAAAAATGACAAA

ATACTATCAATTTATATAAATGATAATTAAACTCGTCAA

CCAAAGAAATATTCACAAAGTAGATAATAATAGATATT

CAAAAAGTGATATATTATTAGGCAAAAAGTGCAAGAAA

TTAGCGAGTATTCGACAACTTTTTGTCCAATGGTAGAAA

AGAATATTTGTTATCATAAATATAGACAAAGGGCTTTG

ACCAAAACTAAGGAAAAAGTTTGCATAATATAAAAAAT

AAAATAAAATAAAAAAATAAAAATAAAATAAAAGCGA

AAGGAAAAAACAACATCATGGATATGAAAAATTCTAAA

CTATTCTCACCTTTAACAATAGGATCATTAACATTAAAC

AACAGAGTTGGTATGGCACCAATGAGTATGGACTACGA

AGCTGCTGACGGAACAGTTCCAAAAAGATTAGCAGATA

TATTTGTTCGTAGAGCTGAAGGTGGAACAGGATATGTA

ACAATAGACGCGGTAACAATAGATAGTAAATATAAATA

TATGGGTAATACAACTGCTTTAGATTCTGATGATTTAGT

TACTCAGTTCAAAGAATTTGCAACAAGAGTTAGAGAAG

CAGGAAGCACATTAATACCTCAGGTTATACATCCAGGA

CCAGAATCAATATGTGGATACAGACACATAGCACCACT

TGGACCATCAGTTAATACAAATGCTAACTGCCACGTGA

GCCGTGCTATAAGTGTAGATGAAATACATGAAATAATA

AAACAGTTTGGACAGGCTGCTAGAAGAGTTGAAGAAGC

AGGATGCGGTGGTATAGGATTACACTGTGCACATGCTT

ACATGCTACCAGGTTCATTCTTATCTCCATTAAGAAACA

AAAGAATGGATGAATACGGCGGATGTCTAGATAACAGA

GCAAGATTCGTAATAGAAATGATAGAAGAAGTTCGTAG

AAATGTAAGTCCTGATTTCCCAATAATGCTTAGAATATC

TGGGGATGAAAGAATGATAGGAGGAAACTCTTTAGAAG

ATATGTTATACTTAGCTCCAAAATTTGTTGAAGCTGGTG

TAAATATGTTTGAAGTTTCTGGAGGTACTCAGTACGAA

GGATTAGAACACATAATACCAAGTCAGAACAAAAGCAT

AGGTGTAAACGTACACGAAGCATCTGAAATCAAAAAAG

TTGTAGATGTTCCAGTTTACGCTGTTGGTAAAATAAATG

ACATAAGATACGCTGCTGAAATAGTTGAAAGAGGACTA

GTTGATGGGGTATCAATAGGTAGACCATTATTAGCAGA

TCCAGACTTATGTAATAAAGCAAAAGAAAACTTATTTG

ATGAAATAACTCCATGTGCAAGCTGTGGAGGAAGCTGT

ATAAGCCGTACTGCAGATAGACCTCAGTGTCGTTGCCA

TATAAACCCAAGAGTTGGATTCGAATATGATTATCCAG

AAGTTCCAGCTGAAAAATCTAAAAAAGTTCTAGTTGTA

GGTGCTGGACCTGGTGGTATGATGGCAGCAGTTACAGC

AGCTGAAAGAGGACATGATGTAACACTTTGGGAAGCTG

ACACTCAGATAGGTGGACAGATAAACTTAGCAGTAGTA

GCTCCAGGTAAACAGGAAATGACTAAATGGTTATCTCA

CTTAAACTACAGAGCTAAAAAAGCTGGAGTTAAAATGG

TATTAGGAAAAGAAGCTACAGTAGAAAACATAAAAGA ATTTGCTCCAGAAGCAGTTATAGTTGCAACAGGTGCTA

GACCATTAGTTCCACCAATAAAAGGAACTCAGGACTAC

CCAGTTCTTACAGCTCATGACTTCTTAAGAGGAAAATTC

GTTATACCAAAAGGAAAAGTTTGTGTACTAGGTGGAGG

AGCTGTTGCTTGTGAAACTGCAGAAACAGTATTAGAAA

ACGCTAGACCAAACGCATTCACTAGAGGATTTGATGCT

AGTATCGGTGATGTAGATGTTACATTAGTAGAAATGTT

ACCACAGTTATTAACAGGAGTATGTGCTCCAAATAGAA

CTCCATTAATAAGAAAACTTAAAAACAAAGGTGTTCAT

ATAAATGTAAATACTAAAATATTAGAAGTAACTGACCA

CGACGTTAAAGTTCAGAGAGCTGACGGTGCAGAAGAAT

GGTT A A A AGG ATTC G ACT AC AT ACT ATTC GG ACTTGGTT

CTAGAAACTACGATCCAATATCTGAACAGATAAAAGAA

TTCGTTCCAGAAGTACACGTTGTTGGGGATGCTAAGAG

AGCTAGACAGGCAAGCTTTGCAATGTGGGAAGCTTTCG

AAGCAGCATACAGCTTATAA

112 Clostridium ATGAATGATGTGAAATGTAAATATTTTAATAAATTTAAT hiranonis TO-931 ACAGGAATGTCAGATTTTGTTACTCCAGGAAAACAGTT BaiB AGAATATGTAGCAAAATGCAAGCCAGATGAAAAAGCT

ATCATATATATAGATAAAGAAGACAATGTGAGAGATAT

CACTTGGAAGGAACTTCACATAGCTTCAAATAAACTAG

CTTGGCATTTAATGAAAAAGGGATTTGGAAAAGGTCAG

GTAGCAATGGTATCTTTCCCAAATGGTATAGAACATAT

ATTAGCAACATTAGCTGTTTGGAAAACAGGAGGTTGCT

ACATGCCAGTTTCTTGTAAGATAACAGATACAGAGCTT

GGTGATATATGCAGAATAATAAAACCAACAGTTTCTTTT

ACAGATAAAGAAATGCCTTGTAGAACAGAAAGTATAAA

AATAGGATCAGTATTCGATGTTTGTAAAGACGAATCAG

AAGAAATGCCAGAAGATATAGCTGCAAATCCAAATATG

ATTTCTCCATCTGGAGGAACAACAGGAGAGCCTAAGTT

CATAAAACAGAATGTGGCAAGTGGCTTATCTGATGAAA

TTATAAAAAGCTGGTTTGAAATGTCAGGTATGGAATTT

GAACAAAGACAATTATTAGTAGGACCACTTTTCCATGG

TGCTCCTCATACAGCAGCATTTAATGGATTATTTGTAGG

AAATACATTGATAATACCTAGAAATTTAAGACCTGAAA

GTATAGTTAGATATATAAAAGAATACAAAATAGAATTT

ATACAGATGATCCCAACATTAATGAATAGAATAATAAA

ATTAGCTGATGTTGATAAAGAAGATTTTAAATCAATAA

AAGCACTACACCATACTGGTGGATATTGTTCTCCATATT

TAAAAGAAAAGTGGATCGATATAATAGGAGCTGAAAA

AGTTCACGAAATGTACTCTATGACAGAGGCAATCGGTA

TCACTTGTATAAGAGGAGATGAATGGCTTAAACACTAT

GGAAGCGTAGGACTTCCACTAGGAGGAAGCAGAATATC

AATAAGAGATGAAGAAGGAAATGAATTAGGACCACAT

GAGGTTGGAGAAATTCATATGACTTCACCAAGTGCTTG

TTGCATGACAGAATACATAAACCATAAACCACTTGAAA

CTAAAGATGGTGGATTTAGAAGTGTTGGTGATTTCGGTT

ATGTAGATGAAGATGGATACCTTTACTTCTCAGATAGA

AGAAGCGACATGCTTGTTATAGGTGGAGAAAACGTATT

TGCGACTGAAGTTGAACCAGTACTACCAGCTTATGAAA AAGTAGTTGATGCTGTGGTAGTTGGAATACCTGATGAA

GAGTGGGGAAGAAGATTACACGCAATAGTACAGAAGA

AAGAAGAAGTTTCAGCAGAAGAATTAATCGAGTACTTA

GGAAAACACTTATTACCATATAAAGTTCCAAAGAGCTT

TACATTTGTTCCTTGCATACCAAGAGGTGACAATGGAA

AGGTAAACAGAGATAAGATGCTAAAAGGCTTAATAGA

AAAAAATCTAGTTAATAAAGTTTGCTAG

113 Clostridium MNDVKCKYFNKFNTGMSDFVTPGKQLEYVAKCKPDEKAI hiranonis TO-931 IYIDKEDNVRDITWKELHIASNKLAWHLMKKGFGKGQVA BaiB protein MVSFPNGIEHILATLAVWKTGGCYMPVSCKITDTELGDIC

RIIKPTVSFTDKEMPCRTESIKIGSVFDVCKDESEEMPEDIA

ANPNMISPS GGTTGEPKFIKQNVAS GLSDEIIKS WFEMS GM

EFEQRQLLVGPLFHGAPHTAAFNGLFVGNTLIIPRNLRPESI

VRYIKEYKIEFIQMIPTLMNRIIKLADVDKEDFKSIKALHHT

GGYCSPYLKEKWIDIIGAEKVHEMYSMTEAIGITCIRGDE

WLKH YGS VGLPLGGS RIS IRDEEGNELGPHE VGEIHMTS PS

ACCMTEYINHKPLETKDGGFRSVGDFGYVDEDGYLYFSD

RRSDMLVIGGENVFATEVEPVLPAYEKVVDAVVVGIPDEE

WGRRLHAIVQKKEEVSAEELIEYLGKHLLPYKVPKSFTFV

PCIPRGDNGKVNRDKMLKGLIEKNLVNKVC

114 Clostridium ATGAGTTACGACGCACTTTTTTCACCATTTAAAATCAGA hiranonis TO-931 GGATTAGAACTTAAAAACAGAATAGTTCTACCAGGTAT BaiCD GAATACAAAAATGGCAAAAAATAAACATGATTTAAGCG

ATGATATGATAGCTTACCATGTTGCAAGAGCAAAAGCA

GGTTGTGCATTAAATATATTTGAATGTGTTGCGCTATGT

CCAGCACCTCATGCATATATGTACATGGGATTATACAAT

GACAATCATGTAGCTCAGTTAAAAAAATTAACAGATGC

TGTTCACGAAGTTGGCGGTAAAATGGCTGTTCAGTTATG

GCATGGTGGTTTCAGCCCACAGATGTTCTTTGATAAAAC

AAATACATTAGAAACACCAGATACTATAACAGTTGAAC

GTATTCATGAAATAGTTAAAGAGTTTGGAGAAGGTGCA

AGAAGAGCTGTTGAAGCTGGATTCGATGCAGTTGAATT

CCATGCAGCACACAGTTACTTACCTCACGAATTCCTAAG

TCCAGGAATGAACAAAAGAACTGACGAATATGGTGGA

AACTTCGAAAATCGTTGCAGATTCTGCTTCGAAGTAGTT

GAAGCTATACGTGCAAATATACCAGAAGATATGCCATT

CTTCATGAGAGTTGACTGCATAGATGAGTTAATGGATG

AAGTAATGACAGAAGAAGAAATAGTAGAATTCATAAAT

AGATGTGCTGATCTAGGAGTAGACGTAGCTGACTTATC

AAGAGGTAATGCTCAGTCATTCGCAACAGTTTACGAAG

TTCCTCCTTTCAACTTACAGCACGGTTTCAATATAGAAA

ACATATACAACATCAAAAAACAGATAAAAATACCAGTA

ATGGGTGTTGGACGTATAAACACAGGAGAAATGGCTAA

CCAGGTAATAGCAGATGGAAAATTTGACTTAGTTGGTA

TAGGTCGTGCTCAGTTAGCAGATCAGGATTGGGTTGCT

AAAGTTAGAGAAGGTAAAGAAGATTTAATACGTCATTG

TATAGGATGTGACCAGGGATGCTACGATGCAGTTATAA

ACCCTCAGATGACTCATATAACTTGTACAAGAAACCCT

CACTTATGCTTAGAATACAAAGGTATGCCAAAAACTGA

TGAACCTAAAAAAGTTATGATAATCGGTGGTGGTATGG CTGGTATATTAGCAGCTGAAGTACTTAAAAAACGTGGA

CATGAACCAGTTATATTCGAAGCTTCTGATCACTTAGCA

GGACAGTTCGTATTAGCAGGTAAAGCTCCAATGAAAGA

AGACTGGGCAGCTGCAGCTAAATGGGAAGCTGAAGAA

GTAGCTCGTTTAGGAATAGAAGTTAGATACAATACAAA

AGTTACTCCAGAATTAATAGAAGAATTCGCTCCAGACC

ACGTTGTTATAGCTATAGGATCTGATTACGTAGCTCCAG

CTATACCAGGTATAGATAGTGACAAAGTTTACACTCAG

TATCAGGTATTAAAAGGTGAAGTAGAACCAAAAGGACA

TGTAGCAGTAGTTGGTTGTGGATTAGTTGGTACAGAAG

TTGCTCAGTACTTAGCAGCTAGAGGAGCTCAGGTAACA

GCTATAGAAAGAAAAGGTGTTGGTACAGGTCTAAGCAT

GCTTAGAAGAATGTTCATGAACCCAGAATTCAAATACT

ACAAAATAAACAAAATGTCTGGAACTAACATAGTTGGT

ATAGAACCAGGAAAACTTCACTACATAATGACTAACAA

GAAAACTCAGGAAGTTACTGAAGGTGTGTTAGAATGTG

ATGCAGCAGTAATCTGTACAGGTATAACTGCTAGACCA

AGTGAAGATTTACAGGAAAAATGTAAAGAATTAGGTGT

TCCATTCAACGTAATAGGTGACGCAGCTGGTGCTAGAG

ATGCTAGAATAGCTACTCAGGAAGGTTACGAAGTAGGT

ATGAGTATATAA

115 Clostridium MSYDALFSPFKIRGLELKNRIVLPGMNTKMAKNKHDLSD hiranonis TO-931 DMIAYHVARAKAGCALNIFECVALCPAPHAYMYMGLYN BaiCD protein DNHVAQLKKLTDAVHEVGGKMAVQLWHGGFSPQMFFD

KTNTLETPDTITVERIHEIVKEFGEGARRAVEAGFDAVEFH

AAHSYLPHEFLSPGMNKRTDEYGGNFENRCRFCFEVVEAI

RANIPEDMPFFMRVDCIDELMDEVMTEEEIVEFINRCADL

GVDVADLSRGNAQSFATVYEVPPFNLQHGFNIENIYNIKK

QIKIPVMGVGRINTGEMANQVIADGKFDLVGIGRAQLADQ

DWVAKVREGKEDLIRHCIGCDQGCYDAVINPQMTHITCT

RNPHLCLEYKGMPKTDEPKKVMIIGGGMAGILAAEVLKK

RGHEPVIFEASDHLAGQFVLAGKAPMKEDWAAAAKWEA

EEVARLGIEVRYNTKVTPELIEEFAPDHVVIAIGSDYVAPAI

PGIDSDKVYTQYQVLKGEVEPKGHVAVVGCGLVGTEVAQ

YLAARGAQVTAIERKGVGTGLSMLRRMFMNPEFKYYKIN

KMSGTNIVGIEPGKLHYIMTNKKTQEVTEGVLECDAAVIC

TGITARPSEDLQEKCKELGVPFNVIGDAAGARDARIATQE

GYEVGMSI

116 Clostridium ATGACTTTAGAAGCAAGAATAGAAGCATTAGAAAAAG hiranonis TO-931 AAATACAGAGATTAAACGATATAGAAGCTATAAAACAG BaiE TTAAAAGCTAAATATTTCCGTTGCCTAGATGGAAAATTA

TGGGATGAATTAGAAACTACTCTTTCTCCTAACATAGAA

ACTTCTTACTCTGATGGAAAATTAGTATTCCACAGCCCA

AAAGAAGTAACTGAATATTTAGCAGCAGCAATGCCTAA

AGAAGAAATAAGTATGCACATGGGACATACTCCAGAAA

TAACTATAGACAGCGAAAATACTGCTACAGGAAGATGG

TACTTAGAAGATAACCTAATATTCACAGACGGAAAATA

CAAAAACGTTGGAATAAACGGTGGAGCATTCTACACAG

ATAAATATGAAAAAATAGACGGACAGTGGTACATAAA

AGAAACTGGATATGTTCGTATATTTGAAGAACATTTCAT GAGAGATCCAAAAATACATATAACTAGCAACATGCATA AAGAAAAATAA

117 Clostridium MTLEARIEALEKEIQRLNDIEAIKQLKAKYFRCLDGKLWD hiranonis TO-931 ELETTLS PNIETS YS D GKLVFHS PKE VTE YL A A AMPKEEIS BaiE protein MHMGHTPEITIDSENTATGRWYLEDNLIFTDGKYKNVGIN

GGAFYTDKYEKIDGQWYIKETGYVRIFEEHFMRDPKIHITS NMHKEK

118 Clostridium ATGAACTTAGTACAGGACAAAATAGTTATAATAACAGG hiranonis TO-931 TGGAACAAGTGGTATAGGTCTTTGCGCAGCAAAAATAT BaiA TCATGGATAACGGTGCAACAGTTTCTATATTCGGAAAA

ACTCAGGAAGAAGTAGATGCTGCTAAAGCAGAATTAAA

AGAAACTCACCCAGATAAAGAAGTATTAGGATTTGCTC

CAGATTTAACTAATAGAGATGAAGTTATGGCTGCAGTT

GGTGCAGTAGCTGAAAAATACGGAAGATTAGACGTTAT

GATAAACAATGCTGGTGTTACTAGCTCAAACGTATTCTC

AAGAGTTAGCCCAGAAGAATTCACATATTTAATGGATA

TAAACGTTACAGGTGTATTCCATGGTGCTTGGGCTGCTT

ACCACTGCCTGAAAGGTGAAAAGAAGATTATAATAAAT

ACTGCTTCAGTAACAGGAATACACGGATCATTATCAGG

AGTTGGATACCCAACAAGTAAATCAGCTGTTGTAGGAT

TCACTCAGGCTCTTGGTAGAGAAATAATACGTAAAAAC

ATAAGAGTTGTTGGTGTTGCACCAGGTGTTGTTAACACT

CCAATGGTTGGTAATATACCAGATGAAATATTAGATGG

ATACCTAAGCTCATTCCCAATGAAGAGAATGTTAGAAC

CAGAAGAAATAGCTAACACTTACTTATTCTTAGCTTCTG

ACTTAGCTAGTGGTATAACAGCTACAACTGTAAGCGTT

GACGGTGCTTATAGACCATCATAA

119 Clostridium MNLVQDKIVIITGGTSGIGLCAAKIFMDNGATVSIFGKTQE hiranonis TO-931 EVDAAKAELKETHPDKEVLGFAPDLTNRDEVMAAVGAV BaiA protein AEKYGRLDVMINNAGVTSSNVFSRVSPEEFTYLMDINVTG

VFHGAWAAYHCLKGEKKIIINTASVTGIHGSLSGVGYPTS

KSAVVGFTQALGREIIRKNIRVVGVAPGVVNTPMVGNIPD

EILD G YLS S FPMKRMLEPEEIANT YLFLAS DLAS GIT ATT VS

VDGAYRPS

120 Clostridium ATGGCTGGATTAAAAGATTTTCCTAAATTTGGTGCACTT hiranonis TO-931 TCTGGATTAAAAATATTAGATAGTGGATCTAACATAGC BaiF TGGACCTCTAGGTGGTGGACTTTTAGCAGAATGTGGTG

CTACAGTTATACACTTCGAAGGACCTAAAAAACCTGAC

AACCAGAGAGGTTGGTATGGATACCCTCAGAACCACAG

AAACCAGTTATCAATGGTTGCTGATATAAAATCTGAAG

AAGGTAGAAAAATATTCTTAGACTTAATAAAATGGGCT

GACATATGGGTTGAATCATCAAAAGGTGGACAGTACGA

CAGACTAAGTCTTTCTGATGAAGTTATATGGTCAGTAAA

CCCTAAAATAGCTATAGTTCACGTTTCTGGATACGGACA

GGTTGGAGATCCATCATACGTAACAAAAGCTTCTTATG

ATGCTGTTGGACAGGCATTCAGTGGATACATGTCATTA

AATGGTGTTAATGAAGCATTAAAAATAAATCCTTACCT

AAGTGACTTCGTATGTGTTCTTACTACTTGCTGGGCAAT

GTTAGCATGCTACGTAAGTACTCAGTTAACTGGAAAAG

GAGAATCTGTAGACGTTGCTCAGTACGAAGCATTAGCT CGTATAATGGACGGACGTATGATACAGTACGCTACTGA

TGGTGTAAGTGTTCCAAAAACTGGTAACAAAGATGCTC

AGGCAGCTCTATTCAGCTTCTATACTTGTAAAGATGGAA

GAACTATATTCATAGGTATGACTGGTGCTGAAGTATGT

AAGAGAGGATTCCCTGTAATAGGGCTTCCAGTTCCTGG

TACAGGTGACCCTGACTTCCCAGAAGGATTCACAGGAT

GGATGATAAATACTCCAGTTGGACAGAGAATGGAAAAA

GCTATGGAAGCATTCGTTGCTGAAAGAACTATGCCAGA

AGTTGAAAAAGCTATGATAGATGCTCAGATACCATGCC

AGAGAGTTTATGATCTTGAAGACTGCTTAAACGACCCT

CACTGGAATGCTCGTGGAACTATAATGGAATGGGATGA

CCCAATGATGGGACACATAAAAGGTCTTGGATTAATAA

ACAAATTCAAAAACAACCCTTCTGAAATATGGAGAGGT

GCTCCATTATTCGGTATGGACAACAGAGACATAATTAG

AGACCTTGGATATTCTGAGGAGGAAGTTAACGATTTAT

ACGCTAAAGGTATTGTAAACGAATTCGACCTTGAAACA

ACTATAAAACGTTACAAACTTGATCAGGTTATACCTCAC

ATGGCTAAAAAAGATAAATAA

121 Clostridium MAGLKDFPKFGALSGLKILDSGSNIAGPLGGGLLAECGAT hiranonis TO-931 VIHFEGPKKPDNQRGWYGYPQNHRNQLSMVADIKSEEGR BaiF protein KIFLDLIKWADIWVESSKGGQYDRLSLSDEVIWSVNPKIAI

VH VS G YGQ VGDPS Y VTKAS YD A VGQ AFS GYMS LNG VNE

ALKINP YLS DF VC VLTTC W AMLAC Y VS TQLTGKGES VD V

AQYEALARIMDGRMIQYATDGVSVPKTGNKDAQAALFSF

YTCKDGRTIFIGMTGAEVCKRGFPVIGLPVPGTGDPDFPEG

FTGWMINTPVGQRMEKAMEAFVAERTMPEVEKAMIDAQI

PCQRVYDLEDCLNDPHWNARGTIMEWDDPMMGHIKGLG

LINKFKNNPSEIWRGAPLFGMDNRDIIRDLGYSEEEVNDLY

AKGIVNEFDLETTIKRYKLDQVIPHMAKKDK

122 Clostridium ATGACAGCAACAAACGCAAACTATAAAAAAGGCTTTAT hiranonis TO-931 CCCATTTGCTATAGCAGCGTTACTAGTAGGTCTTATAGG BaiG TGGTTTCACAGCCGTTCTAGCACCTGCATTCGTAGCAGA

TATGGGTCTTAACGATAACAATACTACATGGATAGCAC

TAGCGCTTGCAATGTCTACAGCTGCATGTGCTCCAATAC

TTGGTAAATTAGGTGACGTACTTGGACGTCGTAAAACTT

TATTATTAGGAATCATAGTATTCACAATAGGTAACGTAT

TAACAGCAATAGCATCTTCATTAATATTCATGCTAGGTG

CAAGATTTATAGTTGGGGTTGGTACAGCGGCTATAGCT

CCAGTTATAATGGCTTACATAGTTACAGAATATCCACCA

GAAGAAACTGGTAAGGGATTCGCTCTTTATATGTTAAT

ATCAAGTGCTGCAGTTGTTGTTGGTCCAACTTGTGGTGG

ATTAATAATGCAGGCATTTGGATGGAGAATGATGATGT

GGGTTTGTGTTGCCCTTTGTGTAGTAACATTCTTCATAT

GTTCAGTAATGATTAAGAAAACAGACTTTGAAAAGAAA

AGTCTTGATAACTTCGATAAAAAAGGTGCAGTATGCGT

ACTAATATTCTTCAGTTTAGTATTATGTATACCATCATTT

GGACAGAATATAGGTTGGACATCAGCGCCATTCCTAGG

TGTTACAGCAGTAGCTTTAGTAACATTATTCTTATTAAT

AAAAGCTGAAAGCAGTGCAGAAAACCCAATATTAAGTG

GTAAATTTATGAAACGTAAAGAATTCATATTACCAGTA TTAATATTATTCCTTACTCAGGGATTAATGCAGGCTAAC

ATGACTAACGTAATATTATTCGTTAGAGCTACTCAGCCA

GAAAATACAATAATATCAAGTTTCGCAATATCAATCCTT

TACATAGGTATGTCTTTAGGTTCAGTATTCATAGGACCT

ATGGCAGATAAAAAAGAACCAAAAACTGTACTTACAGG

ATCACTTCTATTCACTGGTATAGGTTGTGCAATGATGTA

CTTCTTCACAGAAACTGCACCATTCGCAATGTTAGCTGG

ATCTCTAGGAATGTTAGGTATAGGACTTGGAGGAAATG

CTACAATACTAATGAAAGTTTCATTATCTGGATTATCTC

AGGCAGAAGCTGGATCAGGAACAGGAACATACGGATT

ATTCAGAGATATATCAGCTCCATTTGGTGTTGCGGTATT

CGTACCACTATTTGCAAACACAGTTACAACAAGAATGG

CTGGAGTAATGGCTAACGGAACTGCAGAAGCTGCTGCT

AAATCATTAGCATCTGTTTCTTCTATACATACATTAGCA

TTAGTTGAAGTATGCTGTGTAATATTAGCAATAGTTGCA

GTTAGAATGCTACCAAAAATACACAATAAATAA

123 Clostridium MTATNANYKKGFIPFAIAALLVGLIGGFTAVLAPAFVADM hiranonis TO-931 GLNDNNTTWIALALAMSTAACAPILGKLGDVLGRRKTLL BaiG protein LGIIVFTIGN VLT AIAS S LIFMLG ARFIVG VGT A AIAP VIM A Y rVTEYPPEETGKGFALYMLISSAAVVVGPTCGGLIMQAFG WRMMMWVCVALCVVTFFICSVMIKKTDFEKKSLDNFDK KGAVCVLIFFSLVLCIPSFGQNIGWTSAPFLGVTAVALVTL FLLIKAESSAENPILSGKFMKRKEFILPVLILFLTQGLMQAN MTN VILF VR ATQPENTIIS S FAIS ILYIGMS LGS VFIGPM ADK KEPKTVLTGSLLFTGIGCAMMYFFTETAPFAMLAGSLGML GIGLGGN ATILMKVS LS GLS Q AE AGS GTGT YGLFRDIS APF GV A VFVPLFANT VTTRM AG VM ANGT AE A A AKS LAS VS S I HTLALVEVCCVILAIVAVRMLPKIHNK

124 Clostridium ATGGATATGAAAAATTCTAAACTATTCTCACCTTTAACA hiranonis TO-931 ATAGGATCATTAACATTAAACAACAGAGTTGGTATGGC BaiH ACCAATGAGTATGGACTACGAAGCTGCTGACGGAACAG

TTCCAAAAAGATTAGCAGATATATTTGTTCGTAGAGCTG

AAGGTGGAACAGGATATGTAACAATAGACGCGGTAAC

AATAGATAGTAAATATAAATATATGGGTAATACAACTG

CTTTAGATTCTGATGATTTAGTTACTCAGTTCAAAGAAT

TTGCAACAAGAGTTAGAGAAGCAGGAAGCACATTAATA

CCTCAGGTTATACATCCAGGACCAGAATCAATATGTGG

ATACAGACACATAGCACCACTTGGACCATCAGTTAATA

CAAATGCTAACTGCCACGTGAGCCGTGCTATAAGTGTA

GATGAAATACATGAAATAATAAAACAGTTTGGACAGGC

TGCTAGAAGAGTTGAAGAAGCAGGATGCGGTGGTATAG

GATTACACTGTGCACATGCTTACATGCTACCAGGTTCAT

TCTTATCTCCATTAAGAAACAAAAGAATGGATGAATAC

GGCGGATGTCTAGATAACAGAGCAAGATTCGTAATAGA

AATGATAGAAGAAGTTCGTAGAAATGTAAGTCCTGATT

TCCCAATAATGCTTAGAATATCTGGGGATGAAAGAATG

ATAGGAGGAAACTCTTTAGAAGATATGTTATACTTAGC

TCCAAAATTTGTTGAAGCTGGTGTAAATATGTTTGAAGT

TTCTGGAGGTACTCAGTACGAAGGATTAGAACACATAA

TACCAAGTCAGAACAAAAGCATAGGTGTAAACGTACAC GAAGCATCTGAAATCAAAAAAGTTGTAGATGTTCCAGT

TTACGCTGTTGGTAAAATAAATGACATAAGATACGCTG

CTGAAATAGTTGAAAGAGGACTAGTTGATGGGGTATCA

ATAGGTAGACCATTATTAGCAGATCCAGACTTATGTAA

TAAAGCAAAAGAAAACTTATTTGATGAAATAACTCCAT

GTGCAAGCTGTGGAGGAAGCTGTATAAGCCGTACTGCA

GATAGACCTCAGTGTCGTTGCCATATAAACCCAAGAGT

TGGATTCGAATATGATTATCCAGAAGTTCCAGCTGAAA

AATCTAAAAAAGTTCTAGTTGTAGGTGCTGGACCTGGT

GGTATGATGGCAGCAGTTACAGCAGCTGAAAGAGGACA

TGATGTAACACTTTGGGAAGCTGACACTCAGATAGGTG

GACAGATAAACTTAGCAGTAGTAGCTCCAGGTAAACAG

GAAATGACTAAATGGTTATCTCACTTAAACTACAGAGC

TAAAAAAGCTGGAGTTAAAATGGTATTAGGAAAAGAA

GCTACAGTAGAAAACATAAAAGAATTTGCTCCAGAAGC

AGTTATAGTTGCAACAGGTGCTAGACCATTAGTTCCACC

AATAAAAGGAACTCAGGACTACCCAGTTCTTACAGCTC

ATGACTTCTTAAGAGGAAAATTCGTTATACCAAAAGGA

AAAGTTTGTGTACTAGGTGGAGGAGCTGTTGCTTGTGA

AACTGCAGAAACAGTATTAGAAAACGCTAGACCAAACG

CATTCACTAGAGGATTTGATGCTAGTATCGGTGATGTAG

ATGTTACATTAGTAGAAATGTTACCACAGTTATTAACAG

GAGTATGTGCTCCAAATAGAACTCCATTAATAAGAAAA

CTTAAAAACAAAGGTGTTCATATAAATGTAAATACTAA

AATATTAGAAGTAACTGACCACGACGTTAAAGTTCAGA

GAGCTGACGGTGCAGAAGAATGGTTAAAAGGATTCGAC

TACATACTATTCGGACTTGGTTCTAGAAACTACGATCCA

ATATCTGAACAGATAAAAGAATTCGTTCCAGAAGTACA

CGTTGTTGGGGATGCTAAGAGAGCTAGACAGGCAAGCT

TTGCAATGTGGGAAGCTTTCGAAGCAGCATACAGCTTA

TAA

125 Clostridium MDMKNSKLFSPLTIGSLTLNNRVGMAPMSMDYEAADGT hiranonis TO-931 VPKRLADIFVRRAEGGTGYVTIDAVTIDSKYKYMGNTTAL BaiH protein DSDDLVTQFKEFATRVREAGSTLIPQVIHPGPESICGYRHIA

PLGPS VNTN ANCH VS RAIS VDEIHEIIKQFGQ A ARRVEE AG

CGGIGLHCAHAYMLPGSFLSPLRNKRMDEYGGCLDNRAR

FVIEMIEE VRRN VS PDFPIMLRIS GDERMIGGNS LEDMLYL

APKFVEAGVNMFEVSGGTQYEGLEHIIPSQNKSIGVNVHE

ASEIKKVVDVPVYAVGKINDIRYAAEIVERGLVDGVSIGRP

LLADPDLCNKAKENLFDEITPCASCGGSCISRTADRPQCRC

HINPRVGFEYDYPEVPAEKSKKVLVVGAGPGGMMAAVT

AAERGHDVTLWEADTQIGGQINLAVVAPGKQEMTKWLS

HLNYRAKKAGVKMVLGKEATVENIKEFAPEAVIVATGAR

PLVPPIKGTQDYPVLTAHDFLRGKFVIPKGKVCVLGGGAV

ACETAETVLENARPNAFTRGFDASIGDVDVTLVEMLPQLL

TGVCAPNRTPLIRKLKNKGVHINVNTKILEVTDHDVKVQR

ADGAEEWLKGFDYILFGLGSRNYDPISEQIKEFVPEVHVV

GD AKR ARQ AS F AMWE AFE A A YS L

Claims

1. A recombinant bacterial cell, wherein the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter that is not associated with the bile salt hydrolase gene in nature.

2. The recombinant bacterial cell of claim 1, wherein the first promoter is a directly or indirectly inducible promoter.

3. The recombinant bacterial cell of claim 1 or claim 2, wherein the first promoter is directly or indirectly induced by environmental conditions.

4. The recombinant bacterial cell of any of claims 1-3, wherein the first promoter is directly or indirectly induced by environmental conditions specific to the gut of a mammal.

5. The recombinant bacterial cell of any of claims 1-4, wherein the first promoter is directly or indirectly induced by low oxygen or anaerobic conditions.

6. The recombinant bacterial cell of any of claims 1-5, wherein the first promoter is an FNR responsive promoter.

7. The recombinant bacterial cell of claim 1, wherein the first promoter is a constituve promoter.

8. The recombinant bacterial cell of any of claims 1-7 further comprising a heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s).

9. The recombinant bacterial cell of claim 8 wherein the heterologous gene sequence encoding one or more 7a-dehydroxylating enzyme(s) comprises baiB, baiCD, baiE, baiAl, baiA2, baiA3, baiF, baiG, baiH, and/or bail genes.

10. The recombinant bacterial cell of any of claims 1-9 wherein the heterologous gene encoding the one or more 7a-dehydroxylating enzyme(s) is operably linked to a second promoter that is not associated with the 7a-dehydroxylating enzyme gene(s) in nature.

11. The recombinant bacterial cell of any of claims 1-10, wherein the second promoter is a directly or indirectly inducible promoter.

12. The recombinant bacterial cell of any of claims 1-11, wherein the second promoter is directly or indirectly induced by environmental conditions.

13. The recombinant bacterial cell of any of claims 1-12, wherein the second promoter is directly or indirectly induced by environmental conditions specific to the gut of a mammal.

14. The recombinant bacterial cell of any of claims 1-13, wherein the second promoter is directly or indirectly induced by low oxygen or anaerobic conditions.

15. The recombinant bacterial cell of any of claims 1-14, wherein the second promoter is an FNR responsive promoter.

16. The recombinant bacterial cell of any of claims 1-10, wherein the second promoter is a constituve promoter.

17. The recombinant bacterial cell of any of claims 1-16 further comprising a heterologous gene encoding one or more bile salt and/or bile acid transporter(s).

18. The recombinant bacterial cell of any of claims 1-17 wherein the heterologous gene encoding the one or more bile salt and/or bile acid transporter(s) is operably linked to a third promoter that is not associated with the bile salt and/or bile acid transporter gene in nature.

19. The recombinant bacterial cell of any of claims 1-18, wherein the third promoter is a directly or indirectly inducible promoter.

20. The recombinant bacterial cell of any of claims 1-19, wherein the third promoter is directly or indirectly induced by environmental conditions.

21. The recombinant bacterial cell of any of claims 1-20, wherein the third promoter is directly or indirectly induced by environmental conditions specific to the gut of a mammal.

22. The recombinant bacterial cell of any of claims 1-21, wherein the third promoter is directly or indirectly induced by low oxygen or anaerobic conditions.

23. The recombinant bacterial cell of any of claims 1-22, wherein the third promoter is an FNR responsive promoter.

24. The recombinant bacterial cell of any of claims 1-18, wherein the third promoter is a constituve promoter.

25. The recombinant bacterial cell of any of claims 1-24 further comprising a

heterologous gene encoding one or more bile acid exporter(s).

26. The recombinant bacterial cell of any of claims 1-25 wherein the heterologous gene encoding the one or more bile acid exporter(s) is operably linked to a fourth promoter that is not associated with the bile acid exporter gene in nature.

27. The recombinant bacterial cell of any of claims 1-26, wherein the fourth promoter is a directly or indirectly inducible promoter.

28. The recombinant bacterial cell of any of claims 1-27, wherein the fourth promoter is directly or indirectly induced by environmental conditions.

29. The recombinant bacterial cell of any of claims 1-28, wherein the fourth promoter is directly or indirectly induced by environmental conditions specific to the gut of a mammal.

30. The recombinant bacterial cell of any of claims 1-29, wherein the fourth promoter is directly or indirectly induced by low oxygen or anaerobic conditions.

31. The recombinant bacterial cell of any of claims 1-30, wherein the fourth promoter is an FNR responsive promoter.

32. The recombinant bacterial cell of any of claims 1-26, wherein the fourth promoter is a constituve promoter.

33. The recombinant bacterial cell of any one of claims 1-32, wherein the heterologous gene encoding the bile salt hydrolase enzyme is from Lactobacillus.

34. The recombinant bacterial cell of any one of claims 1-33, wherein the heterologous gene encoding the bile salt hydrolase enzyme is from Methanobrevibacter smithii.

14.

35. The recombinant bacterial cell of any one of claims 1-34 wherein the heterologous gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell.

36. The recombinant bacterial cell of any one of claims 1-35, wherein the heterologous gene encoding the bile salt hydrolase enzyme is located on a chromosome in the bacterial cell.

37. The recombinant bacterial cell of any one of claims 1-36 wherein the heterologous gene encoding the 7a-dehydroxylating enzyme is located on a plasmid in the bacterial cell.

38. The recombinant bacterial cell of any one of claims 1-37, wherein the heterologous gene encoding the 7a-dehydroxylating enzyme is located on a chromosome in the bacterial cell.

39. The recombinant bacterial cell of any one of claims 1-38 wherein the heterologous gene encoding the bile salt and/or bile acid transporter is located on a plasmid in the bacterial cell.

40. The recombinant bacterial cell of any one of claims 1-39, wherein the heterologous gene encoding the bile salt and/or bile acid transporter is located on a chromosome in the bacterial cell.

41. The recombinant bacterial cell of any one of claims 1-40 wherein the heterologous gene encoding the bile acid exporter is located on a plasmid in the bacterial cell.

42. The recombinant bacterial cell of any one of claims 1-41, wherein the heterologous gene encoding the bile acid exporter is located on a chromosome in the bacterial cell.

43. The recombinant bacterial cell of any one of the previous claims, wherein the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter.

44. The recombinant bacterial cell of any one of the preceding claims, wherein the first inducible promoter and the second inducible promoter are different promoters.

45. The recombinant bacterial cell of any one of claims 1-44, wherein the recombinant bacterial cell is a recombinant probiotic bacterial cell.

46. The recombinant bacterial cell of any one of claims 1-45, wherein the recombinant bacterial cell is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus and Lactococcus.

47. The recombinant bacterial cell of claim 46, wherein the recombinant bacterial cell is of the genus Escherichia.

48. The recombinant bacterial cell of claim 47, wherein the recombinant bacterial cell is of the species Escherichia coli strain Nissle.

49. The recombinant bacterial cell of any one of claims 1-48, wherein the recombinant bacterial cell is an auxotroph in a gene that is complemented when the recombinant bacterial cell is present in a mammalian gut.

50. The recombinant bacterial cell of claim 49, wherein the mammalian gut is a human gut.

51. The recombinant bacterial cell of claim 49 or claim 50, wherein the recombinant bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine

biosynthetic pathway.

52. A pharmaceutical composition comprising the recombinant bacterial cell of any of claims 1-51 and a pharmaceutically acceptable carrier.

53. A method for treating a disease associated with bile salts in a subject, the method comprising administering the pharmaceutical composition of claim 52 to the subject.

54. A method for decreasing a level of bile salts in the gut of a subject, the method comprising administering the pharmaceutical composition of claim 52 to the subject, thereby decreasing the level of bile salts in the gut of the subject.

55. A method for decreasing a level of conjugated bile acids in a subject, the method comprising administering the pharmaceutical composition of claim 52 to the subject, thereby decreasing the level of conjugated bile acids in the subject.

56. A method for increasing a level of unconjugated bile acids in a subject, the method comprising administering the pharmaceutical composition of claim 52 to the subject, thereby increasing the level of unconjugated bile acids in the subject.

57. A method for increasing the rate of deconjugation of bile salts in a subject, the method comprising administering the pharmaceutical composition of claqim 52 to the subject, thereby increasing the rate of deconjugation of bile salts in the subject.

58. The method of claim53, wherein the disorder associated with bile salts is a metabolic disease or a liver disease.

59. The method of claim 53, wherein the disorder associated with bile salts is a cardiovascular disease.

60. The method of claim 59, wherein the cardiovascular disease is hypercholesterolemia.

61. The method of claim 53, wherein the disorder associated with bile salts is diabetes or obesity.