WO2023245168A1

WO2023245168A1 - Bacteria engineered to treat diseases associated with bile acid metabolism and methods of use thereof

Info

Publication number: WO2023245168A1
Application number: PCT/US2023/068583
Authority: WO
Inventors: Jillian Marie MEANS; Analise Zaunbrecher REEVES; Chengyi Jenny Shu; Douglas KENNY; Afrand KAMALI SARVESTANI; Michael James; Abhi SHAH
Original assignee: Synlogic Operating Company, Inc.
Priority date: 2022-06-17
Filing date: 2023-06-16
Publication date: 2023-12-21

Abstract

The present disclosure provides recombinant bacterial cells that have been engineered with genetic circuitry which allow the recombinant bacterial cells to sense a patient's internal environment and respond by turning an engineered metabolic pathway on or off. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject. These recombinant bacterial cells are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome. Specifically, the present disclosure provides recombinant bacterial cells that comprise a bile acid catabolism enzyme for the treatment of diseases and disorders associated with bile acid metabolism, such as inflammatory bowel disease. The disclosure further provides pharmaceutical compositions and methods of treating disorders associated with bile acid metabolism, such as inflammatory bowel disease.

Description

BACTERIA ENGINEERED TO TREAT DISEASES ASSOCIATED WITH BILE ACID METABOLISM AND METHODS OF USE THEREOF

Related Applications

[01] This application claims priority to U.S. Provisional Application No. 63/353,174, filed on June 17, 2022, and U.S. Provisional Application No. 63/402,134, filed on August 30, 2022. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties.

Background

[02] Inflammatory bowel diseases (IBDs) are a group of diseases characterized by significant local inflammation in the gastrointestinal tract typically driven by T cells and activated macrophages and by compromised function of the epithelial barrier that separates the luminal contents of the gut from the host circulatory system (Ghishan et al. , 2014). IBD pathogenesis is linked to both genetic and environmental factors and may be caused by altered interactions between gut microbes and the intestinal immune system.

[03] Current approaches to treat IBD are focused on therapeutics that modulate the immune system and suppress inflammation. These therapies include steroids, such as prednisone, and tumor necrosis factor (TNF) inhibitors, such as Humira® (Cohen et al. , 2014). Drawbacks from this approach are associated with systemic immunosuppression, which includes greater susceptibility to infectious disease and cancer. Thus, there remains a great need for additional therapies to reduce gut inflammation, enhance gut barrier function, and/or treat autoimmune disorders, and that avoid undesirable side effects.

Summary

[04] The present disclosure provides recombinant bacteria for catabolism of bile acids, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with elevated levels of bile acids, e.g., secondary bile acids, e.g., IBD and metabolic disorders, and primary bile acids. The recombinant bacteria are capable of catabolizing in low-oxygen environments, e.g., the gut of a subject. Thus, the recombinant bacteria and pharmaceutical compositions comprising those bacteria are non-pathogenic, and can be used in order to treat and/or prevent conditions associated with autoimmune and inflammatory diseases and disorders, such as IBD (e.g., ulcerative colitis and Crohn’s disease), metabolic disorders (e.g., liver disease and type II diabetes).

[05] In some embodiments, the bile acid catabolism enzyme is a catabolizes a secondary bile acid, e.g., lithocholic acid (LCA), or a primary bile acid, e.g., cholic acid (CA). In some embodiments, the bile acid catabolism enzyme is a sulfotransferase, e.g., a human sulfotransferase. In some embodiments, the sulfotransferase is a SULT1 or SULT2 sulfotransferase. In some embodiments, the sulfotransferase is SULT2A1, e.g., human SULT2A1, or SULT2A8, e.g., mouse SULT2A8.

[06] In one aspect, the disclosure provides for a recombinant bacterium comprising a gene encoding a bile acid catabolism enzyme, wherein the gene encoding the bile acid catabolism enzyme is operably linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature. In some embodiments, the promoter is constitutive or is a directly or indirectly inducible promoter, which optionally is induced by exogenous environmental conditions.

[07] In some embodiments, the inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions. In some embodiments, the inducible promoter is an FNR- inducible promoter. In some embodiments, the inducible promoter is induced by temperature. In some embodiments, the inducible promoter is a cI857 promoter.

[08] In some embodiments, the gene encoding the bile acid catabolism enzyme is present on a plasmid in the bacterium. In some embodiments, the gene encoding the bile acid catabolism enzyme is present on a chromosome in the bacterium.

[09] In some embodiments, the bacterium is a non-pathogenic bacterium. In some embodiments, the bacterium is a probiotic or a commensal bacterium. In some embodiments, the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus . In some embodiments, the bacterium is Escherichia coli strain Nissle.

[010] In some embodiments, the sulfotransferase gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 500. In some embodiments, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 501.

[Oi l] In some embodiments, the sulfotransferase gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 516. In some embodiments, the gene sequence encoding the sulfotransferase encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 517.

[012] In some embodiments, the bacterium further comprises a heterologous gene encoding a bile acid importer or transporter. Nonlimiting examples of importers or transporters include homologs of human ASBT, baiG or PanS. In some embodiments, the homolog of human ASBT is from Neisseria meningitidis (ASBTNM), from Yersinia frederiksenii (ASBTYf), from E. coli M34 or from E. coli VREC0334. In some embodiments, the bile acid importer or transporter is baiG from Eubacterium sp. strain VPI 12708. In some embodiments, the bile acid importer or transporter is PanS is from Escherichia coli EC23. [013] In some embodiments, the bile acid transporter gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 502, 504, 506, 508, 510, or 512. In some embodiments, the gene sequence encoding a bile acid transporter encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 503, 505, 507, 509, 511 or 513.

[014] In some embodiments, the bile acid transporter gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 510, In some embodiments, the gene sequence encoding a bile acid transporter encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 511.

[015] In some embodiments, the bile acid transporter gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 512. In some embodiments, the gene sequence encoding a bile acid transporter encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 513.

[016] In some embodiments, the bacterium further comprises a heterologous gene encoding a sulfate importer or transporter. In some embodiments, the sulfate transporter is CysZ. In some embodiments, CysZ is from E. coli. In some embodiments, CysZ has a sequence comprising MVSSFTSAPRSGFYYFAQGWKLVSQPGIRRFVILPLLVNILLMGGAFWWLFTQLDVWIPTLM SYVPDWLQWLSYLLWPLAVISVLLVFGYFFSTIANWIAAPFNGLLAEQLEARLTGATPPDTGI FGIMKDVPRIMKREWQKFAWYLPRAIVLLILYFIPGIGQTVAPVLWFLFSAWMLAIQYCDYPF DNHKVPFKEMRTALRTRKITNMQFGALTSLFTMIPLLNLFIMPVAVCGATAMWVDCYRDKH AMWR (SEQ ID NO: 524).

[017] In some embodiments, the bacterium further comprises a genetic modification that reduces the reduction of 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS) in the cell from the bacterial cell. In some embodiments, the genetic modification is a knock-out of an endogenous PAPS reductase. In some embodiments, the endogenous PAPS reductase is encoded by a cysH gene.

[018] In some embodiments, the bacterium further comprises an insertion, deletion or mutation of an endogenous phage gene. In some embodiments, the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.

[019] In some embodiments, the bacterium further comprises a modified endogenous colibactin island. In some embodiments, the modified endogenous colibactin island comprises one or more modified clb sequences selected from the group consisting of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), clbJ (SEQ ID NO: 303), c/W (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), clbR (SEQ ID NO: 311), and clbS (SEQ ID NO: 312).

[020] In some embodiments, the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), c»J(SEQ ID NO: 303), c»X (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), c/6<2 (SEQ ID NO: 310), and clbR (SEQ ID NO: 311).

[021] In some embodiments, the bacterium is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut. In some embodiments, the auxotrophy is in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

[022] In another aspect, the present disclosure provides for a pharmaceutically acceptable composition comprising the recombinant bacterium as provided herein; and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for oral administration.

[023] In another aspect, the present disclosure provides for a method of treating a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition as provided herein, thereby treating the disease or disorder. In some embodiments, the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.

[024] In some embodiments, the disease or disorder is a metabolic disease selected from the group consisting of liver disease; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); liver cirrhosis; obesity; type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Boijeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.

[025] In some embodiments, the disease or disorder selected an autoimmune disease selected from the group consisting of multiple sclerosis, central nervous system inflammation (CNS) inflammation, 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced colitis, T cell-induced colitis, T cell-induced small bowel inflammation, chronic colitis, rheumatoid arthritis, celiac disease, myasthenia gravis, and B-cell-mediated T-cell-dependent autoimmune disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet’s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage -Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s. In some embodiments, the disease or disorder is ulcerative colitis or Crohn’s disease.

[026] In another aspect, the present disclosure provides a method of treating, reducing, or ameliorating symptoms of a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition as provided herein, wherein the symptom of the disease or disorder is inflammation.

[027] In some embodiments, the subject is a human.

[028] In another aspect, the disclosure provides for a recombinant bacterium comprising: (i) a heterologous gene encoding a bile acid catabolism enzyme, wherein the bile acid catabolism enzyme is a human SULT2A1 sulfotransferase or a mouse SULT2A8 sulfotransferase, and wherein the gene encoding the bile acid catabolism enzyme is operably linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature; (ii) a heterologous gene encoding a bile acid transporter, wherein the bile acid transporter is an ASBT transporter from Yersinia frederiksenii, and wherein the gene encoding the bile acid transporter is operably linked to a promoter that is not associated with the gene encoding the bile acid transporter in nature; and (iii) a knock-out of an endogenous cysH gene. In one embodiment, the bile acid catabolism enzyme is a human SULT2A1 sulfotransferase. In another embodiment, the bile acid catabolism enzyme is a mouse SULT2A8 sulfotransferase.

[029] In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA) and/or cholic acid (CA). In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA).

[030] In some embodiments, the bile acid catabolism enzyme catabolizes cholic acid (CA). In some embodiments, the bile acid catabolism enzyme catabolizes ursodeoxycholic acid (UDCA). In some embodiments, the bile acid catabolism enzyme catabolizes deoxycholic acid (DCA). In some embodiments, the bile acid catabolism enzyme catabolizes chenodeoxycholic acid (CDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glyco-lithocholic acid (GLCA). In some embodiments, the bile acid catabolism enzyme catabolizes tauro-lithocholic acid (TLCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycoursodeoxycholic acid (GUDCA). In some embodiments, the bile acid catabolism enzyme catabolizes tauroursodeoxycholic acid (TUDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycochenodeoxy cholic acid (GCDCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurochenodeoxycholic acid (TCDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycodeoxycholic acid (GDCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurodeoxycholic acid (TDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycocholic acid (GCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurocholic acid (TCA). In some embodiments, the bile acid catabolism enzyme catabolizes dehydroepiandrosterone (DHEA).

[031] In some embodiments, the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are operably linked to different promoters, wherein the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are operably linked to different copies of the same promoter, or wherein the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are present in a gene cassette linked to the same promoter.

[032] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is an inducible promoter or a constitutive promoter; and/or wherein the promoter operably linked to the bile acid transporter is an inducible promoter or a constitutive promoter.

[033] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is induced by a chemical inducer; and/or wherein the promoter operably linked to the bile acid transporter is induced by a chemical inducer.

[034] In some embodiments, the chemical inducer is isopropylthio-beta-galactoside (IPTG).

[035] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is induced by exogenous environmental conditions; and/or wherein the promoter operably linked to the bile acid transporter is induced by exogenous environmental conditions.

[036] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is induced by low-oxygen or anaerobic conditions; and/or wherein the promoter operably linked to the bile acid transporter is induced by low-oxygen or anaerobic conditions.

[037] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is an FNR-inducible promoter; and/or wherein the promoter operably linked to the bile acid transporter is an FNR-inducible promoter.

[038] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is induced by temperature; and/or wherein the promoter operably linked to the bile acid transporter is induced by temperature.

[039] In some embodiments, the promoter operably linked to the bile acid catabolism enzyme is a cI857 promoter; and/or wherein the promoter operably linked to the bile acid transporter is a cI857 promoter.

[040] In some embodiments, the human SULT2A1 sulfotransferase gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 500. In some embodiments, the gene sequence encoding the human SULT2A1 sulfotransferase encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 501. [041] In some embodiments, the bile acid transporter gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 504.

[042] In some embodiments, the gene sequence encoding the bile acid transporter encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 505.

[043] In some embodiments, the gene encoding the bile acid catabolism enzyme is present on a plasmid in the recombinant bacterium. In some embodiments, the gene encoding the bile acid catabolism enzyme is present on a chromosome in the recombinant bacterium.

[044] In some embodiments, the recombinant bacterium is a non-pathogenic bacterium. In some embodiments, the recombinant bacterium is a probiotic or a commensal bacterium. In some embodiments, the recombinant bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus . In some embodiments, the recombinant bacterium is Escherichia coli strain Nissle.

[045] In some embodiments, the recombinant bacterium further comprising an insertion, deletion or mutation of an endogenous phage gene. In some embodiments, the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.

[046] In some embodiments, the recombinant bacterium further comprising a modified endogenous colibactin island. In some embodiments, the modified endogenous colibactin island comprises one or more modified clb sequences selected from the group consisting of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbE (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), c//?./ (SEQ ID NO: 303), c/W (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), c/WV(SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), clbR (SEQ ID NO: 311), and clbS (SEQ ID NO: 312). In some embodiments, the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbE (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), c//i./ (SEQ ID NO: 303), c/Zi " (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), and clbR (SEQ ID NO: 311).

[047] In some embodiments, the recombinant bacterium is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut. In some embodiments, the auxotrophy is a in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway. [048] In some embodiments, the bacterium has at least about 65% viability, at least about 70% viability, at least about 75% viability, at least about 80% viability, at least about 85% viability, at least about 90% viability, or at least about 95% viability.

[049] In some embodiments, the bacterium is capable of sulfonating chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), tauro-litocholic acid (TLCA), glycol-litocholic acid (GLCA), and/or lithocholic acid (LCA).

[050] In some embodiments, the bacterium is capable of sulfonating lithocholic acid (LCA). In some embodiments, the bacterium is capable of sulfonating cholic acid (CA). In some embodiments, the bacterium is capable of sulfonating ursodeoxycholic acid (UDCA). In some embodiments, the bacterium is capable of sulfonating deoxy cholic acid (DCA). In some embodiments, the bacterium is capable of sulfonating chenodeoxycholic acid (CDCA). In some embodiments, the bacterium is capable of sulfonating glyco -lithocholic acid (GLCA). In some embodiments, the bacterium is capable of sulfonating tauro-lithocholic acid (TLCA). In some embodiments, the bacterium is capable of sulfonating glycoursodeoxy cholic Acid (GUDCA). In some embodiments, the bacterium is capable of sulfonating tauroursodeoxy cholic Acid (TUDCA). In some embodiments, the bacterium is capable of sulfonating glycochenodeoxycholic acid (GCDCA). In some embodiments, the bacterium is capable of sulfonating taurochenodeoxycholic acid (TCDCA). In some embodiments, the bacterium is capable of sulfonating glycodeoxycholic acid (GDCA). In some embodiments, the bacterium is capable of sulfonating taurodeoxycholic acid (TDCA). In some embodiments, the bacterium is capable of sulfonating glycocholic acid (GCA). In some embodiments, the bacterium is capable of sulfonating taurocholic acid (TCA). In some embodiments, the bacterium is capable of sulfonating dehydroepiandrosterone (DHEA).

[051] In some embodiments, the bacterium is capable of sulfonating lithocholic acid (LCA) into lithocholic acid-3 -sulfate or LCA3 Sulfate (LCA3S). In some embodiments, the bacterium is capable of sulfonating cholic acid (CA) into CA-3 -sulfate (CA3S) or CA-7 -sulfate (CA7S). In some embodiments, the bacterium is capable of sulfonating ursodeoxycholic acid (UDCA) into UDCA -3- sulfate (UDCA3S) or UDCA -7 -sulfate (UDCA7S). In some embodiments, the bacterium is capable of sulfonating deoxy cholic acid (DCA) into DCA -3 -sulfate (DCA3S). In some embodiments, the bacterium is capable of sulfonating chenodeoxycholic acid (CDCA) into CDCA-3 -sulfate (CDCA3S) or CDCA-7-sulfate (CDCA7S). In some embodiments, the bacterium is capable of sulfonating glyco- lithocholic acid (GLCA) into GLCA -3 -sulfate (GLCA3S). In some embodiments, the bacterium is capable of sulfonating tauro-lithocholic acid (TLCA) into TLCA-3 -sulfate (TLCA3S). In some embodiments, the bacterium is capable of sulfonating glycoursodeoxycholic Acid (GUDCA) into GUDCA -3 -Sulfate (GUDCA3S) or GCDCA-7-sulfate (CDCA7S). In some embodiments, the bacterium is capable of sulfonating tauroursodeoxycholic Acid (TUDCA) into TUDCA-3 -sulfate (TUDCA3S) or TCDCA-7-sulfate (TCDCA7S). In some embodiments, the bacterium is capable of sulfonating glycochenodeoxycholic acid (GCDCA) into GCDCA-3 -sulfate (GCDCA3S). In some embodiments, the bacterium is capable of sulfonating taurochenodeoxycholic acid (TCDCA) into TCDCA-3 -sulfate (TCDCA3S). In some embodiments, the bacterium is capable of sulfonating glycodeoxy cholic acid (GDCA) into GDCA -3 -sulfate (GDCA3S). In some embodiments, the bacterium is capable of sulfonating taurodeoxycholic acid (TDCA) into TDCA-3 -sulfate (TDCA3S). In some embodiments, the bacterium is capable of sulfonating glycocholic acid (GCA) into GCA-3 - sulfate (GCA3S) or GCA-7-sulfate (GCA7S). In some embodiments, the bacterium is capable of sulfonating taurocholic acid (TCA) into TCA-3-sulfate (TCA3S) or TCA-7-sulfate (TCA7S). In some embodiments, the bacterium is capable of sulfonating dehydroepiandrosterone (DHEA) into dehydroepiandrosterone sulfate (DHEAS).

[052] In some embodiments, the bacterium is further capable of sulfonating cholic acid (CA).

[053] In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of at least about 0.1 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.5 nmol/h/le9 cells, at least about 0.6 nmol/h/le9 cells, at least about 0.7 nmol/h/le9 cells, at least about 0.8 nmol/h/le9 cells, at least about 0.9 nmol/h/le9 cells, at least about 1.0 nmol/h/le9 cells in vitro, at least about 1.1 nmol/h/le9 cells at least about 1.2 nmol/h/le9 cells, at least about 1.3 nmol/h/le9 cells, at least about 1.4 nmol/h/le9 cells, or at least about 1.5 nmol/h/le9 cells in vitro. In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of at least about 0.5 nmol/h/le9 cells in vitro.

[054] In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of about 0.1 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.3 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.3 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, or about 1.4 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells in vitro.

[055] In some embodiments, the bacterium has an LCA sulfonation rate of about 0.5 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells.

[056] In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.001 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, or about 0.001 nmol/hr/le9 cells to about 0.002 nmol/hr/le9 cells.

[057] In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells.

[058] In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells, or about 0.004 nmol/hr/le9 cells.

[059] In some embodiments, the bacterium has a CA sulfonation rate of about 0.003 nmol/hr/le9 cells.

[060] In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.002 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, or about 0.006 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells.

[061] In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.004 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells.

[062] In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.002 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells, about 0.006 nmol/hr/le9 cells, or about 0.007 nmol/hr/le9 cells. [063] In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.005 nmol/hr/le9 cells.

[064] In any embodiment disclosed herein, the sulfonation rate may be determined using a bacterium grown in an AMBR bioreactor. In any embodiment disclosed herein, the sulfonation rate may be determined using a bacterium grown in a flask.

[065] In another aspect, the disclosure provides for a pharmaceutically acceptable composition comprising the recombinant bacterium as provided herein, and a pharmaceutically acceptable carrier.

[066] In some embodiments, the composition is formulated for oral administration.

[067] In another aspect, the disclosure provides for a method for decreasing a level of a bile acid in the gut of a subject, the method comprising a step of administering to the subject the pharmaceutical composition as provided herein, thereby decreasing the level of the bile acid in the gut of the subject.

[068] In another aspect, the disclosure provides for a method of treating a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition as provided herein, thereby treating the disease or disorder.

[069] In some embodiments, the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.

[070] In some embodiments, the disease or disorder is a metabolic disease selected from the group consisting of liver disease; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); liver cirrhosis; obesity; type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Boijeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.

[071] In some embodiments, the disease or disorder selected an autoimmune disease selected from the group consisting of multiple sclerosis, central nervous system inflammation (CNS) inflammation, 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced colitis, T cell-induced colitis, T cell-induced small bowel inflammation, chronic colitis, rheumatoid arthritis, celiac disease, myasthenia gravis, and B-cell-mediated T-cell-dependent autoimmune disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet’s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage -Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s disease.

[072] In some embodiments, the disease or disorder is ulcerative colitis or Crohn’s disease.

[073] In another aspect, the disclosure provides for a method of treating, reducing, or ameliorating symptoms of a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition as provided herein, wherein the symptom of the disease or disorder is inflammation.

[074] In some embodiments, the subject has an decreased level of a secondary bile acid in the gut after the composition is administrated.

[075] In some embodiments, the secondary bile acid is lithocholic acid (LCA). In some embodiments, the secondary bile acid is deoxycholic acid (DCA). In some embodiments, the secondary bile acid is ursodeoxycholic acid (UDCA). In some embodiments, the secondary bile acid is chenodeoxycholic acid (CDCA). In some embodiments, the secondary bile acid is glyco- lithocholic acid (GLCA). In some embodiments, the secondary bile acid is tauro-lithocholic acid (TLCA). In some embodiments, the secondary bile acid is tauroursodeoxycholic Acid (TUDCA). In some embodiments, the secondary bile acid is glycochenodeoxy cholic acid (GCDCA). In some embodiments, the secondary bile acid is glycodeoxycholic acid (GDCA). In some embodiments, the secondary bile acid is taurodeoxycholic acid (TDCA).

[076] In some embodiments, the subject has a decreased level of a primary bile acid in the gut after the composition is administered. In some embodiments, the primary bile acid is cholic acid (CA). In some embodiments, the primary bile acid is chenodeoxycholic acid (CDCA).

[077] In some embodiments, the subject has an decreased level of a conjugated primary bile acid in the gut after the composition is administered.

[078] In some embodiments, the conjugated primary bile acid is glycoursodeoxy cholic Acid (GUDCA). In some embodiments, the conjugated primary bile acid is taurochenodeoxycholic acid (TCDCA). In some embodiments, the conjugated primary bile acid is glycocholic acid (GCA). In some embodiments, the conjugated primary bile acid is taurocholic acid (TCA).

[079] In some embodiments, the subject is a human.

Brief Description of the Drawings

[080] FIG. 1A provides a schematic showing a genetically engineered bacterium which is capable of converting a toxic bile acid into a non-toxic bile acid.

[081] FIG. IB provides a schematic showing a genetically engineered bacterium comprising a gene encoding a sulfotransferase and a bile acid transporter (for bile acid import), such as bacterial homologues of the Apical Sodium dependent Bile acid Transporter (ASBT), as described herein.

[082] FIG. 2A and 2B provides a schematic showing lithocholic Acid (LCA) Sulfation by SULT2A1 (FIG. 2A) and chenodeoxycholic Acid (CDCA) sulfation by SULT2A8 (FIG. 2B).

[083] FIG. 3 is a schematic of an exemplary embodiment of the disclosure. A genetically engineered bacterium expresses SULT2A1 from an inducible promoter. The bacterium further comprises gene sequences designed to express a BA transporter from an inducible promoter, to enable additional uptake and sulfonation of BA substrates. An exemplary transporter ASBT-Yf from Y. frederiksenii is shown.

[084] FIG. 4A is a schematic showing the cycle of the sulfur donor, PAPS, in E. coli. Deletion of the cysH gene (encoding PAPS reductase; red X) prevents the degradation of PAPS, leading to intracellular accumulation of the sulfur donor.

[085] FIG. 4B is a schematic showing an exemplary embodiment of the disclosure.

[086] FIG. 5 is a graph showing in vitro conversion of LCA toLCA-3 -sulfate over time by engineered E. coli Nissle cells. SYN8876: Logic2868 encoding IPTG-inducible SULT2A1 protein; SYN8978: EcN keys H + Logic2868 encoding IPTG-inducible SULT2A1 protein.

[087] FIG. 6A and 6B are graphs showing in vitro conversion of LCA to LCA-3-sulfate (FIG. 6A) or CA to CA-S (FIG. 6B) over time by engineered E. coli Nissle cells. Graphs illustrate the impact of cysH or cysQ deletion on bile acid sulfonation by strains expressing either SULT2A1 (FIG. 6A) or SULT2A8 (FIG. 6B).

[088] FIG. 7A and 7B are graphs showing rates of sulfonation of strains with either cysH or cysQ deleted, calculated from the conversions shown in FIG. 6A and 6B. FIG. 7A: rate of SULT2A1 activity on LCA. FIG. 7B: rate of SULT2A8 activity on CA.

[089] FIG. 8A is a graph showing LCA sulfonation activity in SULT2A1 expressing strains with added expression of one of the following bile acid transporters: an ABST homolog from Yersinia frederiksenii or Neisseria meningitidis, two homologs of these bacterial ASBTs identified in E. coli via BlastP, and the bile acid importer, BaiG, from Clostridium scindens. Expressing the transporter sourced from Y. frederiksenii results in an increase of sulfonation activity. For this in vitro assay, bacteria were grown in shake flasks.

[090] FIG. 8B is a graph showing rates of LCA sulfonation by SULT2A1 expressing strain SYN8978 and strains additionally expressing a putative bile acid transporter, based on the results shown in FIG. 8A.

[091] FIG. 9A is a graph showing in vitro activity of SULT2A1 expressing strains when grown in AMBR bioreactors.

[092] FIG. 9B is a graph showing viability of the same SULT2A1 expressing strains when grown in AMBR bioreactors as shown in FIG. 9A. [093] FIGs. 1OA is a graph showing in vitro bile acid sulfonation activity over 3 hours by EcN-SULT2Al strains +/- Yf-ASBT (SYN9056, SYN8978) across multiple substrates. CA, cholic acid, CDCA, chenodeoxycholic acid, DCA, deoxycholic acid, TLCA, tauro-lithocholic acid, GLCA, glyco-lithocholic acid, LCA, lithocholic acid. Assay cultures were grown in AMBR bioreactors.

[094] FIG. 10B is a graph showing the rates of bile acid sulfonation as calculated from results shown in FIG. 10A.

[095] FIG. 11A is a schematic showing an experimental set-up to assess and compare in vivo target engagement of SYN9056, (expresses ASBT-Yf) and SYN8978 (no transporter) in a mouse model, m which mice were administered a single dose of deuterium-labeled LCA (D4-LCA) mixed with unlabeled LCA and ileal contents were analyzed.

[096] FIG. 1 IB is a graph showing ileal production of D4-LCA-3S.

[097] FIG. 11C is a graph showing ileal production of LCA-3S.

[098] FIG. 12A is a graph showing in vitro bile acid sulfonation activity over 3 hours by

EcN-SULT2A8 strain SYN9018, which sulfonates CA (cholic acid) and CDCA (chenodeoxycholic acid).

[099] FIG. 12B is a graph showing rates of sulfonation by SULT2A8 expressing strain, SYN9018, on CA and CDCA, based on the results shown in FIG. 12A.

Detailed Description

[0100] Disclosed herein are genetically engineered bacteria that are capable of detoxifying select classes of disease-causing bile acids (BA), e.g., in the small intestine.

[0101] BA play fundamental roles in GI physiology, acting as surfactants in the small intestine to aid in lipid digestion and protect against infection. 95% of BA are actively reabsorbed by specialized enterocytes in the terminal ileum, after which they recirculate to the liver in a process known as enterohepatic circulation. BA that escape reabsorption in the ileum enter the colon and are metabolized into (more hydrophobic) secondary BA by the intestinal microbiota and may be passively reabsorbed in the colon to re-enter the circulating BA pool. Perturbations in BA synthesis, enterohepatic circulation and/or bacterial metabolism have become increasingly associated with GI disorders. Alterations in the size and/or composition of the circulating BA pool have been linked to IBD. Reductions in key BA -metabolizing bacterial species in IBD patients have been speculated to increase the levels of conjugated primary BA, while reducing levels of secondary BA. Mounting evidence indicates that BA can have direct, profound, and often opposing influences on immune activation and inflammation in the ileum. BA circulation through the ileal mucosa has generally pro- inflammatory consequences, which need to be subverted through active ‘adaptations’ by local lymphocytes, whereas lower concentrations of BA in the colon tend to have protective and antiinflammatory properties. Inhibition of BA intestinal reabsorption has become a key therapeutic goal of new therapies for cholestatic liver diseases. However, these approaches, which include direct sequestration of BA in the intestinal lumen or inhibition of the ileal BA reuptake transporter (ASBT) can have serious side effects due to blocking beneficial BA-dependent hormone signaling in ileal enterocytes.

[0102] Enterohepatic circulation of primary and secondary bile acids (BA) provides a target for designing genetically engineered bacteria that transform and de-toxify BA causing inflammation and disease. Aside from re-absorption, elimination of BA from the circulating pool may be influenced by changes in their physio-chemical properties and manipulated by different modifications.

[0103] Sulfonation is the primary mechanism of BA detoxification and elimination. BA sulfonation decreases passive absorption in the small intestine and colon, and sulfonated BA are poor substrates for ASBT48. As a result, BA sulfonation increases fecal clearance and a high fraction of urinary BA are sulfonated compared to the circulating BA pool. Due to the properties of sulfonated BA and given that sulfonation is reduced during inflammation, local sulfonation of BA in the small intestine is a potential approach to reducing BA reabsorption and enhancing elimination.

[0104] The present disclosure provides recombinant bacterial cells that have been engineered with optimized genetic circuitry which allow the recombinant bacterial cells to turn on and off an engineered metabolic pathway by sensing a patient’s internal environment or by chemical induction during, for example, manufacturing. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject and are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome.

[0105] The present disclosure provides recombinant bacteria for catabolism of certain bile acids which cause inflammation and disease. Accordingly, the bacteria of the disclosure, which comprise gene cassette(s) or genetic circuit(s) encoding bile acid catabolism enzymes are capable of transforming and de-toxifying these bile acids. The recombinant bacteria are capable of catabolizing in low -oxygen environments, e.g., the gut.

[0106] The present disclosure provides recombinant bacteria for catabolism of certain bile acids which cause inflammation and disease. Accordingly, the bacteria of the disclosure, which comprise gene cassette(s) or genetic circuit(s) encoding bile acid catabolism enzymes are capable of transforming and/or de-toxifying these bile acids. In some embodiments, the bacteria of the disclosure are capable of decreasing the concentration of primary bile acids or increasing the concentration of secondary bile acids. In some embodiments, the bacteria of the disclosure are capable of decreasing the concentration of conjugated primary bile acids or increasing the concentration of deconjugated primary bile acids or secondary bile acids. In some embodiments the genetically engineered bacteria produce secondary bile acids. In some embodiments, the genetically engineered bacteria detoxify inflammatory bile acids. In some embodiments, the genetically engineered bacteria alter receptor signaling or produce a bioactive bile acid, e.g. , a bile acid that can modulate T cell function. In some embodiments, recombinant bacteria are capable of catabolizing in low-oxygen environments and/or at physiological temperature, e.g, in the gut. In some embodiments, recombinant bacteria are of catabolizing in the presence of an inducer.

[0107] Pharmaceutical compositions of recombinant bacteria thereof, and methods of modulating and treating diseases associated with the presence of toxic bile acids are also provided. The recombinant bacteria and pharmaceutical compositions comprising those bacteria are non- pathogenic, and can be used in order to treat and/or prevent conditions associated with autoimmune and inflammatory diseases and disorders.

[0108] Specifically, the present disclosure provides recombinant bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with inflammation. Specifically, the recombinant bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, a bile acid sulfotransferase to treat disease, as well as other circuitry in order to guarantee the safety and non-colonization of the subject that is administered the recombinant bacteria, such as auxotrophies, etc. These recombinant bacteria are safe and well tolerated and augment the innate activities of the subject’s microbiome to achieve a therapeutic effect.

[0109] In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid sulfotransferase(s) and is capable of processing (e.g., metabolizing or catabolizing) and reducing levels of BA, e.g. , lithocholic acid. In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid sulfotransferase and is capable of processing and reducing levels of certain bile acids in low-oxygen environments and/or at physiological temperatures, e.g., in the gut. Thus, the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess BA, e.g., lithocholic acid into a sulfonated counterpart, e.g., lithocholic acid-3- sulfate, in order to treat and/or prevent diseases associated with inflammation, such as IBD, Crohn’s disease or ulcerative colitis.

[0110] 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.

[0111] As used herein, the term “recombinant bacterial cell” or “recombinant bacteria” (also referred to herein as a “genetically engineered bacterial cell”) 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 of the disclosure may comprise exogenous or heterologous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous or heterologous nucleotide sequences stably incorporated into their chromosome.

[0112] As used herein, the term “gene” refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5 ’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence. In one embodiment, a “gene” does not include regulatory sequences preceding and following the coding sequence. A “native gene” refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A “chimeric gene” refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature. As used herein, the term “gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The 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 non-protein coding sequence.

[0113] 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 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.

[0114] 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 a recombinant bacterial cell of the disclosure. [0115] As used herein, the term “bactericidal” refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.

[0116] 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 anti -toxin 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.

[0117] 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.

[0118] “Operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding at least one bile acid catabolism enzyme, e.g., sulfotransferase or other polypeptide described herein, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene(s) encoding the bile acid catabolism enzyme, or other polypeptide of interest. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be “directly linked” to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes. A regulatory region or sequence 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. [0119] 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.

[0120] 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. An “inducible promoter” refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide 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 first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.” Examples of inducible promoters include, but are not limited to, an FNR promoter, a P_arac promoter, a ParaBAD promoter, a propionate promoter, and a P e® promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.

[0121] As used herein, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a bile acid catabolism enzyme, that is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium 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 may be a genetically engineered bacterium comprising an amino acid catabolism gene, in which the plasmid or chromosome carrying the amino acid catabolism gene is stably maintained in the bacterium, such that the bile acid catabolism enzyme can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material. [0122] 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

[0123] 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 disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding at least one bile acid catabolism enzyme.

[0124] 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.

[0125] 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 at least one bile acid catabolism 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.

[0126] 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.

[0127] 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 asparagine. 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 al., Gene, 379: 109-115 (2006)).

[0128] 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.

[0129] “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, the genetically engineered microorganism of the disclosure comprises an oxygen leveldependent 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. 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 diseasestate, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissuespecific 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 exogenous environmental conditions of the disclosure refer to a specific temperature, for example, a temperature between 37 °C and 42 °C.

[0130] 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. Such conditions affect proliferation rate, rate of induction of the payload or gene of interest, e.g. , amino acid catabolism gene, other regulators (e.g., FNRS24Y), and overall viability and metabolic activity of the strain during strain production.

[0131] 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).

[0132] 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.

[0133] 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 etal., 1989; Galimand etal., 1991; Hasegawa et a/., 1998; Hoeren etal., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1.

[0134] In a non -limiting example, a promoter (PfhrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (finrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfhrS promoter is activated under anaerobic and/or low oxygen conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic and/or low oxygen 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 PfinrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfinrS is used interchangeably in this application as FNRS, fnrS, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfhrS.

Table 1. Examples of transcription factors and responsive genes and regulatory regions

[0135] 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 phenylalanine-metabolizing 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 an amino acid metabolism gene.

[0136] “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 o^s 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 o³² promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli o⁷⁰ 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 o^Apromoter (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 o^B 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_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof. [0137] “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.

[0138] In some embodiments, the genetically engineered bacteria are active in the gut. In some embodiments, the genetically engineered bacteria are active in the large intestine. In some embodiments, the genetically engineered bacteria are active in the small intestine, e.g., the ileum. 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 intestine. In some embodiments, the genetically engineered bacteria do not colonize the gut.

[0139] As used herein, the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% O2; <160 torr O2)). 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 (O2) found in a mammalian gut, e.g. , lumen, stomach, small intestine, duodenumjejunum, 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 O2 that is 0-60 mmHg O2 (0-60 torr O2) 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 O2), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O2, 0.75 mmHg O2, 1.25 mmHg O2, 2.175 mmHg O2, 3.45 mmHg O2, 3.75 mmHg O2, 4.5 mmHg O2, 6.8 mmHg O2, 11.35 mmHg 02, 46.3 mmHg O2, 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 O2 or less (e.g., 0 to about 60 mmHg O2). The term “low oxygen” may also refer to a range of O2 levels, amounts, or concentrations between 0-60 mmHg O2 (inclusive), e.g., 0-5 mmHg O2, < 1.5 mmHg O2, 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 incorporated 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 (O2) 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 (O2) 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 (O2) is expressed as the amount of dissolved oxygen (“DO”) which refers to the level of free, noncompound oxygen (O2) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; Img/L = 1 ppm), or in micromoles (pmole) (1 pmole O2 = 0.022391 mg/L O2). 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 (O2) 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 (O2) 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% O2 saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O2 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 O2 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% Ch.etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.

Table 2

[0140] “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.

[0141] “Non-pathogenic bacteria” referto 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 are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, 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, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenbom 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.

[0142] “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. Examples of probiotic bacteria include, but are not limited to, 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). Non-pathogenic 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 to enhance or improve probiotic properties.

[0143] As used herein, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., amino acid metabolism gene, which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and/or propagated. The stable bacterium 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 may be a genetically modified bacterium comprising an amino acid metabolism gene, in which the plasmid or chromosome carrying the amino acid metabolism gene is stably maintained in the host cell, such that amino acid metabolism gene can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material, e.g., an amino acid metabolism gene. In some embodiments, copy number affects the level of expression of the non-native genetic material, e.g., heterologous gene.

[0144] 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 met A).

[0145] 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.

[0146] 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. Treating diseases associated with or involved with gut inflammation, e.g., Crohn’s disease (CD) or ulcerative colitis (UC), may encompass reducing normal levels of certain BAs, reducing excess levels of certain BAs, or eliminating one or more BAs, and does not necessarily encompass the elimination of the underlying disease.

[0147] As used herein the terms “disease associated with inflammation” or a “disorder associated with inflammation” or “autoimmune disease” is a disease or disorder involving the abnormal, e.g., increased, levels of one or more bile acids, in a subject.

[0148] As used herein, “autoimmune disorders” or “disease associated with inflammation” or a “disorder associated with inflammation” include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet’s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha- Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa- Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s granulomatosis.

[0149] Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility. [0150] As used herein, the term “bile acid catabolism” or “bile acid metabolism” refers to the processing, breakdown, modification, conversion and/or degradation of a bile acid molecule (e.g., a secondary bile acid such as lithocholic acid (LCA), or a primary bile acid such as cholic acid (CA)), into other bile acids and compounds that are not associated with the inflammatory or autoimmune disease, or compounds which can be utilized by the bacterial cell.

[0151] As used herein, the term “transporter” is meant to refer to a mechanism, e.g. , protein, proteins, or protein complex, for importing a molecule, e.g., bile acid, peptide (di-peptide, tri-peptide, polypeptide, etc.), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu. For example, a phenylalanine transporter such as PheP imports phenylalanine into the microorganism.

[0152] As used herein, “payload” refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as bacteria or a virus. In some embodiments, the payload is a therapeutic payload, e.g., an amino acid catabolic enzyme or an amino acid transporter polypeptide. 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.

[0153] 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.

[0154] 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. 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 disease or condition associated with excess amino acid levels. 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.

[0155] 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 “tripeptide” refers to a peptide of three linked amino acids. The term “polypeptide” is also intended to refer to the products of postexpression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring 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 current 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 referred 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.

[0156] 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.

[0157] 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, Vai, He, Leu, Met, Ala, Phe, Lys, Arg, His, Phe, Tyr, Trp, His, Asp, and Glu.

[0158] 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 wild-type 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.

[0159] 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.

[0160] 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. 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. 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, inter aha, 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.

[0161] The terms “phage” and “bacteriophage” are used interchangeably herein. Both terms refer to a virus that infects and replicates within a bacterium. As used herein “phage” or bacteriophage” collectively refers to prophage, lysogenic, dormant, temperate, intact, defective, cryptic, and satellite phage, phage tail bacteriocins, tailiocins, and gene transfer agents. As used therein the term “prophage” refers to the genomic material of a bacteriophage, which is integrated into a replicon of the host cell and replicates along with the host. The prophage may be able to produce phages if specifically activated. In some cases, the prophage is not able to produce phages or has never done so (i.e., defective or cryptic prophages). In some cases, prophage also refers to satellite phages. The terms “prophage” and “endogenous phage” are used interchangeably herein. “Endogenous phage” or “endogenous prophage” also refers to a phage that is present in the natural state of a bacterium (and its parental strain). As used herein the term “phage knockout” or “inactivated phage” refers to a phage which has been modified so that it can either no longer produce and/or package phage particles or it produces fewer phage particles than the wild type phage sequence. In some embodiments, the inactivated phage or phage knockout refers to the inactivation of a temperate phage in its lysogenic state, i.e., to a prophage. Such a modification refers to a mutation in the phage; such mutations include insertions, deletions (partial or complete deletion of phage genome), substitutions, inversions, at one or more positions within the phage genome, e.g., within one or more genes within the phage genome. As used herein the adjectives “phage-free”, “phage free” and “phageless” are used interchangeably to characterize a bacterium or strain which contains one or more prophages, one or more of which have been modified. The modification can result in a loss of the ability of the prophage to be induced or release phage particles. Alternatively, the modification can result in less efficient or less frequent induction or less efficient or less frequent phage release as compared to the isogenic strain without the modification. Ability to induce and release phage can be measured using a plaque assay as described herein. As used herein phage induction refers to the part of the life cycle of a lysogenic prophage, in which the lytic phage genes are activated, phage particles are produced and lysis occurs.

[0162] As used herein a "pharmaceutical composition" refers to a preparation of bacterial cells disclosed herein with other components such as a physiologically suitable carrier and/or excipient.

[0163] 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 compound. An adjuvant is included under these phrases.

[0164] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary. For example, as used herein, “a heterologous gene encoding a bile acid catabolism enzyme” should be understood to mean “at least one heterologous gene encoding at least one bile acid catabolism enzyme.” Similarly, as used herein, “a heterologous gene encoding a bile acid transporter” should be understood to mean “at least one heterologous gene encoding at least one bile acid transporter.”

[0165] 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.

[0166] 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.

[0167] The terms “recombinant” and “genetically engineered” are used interchangeably herein. The terms “genetically engineered bacterium” or “genetically engineered bacteria” is used interchangeably with “recombinant bacterium” or “recombinant bacteria” herein.

Bacterial Strains

[0168] The disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile acid catabolism enzyme. In some embodiments, the bacterial cell is a non-pathogenic bacterial cell. In some embodiments, the bacterial cell is a commensal bacterial cell. In some embodiments, the bacterial cell is a probiotic bacterial cell.

[0169] In certain embodiments, the bacterial cell is selected from the group consisting of a Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter formigenes bacterial cell. In one embodiment, the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium animalis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is a Clostridium scindens bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Oxalobacter formigenes bacterial cell. In another embodiment, the bacterial cell does not include Oxalobacter formigenes .

[0170] In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell. [0171] In some embodiments, the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterohacteriaceae family that “has evolved into one of the best characterized probiotics” (Ukena et al., 2007). The strain is characterized by its “complete harmlessness” (Schultz, 2008), and “has GRAS (generally recognized as safe) status” (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle “lacks prominent virulence factors (e.g., E. coli a-hemolysin, P-fimbrial adhesins)” (Schultz, 2008), and E. coli Nissle “does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic” (Sonnenbom et al., 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 al., 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 al., 2004). It is commonly accepted that E. coli Nissle ’s “therapeutic efficacy and safety have convincingly been proven” (Ukena et al., 2007).

[0172] In one embodiment, the recombinant bacterial cell does not colonize the subject.

[0173] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. Furthermore, genes from one or more different species can be introduced into one another, e.g., a bile acid catabolism gene from Homo sapiens can be expressed in Escherichia coli.

[0174] 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.

[0175] In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein. In one aspect, the disclosure provides a recombinant bacterial culture which reduces levels of a bile acid, e.g., lithocholic acid or cholic acid, in the media of the culture. In one embodiment, the levels of an amino acid are reduced by about 50%, about 75%, or about 100% in the media of the cell culture. In another embodiment, the levels of an amino acid are reduced by about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, ninefold, or ten-fold in the media of the cell culture. In one embodiment, the levels of a bile acid, e.g., lithocholic acid or cholic acid, are reduced below the limit of detection in the media of the cell culture.

[0176] In some embodiments of the above described genetically engineered bacteria, the gene encoding a sulfotransferase 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 sulfotransferase 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. [0177] In some embodiments of the above described genetically engineered bacteria, the gene encoding a sulfotransferase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a sulfotransferase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.

[0178] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile acid importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a bile acid importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.

[0179] In some embodiments of the above described genetically engineered bacteria, the gene encoding a sulfotransferase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced and the gene encoding a bile acid importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced. In other embodiments, the gene encoding a sulfotransferase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced and the gene encoding bile acid importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.

[0180] In some embodiments of the above described genetically engineered bacteria, the gene encoding a sulfotransferase boxylase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced and the gene encoding a bile acid importer is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced. In other embodiments, the gene encoding a sulfotransferase is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced and the gene encoding a bile acid importer is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced.

[0181] In some embodiments, the genetically engineered bacteria is an auxotroph. In one embodiment, the genetically engineered bacteria 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 thi l auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a lAthyA and Map A auxotroph.

[0182] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile acid catabolism 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 a bile acid catabolism 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. A, Bile Acid Catabolism Enzymes

1. Sulfotransferases

[0183] The primary mechanism of BA detoxification in humans is mediated by the sulfotransferase, SULT2A1, which sulfonates both primary and secondary BA, increasing their solubility and excretion in urine and feces.

[0184] Sulfotransferases may be expressed or modified in the bacteria disclosed herein in order to enhance catabolism of bile acids, i.e., the transformation of lithocholic acid into lithocholic acid-3 sulfate. For example, the genetically engineered bacteria comprising at least one heterologous gene encoding a Sulfotransferase can catabolize lithocholic acid to treat an autoimmune disease or a disease associated with inflammation in the gut, including but not limited to CD and UC, and others described herein. As used herein, the term “bile acid catabolism enzyme” refers to an enzyme involved in the catabolism, i.e., processing, breakdown, modification, conversion and/or degradation of a bile acid molecule, e.g., a secondary bile acid, such as lithocholic acid. Specifically, when a bile acid catabolism enzyme is expressed in a recombinant bacterial cell, the bacterial cell catabolizes more bile acid, when the bile acid catabolism enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In some embodiments, bile acid transporters may also be expressed or modified in the recombinant bacteria to enhance bile acid import into the cell in order to increase the catabolism of bile acid by the bile acid catabolism enzyme. In other embodiments, bile acid exporters may be knocked-out in the recombinant bacteria to decrease export of bile acid and/or increase cytoplasmic concentration of bile acid.

[0185] As used herein, the term “Sulfotransferase” refers to an enzymes that catalyzes the transfer of a sulfo group from a donor molecule to an acceptor alcohol or amine. The most common sulfo group donor is 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Sulfotransferases are a type of bile acid catabolism enzyme, as the term is used herein.

[0186] Sulfotransferases, such as SULT2A1, e.g., human derived SULT2A1, can sulfonate both primary and secondary BA. SULT2A1 is the major bile acid detoxifying enzyme in humans. The enzyme can convert bile acids into more hydrophilic water-soluble sulfate conjugates that can be easily excreted, thereby increasing clearance of toxic bile acids. SULT2A1 adds a sulfate group to the 3’OH group of bile acids (BAs) and requires the 3 ’-phosphoadenosine 5 ’-phosphosulfate (PAPS) cofactor as sulfur donor. The enzyme is endogenous in both humans and E. coli, including E. coli Nissle. SULT2A1 is a promiscuous enzyme with multiple targets, and has high affinity for LCA. The affinity of the enzyme for various substrates is as follows: dehydroepiandrosterone (DHEA) > lithocolic acid (LCA) > ursodeoxycholic acid (UDCA) > deoxycholic acid (DCA) > chenodeoxy cholic acid (CDCA) > cholic acid (CA).

[0187] In one embodiment contemplated herein, sulfotransferase activity produced by the genetically engineered bacteria is aimed at detoxifying secondary BA (e.g., LCA) in the small intestine. For example, a sulfotransferase may convert or catabolize lithocholic acid (LCA), into lithocholic acid-3 -sulfate or LCA3 Sulfate (LCA3S). Alternatively or in addition, the sulfotransferase may convert UDCA to UDCA-3 -sulfate (UDCA3S), DCA to DCA-3-sulfate (DCA3S), CDCA to CDCA-3 -sulfate (CDCA3S) and/or CA to CA-3 -sulfate (CA3S). Additionally a sulfotransferase may convert or catabolize GLCA to GLCA-3 -sulfate (GLCA3S), TLCA to TLCA-3 -sulfate (TLCA3S), GUDCA to GUDCA-3 -Sulfate (GUDCA3S), TUDCA to TUDCA-3 -sulfate (TUDCA3S), GCDCA to GCDCA-3 -sulfate (GCDCA3S), TCDCA to TCDCA-3 -sulfate (TCDCA3S), GDCA to GDCA-3- sulfate (GDCA3S), TDCA to TDCA-3 -sulfate (TDCA3S), GCA to GCA-3-sulfate (GCA3S), TCA to TCA-3 -sulfate (TCA3S).

[0188] In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA) and/or cholic acid (CA). In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA).

[0189] In some embodiments, the bile acid catabolism enzyme catabolizes cholic acid (CA). In some embodiments, the bile acid catabolism enzyme catabolizes ursodeoxycholic acid (UDCA). In some embodiments, the bile acid catabolism enzyme catabolizes deoxycholic acid (DCA). In some embodiments, the bile acid catabolism enzyme catabolizes chenodeoxycholic acid (CDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glyco-lithocholic acid (GLCA). In some embodiments, the bile acid catabolism enzyme catabolizes tauro-lithocholic acid (TLCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycoursodeoxycholic acid (GUDCA). In some embodiments, the bile acid catabolism enzyme catabolizes tauroursodeoxycholic acid (TUDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycochenodeoxy cholic acid (GCDCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurochenodeoxycholic acid (TCDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycodeoxycholic acid (GDCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurodeoxycholic acid (TDCA). In some embodiments, the bile acid catabolism enzyme catabolizes glycocholic acid (GCA). In some embodiments, the bile acid catabolism enzyme catabolizes taurocholic acid (TCA). In some embodiments, the bile acid catabolism enzyme catabolizes dehydroepiandrosterone (DHEA).

[0190] In some embodiments, the bacterium is capable of exhibiting sulfonation activity of lithocholic acid (LCA). In some embodiments, the bacterium is capable of sulfonating cholic acid (CA). In some embodiments, the bacterium is capable of sulfonating ursodeoxycholic acid (UDCA). In some embodiments, the bacterium is capable of sulfonating deoxy cholic acid (DCA). In some embodiments, the bacterium is capable of sulfonating chenodeoxycholic acid (CDCA). In some embodiments, the bacterium is capable of sulfonating glyco-lithocholic acid (GLCA). In some embodiments, the bacterium is capable of sulfonating tauro-lithocholic acid (TLCA). In some embodiments, the bacterium is capable of sulfonating glycoursodeoxy cholic Acid (GUDCA). In some embodiments, the bacterium is capable of sulfonating tauroursodeoxycholic Acid (TUDCA). In some embodiments, the bacterium is capable of sulfonating glycochenodeoxycholic acid (GCDCA). In some embodiments, the bacterium is capable of sulfonating taurochenodeoxycholic acid (TCDCA). In some embodiments, the bacterium is capable of sulfonating glycodeoxycholic acid (GDCA). In some embodiments, the bacterium is capable of sulfonating taurodeoxycholic acid (TDCA). In some embodiments, the bacterium is capable of sulfonating glycocholic acid (GCA). In some embodiments, the bacterium is capable of sulfonating taurocholic acid (TCA). In some embodiments, the bacterium is capable of sulfonating dehydroepiandrosterone (DHEA).

[0191] In some embodiments, the bacterium is capable of sulfonating lithocholic acid (LCA) into lithocholic acid-3 -sulfate or LCA3 Sulfate (LCA3S). In some embodiments, the bacterium is capable of sulfonating cholic acid (CA) into CA-3 -sulfate (CA3S) or CA-7 -sulfate (CA7S). In some embodiments, the bacterium is capable of sulfonating ursodeoxycholic acid (UDCA) into UDCA -3- sulfate (UDCA3S) or UDCA -7 -sulfate (UDCA7S). In some embodiments, the bacterium is capable of sulfonating deoxy cholic acid (DCA) into DCA -3 -sulfate (DCA3S). In some embodiments, the bacterium is capable of sulfonating chenodeoxy cholic acid (CDCA) into CDCA-3 -sulfate (CDCA3S) or CDCA-7-sulfate (CDCA7S). In some embodiments, the bacterium is capable of sulfonating glyco- lithocholic acid (GLCA) into GLCA -3 -sulfate (GLCA3S). In some embodiments, the bacterium is capable of sulfonating tauro-lithocholic acid (TLCA) into TLCA-3 -sulfate (TLCA3S). In some embodiments, the bacterium is capable of sulfonating glycoursodeoxycholic Acid (GUDCA) into GUDCA -3 -Sulfate (GUDCA3S) or GCDCA-7-sulfate (CDCA7S). In some embodiments, the bacterium is capable of sulfonating tauroursodeoxycholic Acid (TUDCA) into TUDCA-3 -sulfate (TUDCA3S) or TCDCA-7-sulfate (TCDCA7S). In some embodiments, the bacterium is capable of sulfonating glycochenodeoxycholic acid (GCDCA) into GCDCA-3 -sulfate (GCDCA3S). In some embodiments, the bacterium is capable of sulfonating taurochenodeoxycholic acid (TCDCA) into TCDCA-3 -sulfate (TCDCA3S). In some embodiments, the bacterium is capable of sulfonating glycodeoxy cholic acid (GDCA) into GDCA -3 -sulfate (GDCA3S). In some embodiments, the bacterium is capable of sulfonating taurodeoxycholic acid (TDCA) into TDCA-3 -sulfate (TDCA3S). In some embodiments, the bacterium is capable of sulfonating glycocholic acid (GCA) into GCA-3 - sulfate (GCA3S) or GCA-7-sulfate (GCA7S). In some embodiments, the bacterium is capable of sulfonating taurocholic acid (TCA) into TCA-3-sulfate (TCA3S) or TCA-7-sulfate (TCA7S). In some embodiments, the bacterium is capable of sulfonating dehydroepiandrosterone (DHEA) into dehydroepiandrosterone sulfate (DHEAS).

[0192] In some embodiments, the bile acid catabolism enzyme catabolizes a secondary bile acid. In some embodiments, the secondary bile acid is lithocholic acid (LCA). In some embodiments, the secondary bile acid is deoxy cholic acid (DCA). In some embodiments, the secondary bile acid is ursodeoxycholic acid (UDCA). In some embodiments, the secondary bile acid is chenodeoxycholic acid (CDCA). In some embodiments, the secondary bile acid is glyco-lithocholic acid (GLCA). In some embodiments, the secondary bile acid is tauro-lithocholic acid (TLCA). In some embodiments, the secondary bile acid is tauroursodeoxycholic Acid (TUDCA). In some embodiments, the secondary bile acid is glycochenodeoxy cholic acid (GCDCA). In some embodiments, the secondary bile acid is glycodeoxy cholic acid (GDCA). In some embodiments, the secondary bile acid is taurodeoxy cholic acid (TDCA).

[0193] In some embodiments, the bile acid catabolism enzyme catabolizes a primary bile acid. In some embodiments, the primary bile acid is cholic acid (CA). In some embodiments, the primary bile acid is chenodeoxy cholic acid (CDCA).

[0194] In some embodiments, the bile acid catabolism enzyme catabolizes a conjugated primary bile acid. In some embodiments, the conjugated primary bile acid is glycoursodeoxycholic Acid (GUDCA). In some embodiments, the conjugated primary bile acid is taurochenodeoxycholic acid (TCDCA). In some embodiments, the conjugated primary bile acid is glycocholic acid (GCA). In some embodiments, the conjugated primary bile acid is taurocholic acid (TCA).

[0195] In some embodiments, the bile acid catabolism enzyme catabolizes a hormone. In some embodiments, the hormone is dehydroepiandrosterone (DHEA).

[0196] Sulfotransferases, such as SULT2A8, e.g., mouse derived SULT2A8, can sulfonate primary bile acids (BAs). SULT2A8 adds a sulfate group to the 7’ OH group of BAs and requires the PAPS cofactor as sulfur donor. In particular, SULT2A8 is a hepatic sulfotransferse which catalyzes the transfer of sulfonate groups from 3 ’phosphoadenylyl sulfate (PAPS) to the 7-alpha hydroxyl group of primary bile acids to form 7-monosulfate derivatives. SULT2A8 is a promiscuous enzyme with multiple targets, which are not fully characterized and may include CA and CDCA. In one embodiment contemplated herein, sulfotransferase activity produced by the genetically engineered bacteria is aimed at detoxifying primary BA (e.g., cholic acid (CA) and/or CDCA) in the small intestine. For example, a sulfotransferase may convert or catabolize UDCA to UDCA-7-sulfate (UDCA7S), CDCA to CDCA-7-sulfate (CDCA7S) and/or CA to CA-7-sulfate (CA7S). A sulfotransferase may additionally convert or catabolize GCDCA to GCDCA -7 -sulfate (CDCA7S), TCDCA to TCDCA-7-sulfate (TCDCA7S), GCA to GCA-7-sulfate (GCA7S) and/or TCA to TCA-7- sulfate (TCA7S). In some embodiments, the sulfotransferase has high affinity for CA.

[0197] In some embodiments, the sulfotransferase has higher affinity for LCA or CA than for other bile acids. In some embodiments, the sulfotransferase has low affinity for other amino acids. Without wishing to be bound by theory, given that multiple BA species may be present in the gut and capable of competing with LCA or CA for sulfonation by the genetically engineered bacterium of the disclosure, strain affinity (Km) towards BAs may be measured and offers an alternative parameter for strain assessment. The Km for LCA or CA may be compared to the Km for other individual BA species for each strain. Without wishing to be bound by theory, lowest Km for LCA or CA (highest affinity) comparable to other BAs, may be beneficial as these strains would be expected to preferentially catalyze LCA sulfonation in a mixed BA pool. [0198] Sulfotransferases are well known to those of skill in the art (see, e.g., Huang et al., Mar. Drugs, 13(8) :5492-5507, 2015). Sulfate conjugation (sulfation or sulfonation) is a major conjugating pathway responsible for the deactivation, detoxification, and excretion of xenobiotics and endogenous molecules, including bile acids. Among SULTs, the aryl -sulfotransferase (SULT1) and the hydroxysteroid sulfotransferase (SULT2) families are two principal subfamilies of SULTs that are the major contributors to the sulfonation of many xenobiotics, including pharmaceuticals and procarcinogens, and endobiotics, including steroids, thyroid, and neurotransmitters (Xie and Xie, Drug Metab. Dispos 2020, Sept (48 (9): 742-749, and references therein). Cholestasis is reduction or stoppage of bile flow. Bile acids accumulate in the liver due to elevated bile acid production in the liver or insufficient detoxication and elimination of bile acids from the liver. Human SULT2A1 ( HGNC: 11458; NCBI Entrez Gene: 6822; Ensembl: ENSG00000105398; OMIM®: 125263; UniProtKB/Swiss-Prot: Q06520) utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as sulfonate donor to catalyze the sulfonation of steroids in the liver and adrenal glands, including bile acids and many xenobiotics (Suiko et al., Biosci Biotechnol Biochem. 2017). SULT2A1, through bile acid sulfation, therefore can detoxify bile acids and prevent cholestasis. SULT2A1 expression is regulated by several nuclear receptors, such as pregnane X receptor, constitutive androstane receptor, LXRa, HNF4a, and FXR (Runge-Morris et al., Drug Metab Rev. 2013 Feb;45(l): 15-33)..

[0199] In some embodiments, a bile acid catabolism enzyme is encoded by a gene encoding a bile acid catabolism enzyme derived from a bacterial species. In some embodiments, a bile acid catabolism enzyme is encoded by a gene encoding a bile acid catabolism enzyme derived from a non- bacterial species. In some embodiments, a bile acid catabolism enzyme is encoded by a gene derived from a eukaryotic species, e.g., a yeast species or a plant species. In some embodiments, a bile acid catabolism enzyme is encoded by a gene encoding a human bile acid catabolism enzyme.

[0200] In one embodiment, the bile acid catabolism enzyme is a sulfotransferase. In one embodiment, the sulfotransferase gene is a human sulfotransferase. In one embodiment, the sulfotransferase is selected from a SULT1 or SULT2 family member. In some embodiments, the sulfotransferases is a SULT2 family member. In some embodiments, the sulfotransferase is SULT2A1. In some embodiments, the sulfotransferase is SULT2A8.

[0201] In one embodiment, the sulfotransferase gene has at least about 80% identity with the sequence of SEQ ID NO: 500. Accordingly, in one embodiment, the sulfotransferase gene has at least about 90% identity with the sequence of SEQ ID NO: 500. Accordingly, in one embodiment, the sulfotransferase gene has at least about 95% identity with the sequence of SEQ ID NO: 500. Accordingly, in one embodiment, the sulfotransferase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 500. In another embodiment, the sulfotransferase gene comprises the sequence of SEQ ID NO: 500. In yet another embodiment the sulfotransferase gene consists of the sequence of SEQ ID NO: 500. [0202] In one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 501. Accordingly, in one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 501. Accordingly, in one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 501. In some embodiments, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 501. In another embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that comprises the sequence of SEQ ID NO: 501. In yet another embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that consists of the sequence of SEQ ID NO: 501.

[0203] In one embodiment, the sulfotransferase gene has at least about 80% identity with the sequence of SEQ ID NO: 516. Accordingly, in one embodiment, the sulfotransferase gene has at least about 90% identity with the sequence of SEQ ID NO: 516. Accordingly, in one embodiment, the sulfotransferase gene has at least about 95% identity with the sequence of SEQ ID NO: 516. Accordingly, in one embodiment, the sulfotransferase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 516. In another embodiment, the sulfotransferase gene comprises the sequence of SEQ ID NO: 516. In yet another embodiment the sulfotransferase gene consists of the sequence of SEQ ID NO: 516.

[0204] In one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 517. Accordingly, in one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 517. Accordingly, in one embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 517. In some embodiments, the gene sequence encoding a sulfotransferase encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 517. In another embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that comprises the sequence of SEQ ID NO: 517. In yet another embodiment, the gene sequence encoding a sulfotransferase encodes a polypeptide that consists of the sequence of SEQ ID NO: 517.

[0205] Assays for testing the activity of a bile acid catabolism enzyme, i. e. , sulfotransferase, to one of ordinary skill in the art. Activity may be assessed via quantification of LCA or CA sulfonation via LCMS over a set period of time. See e.g., Garcia-Canaveras et al., J Lipid Res 53, 2231-2241.

[0206] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a sulfotransferase, such that the sulfotransferase 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 sulfotransferase. In some embodiments, the gene encoding the sulfotransferase 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 sulfotransferase is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of sulfotransferase. In some embodiments, the gene encoding the sulfotransferase is expressed on a chromosome.

[0207] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MO As), 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 sulfotransferase inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene encoding a particular sulfotransferase inserted at three different insertion sites and three copies of the gene encoding a different sulfotransferase inserted at three different insertion sites.

[0208] In some embodiments, under conditions where the sulfotransferase 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 sulfotransferase, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.

[0209] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the sulfotransferase gene(s). Primers specific for sulfotransferase 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 acid catabolism 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 acid catabolism enzyme gene(s).

[0210] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the bile acid catabolism enzyme gene(s). Primers specific for sulfotransferase 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 sulfotransferase 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 sulfotransferase gene(s).

[0211] In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile acid catabolism enzyme, e.g., sulfotransferase. In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile acid transporter and a heterologous gene encoding a bile acid catabolism enzyme, e.g., a sulfotransferase. In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile catabolism enzyme and a genetic modification that reduces export of bile acids. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of bile acids, a heterologous gene encoding a bile acid catabolism enzyme, e.g., a sulfotransferase, and a genetic modification that reduces export of bile acids. Transporters and exporters are described in more detail in the subsections, below.

[0212] Efficient sulfonation by SULT2A1 requires the presence of a sulfur-donating cofactor, 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS). Accordingly, in some embodiments, modifications to the PAPS biosynthetic pathway will be pursued to increase the amount of sulfur donor in E. coli.

2. Co-factor modifications

[0213] Efficient sulfonation by SULT2A1 or SULT2A8 requires the presence of a sulfur- donating co-factor, 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS). Accordingly, the genetically engineered bacteria may comprise one or more modifications to the PAPS biosynthetic pathway, e.g., to increase the amount of sulfur donor in E. coli. [0214] 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS), which is reduced by PAPS reductase, is present in enteric bacteria and yeast. In one embodiment, a PAPs reductase gene is modified to eliminate or reduce its activity. In one embodiment, the PAPS reductase gene is cysH. In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an PAPS reductase gene, e.g., cysH. In another embodiment, the genetic mutation results in an PAPS reductase having reduced activity as compared to a wild-type exporter protein. In one embodiment, the PAPS reductase activity is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the PAPS reductase is reduced at least two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in a PAPs reductase having no activity and which cannot reduce PAPS within the bacterial cell. In another embodiment, the PAPS reductase gene, cysH, is wild type.

[0215] In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding a PAPS reductase gene, e.g., cysH.

[0216] In yet another embodiment, the genetic modification is an overexpression of a repressor of a PAPs reductase gene, e.g., cysH. In one embodiment, the overexpression of the repressor of the PAPs reductase gene, e.g., cysH is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the PAPs reductase gene, e.g. , cysH 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.

[0217] Alternatively or in addition, the genetically engineered bacteria may comprise gene sequences encoding a sulfate importer, i.e., a sulfate transporter to increase sulfur donor levels in the cell. In one embodiment, the sulfate transporter is CysZ.

[0218] Assays for testing the activity of a bile acid catabolism enzyme, i. e. , sulfotransferase, to one of ordinary skill in the art. Activity may be assessed via quantification of LCA or CA sulfonation via LCMS over a set period of time. See e.g., Garcia-Canaveras et al., J Lipid Res 53, 2231-2241.

[0219] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a sulfate transporter, such that the sulfate transporter 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 sulfate transporter. In some embodiments, the gene encoding the sulfate transporter 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 sulfate transporter is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of sulfate transporter. In some embodiments, the gene encoding the sulfate transporter is expressed on a chromosome.

[0220] In some embodiments, the genetically engineered bacteria comprise gene sequences encoding a sulfate transporter, e.g, CysZ, and a modification, e.g., a mutation or deletion in the endogenous cysH gene. Accordingly, in some embodiments, the genetically engineered bacteria comprising gene sequences encoding a bile acid catabolism enzyme, e.g. sulfotransferase, may further comprise gene sequences encoding a sulfate transporter, e.g., CysZ, and/or a modification, e.g., a mutation or deletion in the endogenous cysH gene.

[0221] In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of at least about 0.1 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.5 nmol/h/le9 cells, at least about 0.6 nmol/h/le9 cells, at least about 0.7 nmol/h/le9 cells, at least about 0.8 nmol/h/le9 cells, at least about 0.9 nmol/h/le9 cells, at least about 1.0 nmol/h/le9 cells , at least about 1.1 nmol/h/le9 cells at least about 1.2 nmol/h/le9 cells, at least about 1.3 nmol/h/le9 cells, at least about 1.4 nmol/h/le9 cells, or at least about 1.5 nmol/h/le9 cells in vitro. In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of at least about 0.5 nmol/h/le9 cells in vitro.

[0222] In some embodiments, the bacterium has an lithocholic acid (LCA) sulfonation rate of about 0.1 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.2 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.5 nmol/h/le9 cells to about 0.6 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.6 nmol/h/le9 cells to about 0.7 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.7 nmol/h/le9 cells to about 0.8 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 0.8 nmol/h/le9 cells to about 0.9 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 0.9 nmol/h/le9 cells to about 1.0 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 1.0 nmol/h/le9 cells to about 1.1 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.1 nmol/h/le9 cells to about 1.2 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, about 1.2 nmol/h/le9 cells to about 1.3 nmol/h/le9 cells, about 1.3 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells, about 1.3 nmol/h/le9 cells to about 1.4 nmol/h/le9 cells, or about 1.4 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells in vitro.

[0223] In some embodiments, the bacterium has an LCA sulfonation rate of about 0.5 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells.

[0224] In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.001 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, or about 0.001 nmol/hr/le9 cells to about 0.002 nmol/hr/le9 cells.

[0225] In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells. In some embodiments, the bacterium has a CA sulfonation rate of about 0.001 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells, or about 0.004 nmol/hr/le9 cells. In some embodiments, the bacterium has a CA sulfonation rate of about 0.003 nmol/hr/le9 cells.

[0226] In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.002 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.002 nmol/hr/le9 cells to about 0.003 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells to about 0.004 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells to about 0.005 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells, or about 0.006 nmol/hr/le9 cells to about 0.007 nmol/hr/le9 cells. In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.004 nmol/hr/le9 cells to about 0.006 nmol/hr/le9 cells. In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.002 nmol/hr/le9 cells, about 0.003 nmol/hr/le9 cells, about 0.004 nmol/hr/le9 cells, about 0.005 nmol/hr/le9 cells, about 0.006 nmol/hr/le9 cells, or about 0.007 nmol/hr/le9 cells. In some embodiments, the bacterium has a CDCA sulfonation rate of about 0.005 nmol/hr/le9 cells.

B, Transporters of Bile acids

[0227] Bile acid transporters, or importers, may be expressed or modified in the recombinant bacteria described herein in order to enhance bile acid transport into the cell. Specifically, when the bile acid transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more bile acid 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 bile acid transporter which may be used to import bile acids into the bacteria so that any gene encoding a bile acid catabolism enzyme expressed in the organism can catabolize the bile acid to treat a disease associated with bile acid metabolism, such CD or UC.

[0228] E. coli intrinsically internalize a variety of BA. Porins facilitate BA transport across the outer membrane, while passive diffusion across the inner membrane is believed to be responsible for intracellular accumulation (Thanassi, D. G., Cheng, L. W. & Nikaido, H. Active efflux of bile salts by Escherichia coli. J Bacteriol 179, 2512-2518, doi: 10.1128/jb.179.8.2512-2518.1997 (1997)). BA- specific transporters have been characterized that may allow for increased intracellular BA transport into EcN (Zhou, X. et al. Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505, 569-573).

[0229] The uptake of bile acids into bacterial cells is mediated by proteins well known to those of skill in the art. At least 2 homologs of human ASBT of bacterial origin have been identified, a ASBT homolog from Neisseria meningitidis (ASBTNM) and a ASBT homolog from Yersinia frederiksenii (ASBTYf). (Hu et al., Nature. 2011 Oct 5; 478(7369): 408-411 and Zhou et al., Nature. 2014 Jan 23; 505(7484): 569-573.) In another example, an inducible bile acid transporter from Eubacterium sp. strain VPI 12708, expressed from the baiG gene has been cloned and characterized (Mallonee and Hylemon, J Bacteriol 178, 7053-7058 (1996)).

[0230] In some embodiments, the bile acid transporter is an ASBT homolog. In some embodiments, the ASBT homolog is from Neisseria meningitidis (ASBTNM). In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 502. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 502. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 502. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 502. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 502. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 502.

[0231] In one embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 503. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 503. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 503. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 503. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 503. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 503.

[0232] In some embodiments, the bile acid transporter is an ASBT homolog from Yersinia frederiksenii (ASBTYf). In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 504. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 504. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 504. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 504. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 504. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 504.

[0233] In one embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 505. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 505. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 505. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 505. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 505. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 505.

[0234] In some embodiment, the bile acid transporter is an ASBT homolog from Escherichia coli. In some embodiments, the bile acid transporter is an ASBT homolog from Escherichia coli M34. In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 506. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 506. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 506. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 506. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 506. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 506. [0235] In one embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 507. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 507. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 507. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 507. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 507. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 507.

[0236] In some embodiments, the bile acid transporter is an ASBT homolog from Escherichia coli VREC0334. In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 508. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 508. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 508. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 508. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 508. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 508.

[0237] In one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 509. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 509. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 509. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 509. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 509. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 509.

[0238] In some embodiment, the bile acid transporter is an from Escherichia coli. In some embodiments, the bile acid transporter is an ketopantoate/pantoate/pantothenate transporter PanS, e.g., from Escherichia coli EC23 . In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 510. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 510. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 510. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 510. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 510. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 510.

[0239] In one embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 511. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 511. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 511. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 511. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 511. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 511.

[0240] In one embodiment, the bile acid transporter is BaiG or a homolog thereof. In some embodiments, the BaiG is from Euhacterium sp. strain VPI 12708. In some embodiments, the gene encoding the bile acid transporter has at least about 80% identity with the sequence of SEQ ID NO: 512. Accordingly, in one embodiment, and the gene encoding the bile acid transporter has at least about 90% identity with the sequence of SEQ ID NO: 512. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 95% identity with the sequence of SEQ ID NO: 512. Accordingly, in one embodiment, the gene encoding the bile acid transporter has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 512. In another embodiment, the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 512. In yet another embodiment the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 512.

[0241] In one embodiment, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, encodes a polypeptide that has at least about 80% identity with the sequence of SEQ ID NO: 513. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 90% identity with the sequence of SEQ ID NO: 513. Accordingly, in one embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least about 95% identity with the sequence of SEQ ID NO: 513. In some embodiments, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 513. In another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that comprises the sequence of SEQ ID NO: 513. In yet another embodiment, the gene sequence encoding a bile acid transporter, e.g., an ASBT analog, encodes a polypeptide that consists of the sequence of SEQ ID NO: 513.

[0242] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a bile acid transporter, such that the bile acid transporter 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 acid transporter. In some embodiments, the gene encoding the bile acid transporter 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 acid transporter is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of bile acid transporter. In some embodiments, the gene encoding the bile acid transporter is expressed on a chromosome.

C. Exporters of Bile acids

[0243] Bile acid exporters may be modified in the recombinant bacteria described herein in order to reduce bile acid export from the cell, e.g. , of certain secondary bile acids from the cell. Specifically, when the recombinant bacterial cells described herein comprise a genetic modification that reduces export of a bile acid, e.g., LCA and/or CA, the bacterial cells retain more of this bile acid 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 the bile acid may be used to retain more bile acid, e.g. , LCA and/or CA in the bacterial cell so that any bile acid catabolism enzyme expressed in the organism, e.g., co-expressed bile acid catabolism enzyme, can catabolize the bile acid. D. Inducible Promoters

[0244] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the polypeptides disclosed herein, e.g., sulfotransferase(s), bile acid transporter(s), and/or sulfate transporter(s) , such that the polypeptides 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 sulfotransferase(s), genes or operons, e.g., two or more genes or operons. In some embodiments, bacterial cell comprises three or more distinct sulfotransferase gene(s) or operons, e.g., three or more sulfotransferase gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct sulfotransferase gene(s) or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more sulfotransferase genes.

[0245] In some embodiments, bacterial cell comprises three or more distinct bile acid transporter gene(s), and/or sulfate transporter gene(s) or operons, e.g., three or more bile acid transporter gene(s), and/or sulfate transporter gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct bile acid transporter genes, and/or sulfate transporter gene(s)or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more s bile acid transporter gene(s), and/or sulfate transporter gene(s).

[0246] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same sulfotransferase bile acid transporter, and/or sulfate transporter gene(s). In some embodiments, the gene encoding a polypeptide described herein, e.g., sulfotransferase, bile acid transporter, and/or sulfate transporter, is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline, arabinose or Isopropyl B-D-l -thiogalactopyranoside (IPTG).

[0247] In some embodiments, the inducible promoter is a IPTG inducible promoter. In one embodiment, the IPTG inducible promoter comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 17. In some embodiments, the recombinant bacterium further comprises a gene sequence encoding a repressor of the Lac promoter. In some embodiments, the gene sequence encoding a repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 15. In some embodiments, the repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 16.

Table 3: IPTG inducible promoter and Lad sequences

[0248] In some embodiments, the promoter that is operably linked to the gene encoding a polypeptide described herein, e.g., sulfotransferase, bile acid transporter, and/or sulfate transporter, is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding sulfotransferase, bile acid transporter, and/or sulfate transporter 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 one embodiment, the inducible promoter is an anhydrotetracycline (ATC)-inducible promoter. In one embodiment, the inducible promoter is an IPTG promoter. In one embodiment, the IPTG promoter is Ptac.

[0249] In certain embodiments, the bacterial cell comprises a gene encoding a polypeptide described herein, e.g., sulfotransferase, bile acid transporter, and/or sulfate transporter, is 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 al., 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.

Table 4

[0250] In one embodiment, the FNR responsive promoter comprises SEQ ID NO: 1. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 2. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 3. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 4. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 5.

[0251] 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 sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter 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.

[0252] In some embodiments, the bacterial cell comprises an oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter from a different bacterial species. The heterologous oxygen-level dependent transcriptional regulator and promoter increase the transcription of genes operably linked to said promoter, e.g., the gene encoding the sulfotransferase, bile acid transporter, and/or sulfate transporter, 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 al., 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.

[0253] 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 sulfotransferase, bile acid transporter, and/or sulfate transporter, 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 sulfotransferase, bile acid transporter, and/or sulfate transporter, 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 al., (2006).

[0254] 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 sulfotransferase, bile acid transporter, and/or sulfate transporter are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the sulfotransferase, bile acid transporter, and/or sulfate transporter. In some embodiments, the transcriptional regulator and the sulfotransferase, bile acid transporter, and/or sulfate transporter are divergently transcribed from a promoter region.

[0255] 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 encoding a sulfotransferase, bile acid transporter, and/or sulfate transporter gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gene(s) integrated into the chromosome allows for greater production of sulfotransferase, bile acid transporter, and/or sulfate transporter 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.

E. Temperature dependent regulation

[0256] In some instances, thermoregulators may be advantageous because of strong transcriptional control without the use of external chemicals or specialized media. Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage X promoters have been used to engineer recombinant bacterial strains. For example, a gene of interest cloned downstream of the X promoters can be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage X. At temperatures below 37°C, cI857 binds to the oL or oR regions of the pR promoter and inhibits 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. In certain instances, it may be advantageous to reduce, diminish, or shut off production of one or more protein(s) of interest. This can be done in a thermoregulated system by growing a bacterial strain at temperatures at which the temperature regulated system is not optimally active. Temperature regulated expression can then be induced as desired by changing the temperature to a temperature where the system is more active or optimally active.

[0257] For example, a thermoregulated promoter may be 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. Bacteria comprising gene sequences or gene cassettes either indirectly or directly operably linked to a temperature sensitive system or promoter may, for example, could be induced by temperatures between 37°C and 42°C. In some instances, the cultures may be grown aerobically. Alternatively, the cultures are grown anaerobically.

[0258] In some embodiments, the bacteria described herein comprise one or more gene sequence(s) or gene cassette(s) which are directly or indirectly operably linked to a temperature regulated promoter. In some embodiments, the gene sequence(s) or gene cassette(s) are induced in vitro during growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the gene sequence(s) are induced upon or during in vivo administration. In some embodiments, the gene sequence(s) are induced during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration and upon or during in vivo administration. In some embodiments, the genetically engineered bacteria further comprise gene sequence (s) encoding a transcription factor which is capable of binding to the temperature sensitive promoter. In some embodiments, the transcription factor is a repressor of transcription.

[0259] In one embodiment, the thermoregulated promoter is operably linked to a construct having gene sequence(s) or gene cassette(s) encoding 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 is induced under a first set of exogenous conditions, and the second promoter is induced 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 or IPTG). 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, one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with an oxygen regulated promoter, e.g., FNR, driving the expression of the same gene sequence(s).

[0260] 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.

[0261] 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 to, comprises, or consists of SEQ ID NO: 19. 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 to, comprises, or consists of SEQ ID NO: 22. 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 to, comprises, or consists of SEQ ID NO: 25. 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 to, comprises, or consists of SEQ ID NO: 20. 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 to, comprises, or consists of SEQ ID NO: 21. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI38 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 to, comprises, or consists of SEQ ID NO: 23. 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 to, comprises, or consists of the polypeptide encoded by SEQ ID NO: 24. 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 to, comprises, or consists of thepolypeptide encoded by SEQ ID NO: 25

[0262] SEQ ID NOs: 19-25 are shown in Table 5.

Table 5: Inducible promoter construct sequences and related elements

F, Phage Deletion

[0263] In some embodiments, the genetically engineered bacteria comprise one or more E. coli Nissle bacteriophage, e.g., Phage 1, Phage 2, and Phage 3. In some embodiments, the genetically engineered bacteria comprise one or mutations in Phage 3. Such mutations include deletions, insertions, substitutions and inversions and are located in or encompass one or more Phage 3 genes. In some embodiments, the one or more insertions comprise an antibiotic cassette. In some embodiments, the mutation is a deletion. In some embodiments, the genetically engineered bacteria comprise one or more deletions, which are located in or comprise one or more genes selected from ECOLIN_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN_09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_09995, ECOLINJOOOO, ECOLIN_10005, ECOLINJOOIO, ECOLIN_10015, ECOLIN_10020, ECOLIN_10025, ECOLIN_10030, ECOLIN_10035, ECOLIN_10040, ECOLIN_10045, ECOLIN_10050, ECOLIN_10055, ECOLIN_10065, ECOLIN_10070, ECOLIN_10075, ECOLIN_10080, ECOLIN_10085, ECOLIN_10090, ECOLIN_10095, ECOLINJOIOO, ECOLIN_10105, ECOLINJOl lO, ECOLIN_10115, ECOLIN_10120, ECOLIN 10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, ECOLIN_10175, ECOLIN_10180, ECOLIN_10185, ECOLIN_10190, ECOLIN_10195, ECOLIN_10200, ECOLIN_10205, ECOLIN_10210, ECOLIN_10220, ECOLIN_10225, ECOLIN_10230, ECOLIN 10235, ECOLIN_10240, ECOLIN_10245, ECOLIN_10250, ECOLIN_10255, ECOLIN_10260, ECOLIN_10265, ECOLIN_10270, ECOLIN_10275, ECOLIN_10280, ECOLIN_10290, ECOLIN_10295, ECOLIN_10300, ECOLIN_10305, ECOLIN_10310, ECOLIN 10315, ECOLIN 10320, ECOLIN 10325, ECOLIN 10330, ECOLIN 10335, ECOLIN_10340, and ECOLIN_10345. In one embodiment, the genetically engineered bacteria comprise a complete or partial deletion of one or more of ECOLIN 10110, ECOLIN 10115, ECOLIN_10120, ECOLIN 10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175. In one specific embodiment, the deletion is a complete deletion of ECOLIN_10110, ECOLIN 10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, and ECOLIN 10170, and a partial deletion of ECOLIN 10175. In one embodiment, the sequence of SEQ ID NO: 292 is deleted from the Phage 3 genome. In one embodiment, a sequence comprising SEQ ID NO: 292 is deleted from the Phage 3 genome.

G. Colibactin Island (also known as pks island)

[0264] In some embodiments, the engineered bacterium further comprises a modified pks island (colibactin island). Non-limiting examples are described in PCT/US2021/061579, the contents of which are herein incorporated by reference in their entirety. Colibactin is a cyclomodulin that is synthetized by enzymes encoded by the pks genomic island. See Fais 2018. The pks genomic island is “highly conserved” in Enterobactericicecie. Id. In Escherichia coli, a 54-kilobase pks genomic island contains 19 genes, clbA to clbS, and encodes various enzymes that have been described as an “assembly line responsible for colibactin synthesis.” Id. The pks genomic island assembly line for colibactin synthesis includes three polyketide synthases (ClbC, Clbl, ClbO), three non-ribosomal peptide synthases (ClbH, ClbJ, ClbN), two hybrid non-ribosomal peptide/polyketide synthases (ClbB, ClbK), and nine accessory, tailoring, and editing proteins. The polyketide synthases, non-ribosomal peptide synthases, and hybrid enzymes “are usually organized in mega-complexes as an assembly line, in which the synthesized compound is transferred from one enzymatic module to the following one.” Id. Colibactin undergoes a prodrug activation mechanism that incorporates an N-terminal structural motif, which is removed during the final stage of biosynthesis.

[0265] In some embodiments, the engineered microorganism, e.g., engineered bacterium, comprises a modified pks island (colibactin island). In some embodiments, the engineered microorganism, e.g., engineered bacterium, comprises a modified clb sequence selected from one or more of the clb A, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbl, clb J, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbR, and clbS gene sequences, as compared to a suitable control, e.g., the native pks island in an unmodified bacterium of the same strain and/or subtype. In some embodiments, the modified clb sequence is an insertion, a substitution, and/or a deletion as compared to the control. In some embodiments, the modified clb sequence is a deletion of the clb island, e.g., clb A, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbl, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbR, and clbS. In one embodiment, the colibactin deletion is the whole island except for the clbS gene, e.g., a deletion of clb A, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbl, clb J, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, and clbR.

[0266] In some embodiments, the modified endogenous colibactin island comprises one or more modified clb sequences selected from clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), c/W (SEQ ID NO: 302), clbJ (SEQ ID NO: 303), clbK (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), clbR (SEQ ID NO: 311), or clbS (SEQ ID NO: 312) gene. In some embodiments, the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), c//?./ (SEQ ID NO: 303), clbK (SEQ ID NO: 304), clbL (SEQ ID NO: 305), clbM (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), and clbR (SEQ ID NO: 311).

Essential Genes and Auxotrophs

[0267] As used herein, the term “essential gene” refers to a gene which 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 al., 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).

[0268] 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.

[0269] 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. 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 an oligonucleotide synthesis gene, for example, thyA. 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. 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 thimidylate 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).

[0270] 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. 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).

[0271] In other embodiments, the genetically engineered bacterium of the present disclosure is a lira A 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 al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. 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).

[0272] 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 comprise a deletion or mutation in two or more genes required for cell survival and/or growth.

[0273] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, 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, ligA, 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, yffB, 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, fab A, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, rib A, 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.

[0274] 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).

[0275] 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, dnaN, tyrS, metG and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN 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.

[0276] 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 (Hl 9 IN, R240C, 131 IS, 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.

[0277] 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). [0278] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.

[0279] 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 recombinant 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 Synthetic Biology (2015) 4: 307-16, 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 (see Wright et al., supra).

Isolated Plasmids

[0280] In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a sulfotransferase, bile acid transporter, and/or sulfate transporter 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 sulfotransferase, bile acid transporter, and/or sulfate transporter. 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. [0281] 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.

Integration

[0282] 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 gene (for example, a sulfotransferase, bile acid transporter, and/or sulfate transporter gene) or gene cassette (for example, a gene cassette comprising a sulfotransferase, and a bile acid transporter, and/or sulfate transporter may be integrated into the bacterial chromosome. Having multiple copies of the gene or gene cassette integrated into the chromosome allows for greater production of the gene of interest, e.g., bile acid transporter, and/or sulfate transporter, and other enzymes of the gene cassette, 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.

[0283] In one non-limiting example, the sulfotransferase and a bile acid transporter and/or sulfate transporter genes are integrated to facilitate bile acid import and metabolism.

Pharmaceutical Compositions and Formulations

[0284] Pharmaceutical compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent a disorder associated with gut inflammation. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[0285] Pharmaceutical compositions comprising the genetically engineered microorganisms of the invention may be used to treat, manage, ameliorate, and/or prevent a disorder associated with amino acid catabolism or symptom(s) associated with diseases or disorders associated with amino acid catabolism. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, and/or one or more genetically engineered virus, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[0286] 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 acid catabolism enzyme alone or in combination with a transporter described herein. 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 acid catabolism enzyme, e.g., a sulfotransferase.

[0287] The pharmaceutical compositions of the invention 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 tableting, 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.

[0288] The genetically engineered microorganisms 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, intravenous, sub-cutaneous, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 104 to 1012 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 on embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[0289] 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.

[0290] The genetically engineered microorganisms may be administered intravenously, e.g. , by infusion or injection. [0291] The genetically engineered microorganisms of the disclosure may be administered intrathecally. In some embodiments, the genetically engineered microorganisms of the invention may be administered orally. The genetically engineered microorganisms 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.

[0292] The genetically engineered microorganisms 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.

[0293] 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., lactose, 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.

[0294] In some embodiments, the genetically engineered microorganisms 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.

[0295] 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); non-aqueous 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 microorganisms described herein.

[0296] In one embodiment, the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to adult subjects or 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-

-TI- 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.

[0297] 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.

[0298] 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.

[0299] In certain embodiments, the genetically engineered microorganisms 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.

[0300] 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. For example, see U.S. 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.

[0301] 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.

[0302] The genetically engineered microorganisms 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.

[0303] The genetically engineered microorganisms 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).

[0304] 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.

[0305] 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.

[0306] 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, polyethylene 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.

[0307] 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.

[0308] 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. [0309] 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.

[0310] In some embodiments, the genetically engineered viruses are prepared for delivery, taking into consideration the need for efficient delivery and for overcoming the host antiviral immune response. Approaches to evade antiviral response include the administration of different viral serotypes as part of the treatment regimen (serotype switching), formulation, such as polymer coating to mask the virus from antibody recognition and the use of cells as delivery vehicles.

[0311] 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, polyethylene 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.

[0312] 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

[0313] Another aspect of the disclosure provides methods of treating diseases and disorders, e.g., autoimmune disorders, diarrheal diseases, IBD, related diseases, metabolic diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the disclosure provides for the use of at least one recombinant species, strain, or subtype of bacteria described herein for the manufacture of a medicament. In some embodiments, the disclosure provides for the use of at least one recombinant species, strain, or subtype of bacteria described herein for the manufacture of a medicament for treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the disclosure provides at least one recombinant species, strain, or subtype of bacteria described herein for use in treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function.

[0314] In some embodiments, the diarrheal disease is selected from the group consisting of acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g., dysentery, and persistent diarrhea. In some embodiments, the IBD or related disease is selected from the group consisting of Crohn’s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet’s disease, intermediate colitis, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis. In some embodiments, the disease or condition is an autoimmune disorder selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal & neuronal neuropathies, Balo disease, Behcet’s disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogan’s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), discoid lupus, Dressier’s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture’s syndrome, granulomatosis with polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis juvenile arthritis uvenile idiopathic arthritis juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere’s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic’s), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud’s phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s granulomatosis. 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 diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, and inflammation of the skin, eyes, joints, liver, and bile ducts. In some embodiments, the disclosure provides methods for reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing a systemic autoimmune disorder, e.g., asthma (Arrieta et al., 2015).

[0315] In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjcson-Forsmann-Lchmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. 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 weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility.

[0316] In some embodiments, the disease or disorder is cholestasis or a disease causing cholestasis. In some embodiments, the disease or disorder is nonalcoholic steatohepatitis (NASH), Nonalchoholic fatty liver disease (NAFLD, biliary atresis, parenteral nutrition-associated cholestasis (PNAC), Gall bladder disease, Alagille syndromes, primary sclerosing cholangitis (PSC), Progressive familial intrahepatic cholestasis (PFIC), or bile acid synthetic defects.

[0317] In some embodiments, the diseases or disorder is one or more genetic defects related to bile acid imbalance. In some embodiments, the disease or disorder is Progressive familial intrahepatic cholestasis (PFIC), or bile acid synthetic defects.

[0318] In some embodiments, the disease or disorder is a disease in which bile acid exacerbates the condition. In some embodiments, the disease or disorder is inflammatory bowel disease (IBD), bile acid diarrhea, liver cancers, GI cancers, portal hypertension, nonalcoholic steatohepatitis (NASH), Nonalchoholic fatty liver disease (NAFLD, biliary atresis, parenteral nutrition-associated cholestasis (PNAC), Gall bladder disease, Alagille syndromes, primary sclerosing cholangitis (PSC), Progressive familial intrahepatic cholestasis (PFIC), or bile acid synthetic defects.

[0319] In some embodiments, the disease is small bowel disease or ileitis. [0320] In some embodiments, the subject to be treated is a human patient. [0321] The method may comprise preparing a pharmaceutical composition with at least one recombinant 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 recombinant bacteria are administered orally, e.g., in a liquid suspension. In some embodiments, the recombinant bacteria are lyophilized in a gel cap and administered orally. In some embodiments, the recombinant bacteria are administered via a feeding tube or gastric shunt. In some embodiments, the recombinant bacteria are administered rectally, e.g., by enema. In some embodiments, the recombinant bacteria are administered topically, intraintestinally, intrajej unally, intraduodenally, intraileally, and/or intracolically.

[0322] In certain embodiments, the recombinant bacteria described herein are administered to treat, manage, ameliorate, or prevent metabolic diseases in a subject. In some embodiments, the method of treating or ameliorating metabolic diseases allows one or more symptoms of the disease to improve by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the symptom (e.g., obesity, insulin resistance) is measured by comparing measurements in a subject before and after administration of the recombinant bacteria. In some embodiments, the subject is a human subject.

[0323] Before, during, and after the administration of the recombinant bacteria in a subject, metabolites level, metabolic symptoms and manifestations may be measured in a biological sample, e.g., blood, serum, plasma, urine, fecal matter, peritoneal fluid, a sample collected from a tissue, such as liver, skeletal muscle, pancreas, epididymal fat, subcutaneous fat, and beige fat. The biological samples may be analyzed to measure symptoms and manifestations of metabolic diseases. Useful measurements include measures of lean mass, fat mass, body weight, food intake, GLP-1 levels, endotoxin levels, insulin levels, lipid levels, HbAlc levels, short-chain fatty acid levels, triglyceride levels, and nonesterified fatty acid levels. Useful assays include, but are not limited to, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests, assays for intestinal permeability, and assays for glycaemia upon multiple fasting and refeeding time points. In some embodiments, the methods may include administration of the compositions to reduce metabolic symptoms and manifestations to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions to reduce metabolic symptoms and manifestations to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject’s levels prior to treatment.

[0324] The recombinant bacteria may be administered alone or in combination with one or more additional therapeutic agents, e.g., insulin. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the recombinant bacteria, e.g. , the agent(s) must not kill the bacteria. The dosage of the recombinant bacteria 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 vivo

[0325] The bacteria of the disclosure may be evaluated in vivo, e.g. , in an animal model. Any suitable animal model of a disease or condition associated with gut inflammation, compromised gut barrier function, and/or an autoimmune disorder may be used (see, e.g., Mizoguchi, 2012). The animal model may be a mouse model of IBD, e.g., a CD45RBHi T cell transfer model or a dextran sodium sulfate (DSS) model. The animal model may be a mouse model of type 1 diabetes (T1D), and T1D may be induced by treatment with streptozotocin.

[0326] Colitis is characterized by inflammation of the inner lining of the colon, and is one form of IBD. In mice, modeling colitis often involves the aberrant expression of T cells and/or cytokines. One exemplary mouse model of IBD can be generated by sorting CD4+ T cells according to their levels of CD45RB expression, and adoptively transferring CD4+ T cells with high CD45RB expression from normal donor mice into immunodeficient mice. Non-limiting examples of immunodeficient mice that may be used for transfer include severe combined immunodeficient (SCID) mice (Morrissey et al., 1993; Powrie et al., 1993), and recombination activating gene 2 (RAG2) -deficient mice (Corazza et al., 1999). The transfer of CD45RBHi T cells into immunodeficient mice, e.g., via intravenous or intraperitoneal injection, results in epithelial cell hyperplasia, tissue damage, and severe mononuclear cell infiltration within the colon (Byrne et al. , 2005; Dohi et al. , 2004; Wei et al. , 2005). In some embodiments, the bacteria of the disclosure may be evaluated in a CD45RBHi T cell transfer mouse model of IBD.

[0327] Another exemplary animal model of IBD can be generated by supplementing the drinking water of mice with dextran sodium sulfate (DSS) (Martinez et al. , 2006; Okayasu et al. , 1990; Whittem et al., 2010). Treatment with DSS results in epithelial damage and robust inflammation in the colon lasting several days. Single treatments may be used to model acute injury, or acute injury followed by repair. Mice treated acutely show signs of acute colitis, including bloody stool, rectal bleeding, diarrhea, and weight loss (Okayasu et al., 1990). In contrast, repeat administration cycles of DSS may be used to model chronic inflammatory disease. Mice that develop chronic colitis exhibit signs of colonic mucosal regeneration, such as dysplasia, lymphoid follicle formation, and shortening of the large intestine (Okayasu et al., 1990). In some embodiments, the bacteria of the disclosure may be evaluated in a DSS mouse model of IBD.

[0328] Alternatively, a Trinitrobenzenesulfonic acid (TNBS) model can be used, leads to the development of an excessive cell mediated immune response reflected by acute Thl inflammation, including a dense colonic tissue infiltration by CD4 T cells and the secretion of various potent pro- inflammatory cytokines, including TNF-alpha and IL-12 (Antoniou et al., Ann Med Surg (Lond). 2016 Nov; 11: 9-15).

[0329] Another suitable IBD model is the IL-10 KO mouse (Cell. 1993 Oct 22;75(2):263- 74). Additionally mice with apyrase deficiency CD39 KO mice or ENTPD8 KO mice may be used. Another suitable model is a.Entpd8 deficient mouse model (Tani et al., PNAS 2021 Vol. 118 No. 39 e2100594118 and Gut. 2022 Jan;71(l):43-54; the contents of which are herein incorporated by reference in its entirety). Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis. In these mice, ATP suppressed apoptosis by inducing metabolic alteration toward glycolysis via P2X4R in neutrophils, causing prolonged survival and elevated reactive oxygen species production in these cells (Tani et al.) This model may be used to assess the effects of administration of a genetically engineered bacterium overexpressing an ATP catabolizing enzyme, e.g., a soluble form of ENTPD8, e.g., in a DSS model.

[0330] In some embodiments, the bacterium of the disclosure is administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by endoscopy, colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. In some embodiments, the animal is sacrificed, and tissue samples are collected and analyzed, e.g., colonic sections are fixed and scored for inflammation and ulceration, and/or homogenized and analyzed for myeloperoxidase activity and cytokine levels (e.g., IL-1J3, TNF-a, IL-6, IFN-y and IL-10).

Examples

[0331] 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.

Example 1: Generation of E. coli Nissle strains that sulfonate primary and secondary bile acids [0332] The cDNA sequence of the bile acid sulfotransferase from human, SULT2A1, or mouse, SULT2A8, was sourced from NCBI sequence database, codon optimized and synthesized (IDT). The synthesized SULT2A1 or SULT2A8 DNA fragment was cloned via Gibson assembly into a pl 5a medium copy plasmid, fused to an IPTG-inducible pTac promoter and ribosome binding site, and to generate Logic2868 or Logic2909 respectively. Logic2868 or Logic2909 were then transformed into E. coli Nissle to generate strains SYN8876 and SYN8965 (Table 6). Expression of SULT2A1 from SYN8876 was confirmed via immunoblotting using an anti-SULT2Al antibody (R&D Systems MAB5828).

[0333] To create a genetic background that enhances the activity of SULT proteins, a strain was engineered to increase the PAPS cofactor and sulfur donor of the enzyme. Deleting the PAPS reductase gene (cysH) gene from EcN using lambda red recombination, allows PAPS to accumulate in the EcN resulting in more readily available sulfur donor presence. The resulting chassis strain is designated SYN8977, and the introduction of Logic2868 or Logic2909 into this strain generates SYN8978 and SYN9018 respectively. CysQ was also deleted from EcN using lambda red as an alternate strategy to attempt to increase PAPS concentration and therefore SULT activity. The resulting chassis strain was designated SYN9074 and the introduction of Logic2868 or Logic2909 into this chassis generates SYN9087 and SYN9088 respectively (Table 6).

[0334] To enhance import of bile acid substrates into the E. coli Nissle prototype cells, the sequence of the Apical Sodium dependent Bile acid Transporter homolog from Yersinia frederiksenii (Y f-ASBT) was sourced from the NCBI sequence database, codon optimized for E. coli expression, and synthesized (IDT). The synthesized Yf-ASBT DNA fragment was cloned using Gibson assembly into an expression vector containing a pSClOl origin of replication, an ampicillin resistance cassette, and a pTac promoter and RBS to generate Logic2887. Introduction of Logic2887 into strain SYN8978 yielded a new strain, SYN9056 that encodes both the SULT2A1 and Yf-ASBT recombinant proteins (Table 1). Similarly, the following putative bile acid transporters were sourced from the NCBI sequence database: an ASBT homolog from Neisseria menigitidis (Nm-ASBT), a homolog of Nm- ASBT identified in E. coli using BlastP (Ec-ASBT), a homolog of Yf-ASBT identified in E. coli using BlastP (PanS), and the bile acid importer BaiG from Clostridium scindens. These were codon optimized for expression in E. coli, synthesized (IDT) and cloned into the above expression vector under control of the IPTG inducible promoter pTac, using Gibson assembly, to generate Logic2888, Logic2889, Logic2891, and Logic2892 respectively. Introduction of these plasmids into SYN8978 yielded new strains expressing both SULT2A1 and putative bile acid importers SYN9057, SYN9058, SYN9059, and SYN9060 respectively.

Table 6. Strains

Example 2. Functional Assays demonstrating 3’ position sulfonation of primary and secondary bile acids via E. coli Nissle strains engineered to express SULT2A1 (EcN-SULT2Al) alone or in combination with a putative bile acid transporter

[0335] For in vitro studies, strains were grown in LB media overnight (~ 18h) at 37°C, back- diluted in LB mediate a starting OD-0.05, grown at 37° C for 2h, and then grown for an additional 4h with the addition of ImM IPTG to induce protein expression.

[0336] Alternatively, cells were back-diluted and grown in an Ambr250 bioreactor (Sartorius) at 37°C in rich media with glycerol, protein expression was induced with ImM IPTG starting at an OD600 of 3.0, then cells were grown for another 4h.

[0337] Cells were then pelleted, resuspended in phosphate buffer with glycerol and frozen for storage. Subsequently cells were thawed on ice, and in some cases, viability was measured by cellometer (Nexcelcom). Cells were then normalized by optical density (OD) at 600nm (ODeoo) and added to assay buffer (M9 minimal salts + 0.5% glucose) containing 15pM of bile acid substrate including one of the following; cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), tauro-lithocholic acid (TLCA), or glyco-lithocholic acid (GLCA). Strains were tested in duplicate and sulfonation was assessed over 3h. At several time points up to 3h, a sample of the reaction is quenched with the addition of 400 pL ice cold methanol. Samples are centrifuged at 15000 rpm for 5 min and supernatant collected for analysis. LCA and LCA-3 -sulfate were quantified by LCMS using established methods.

[0338] Results are shown in FIGs. 5-10.

[0339] Strains comprising plasmids encoding IPTG-inducible SULT2A1 protein with intact PAPS reductase gene (cysH) gene displayed barely detectable LCA sulfonation activity. Deletion of the endogenous cysH gene from EcN (SYN8978), boosted SULT2A1 (FIG. 5 and FIG. 6A) and SULT2A8 activity on CA (FIG. 6B). Addition of exogenous PAPS to the medium however did not further boost LCA sulfonation activity of the SULT2A1 expressing strain (SYN8876) or SYN8978 (AcysH SULT2A1) (FIG. 5).

[0340] CysQ was also deleted from EcN as an alternate strategy to attempt to increase PAPS concentration and therefore SULT activity, however, unlike with the cysH deletion, no boost of LCA- sulfonation activity was observed for SULT2A1 expressing strains and no CA sulfonation activity was observed for SULT2A8 expressing strains (FIG. 6A and FIG. 6B). In vitro SULT2A1 activity on LCA in the presence of the cysH deletion was approximately 0.5 nmol/h/le9 cells (FIG. 7A). SULT2A8 activity on CA in the presence of the cysH deletion was approaching 0.006 nmol/h/le9 cells (FIG. 7B).

[0341] LCA sulfonation activity in SULT2A1 expressing strains with added expression of various bile acid transporters was assessed using cultures grown in shake flasks: an ABST homolog from Yersinia frederiksenii or Neisseria meningitidis, two homologs of the bacterial ASBTs identified in E. coli via BlastP (NCBI), and the bile acid importer BaiG from Clostridium scindens. Expressing the transporter sourced from Y. frederiksenii results in the greatest increase of LCA sulfonation activity (FIG. 8A). SYN9056 (expressing SULT2A1 and ASBT from Y. frederiksenii) had a sulfonation rate of approximately 0.8 nmol/h/le9 cells (FIG. 8B).

[0342] Activity and viability of SULT2A1 expressing prototypes when grown in AMBR bioreactors is shown in FIG. 9A and 9B. When grown in the AMBR bioreactor, SYN9056 (expressing the ASBT transporter homolog) is less active than SYN8978 (expressing SULT2A1 without transporter). However, of note, SYN9056 displays greater viability than SYN8978 when grown in AMBR bioreactors. This is surprising because despite the additional engineering and expression of a transmembrane transporter, potentially causing more stress to the cell, the viability is increased. Often this type of additional engineering is neutral, or even potentially negative, with respect to viability, however, in this instance an improvement in viability was unexpectedly observed.

[0343] In vitro activity is not necessarily representative of in vivo activity. Given that SYN9056 performed better in the flask grown samples, and displayed strong activity, and greater viability in the AMBR batches, SYN9056 having the ASBT transporter was selected as a lead prototype for in vivo experiments.

[0344] Of note, EcN-SULT2Al strains +/- Yf-ASBT (SYN8978, SYN9056) have sulfonation activity across multiple substrates, including CA, CDCA, DCA, TLCA, GLCA, and LCA (FIG. 10A). Sulfonation rates are shown in FIG. 10B.

Example 3. Functional Assays demonstrating sulfonation of bile acids (CA-7-S)

[0345] For in vitro studies, strains were grown in LB media overnight (~ 18h) at 37°C, back- diluted in LB media to a starting OD-0.05, grown at 37° C for 2h, and then grown for an additional 4h with the addition of ImM IPTG to induce protein expression. Cells were then pelleted, resuspended in phosphate buffer with glycerol and frozen for storage. Cells are subsequently thawed on ice, normalized by ODeoo and added to assay buffer (M9 minimal salts + 0.5% glucose) containing 15pM of CA or CDCA. Strains were tested in duplicate and CA and CDCA sulfonation was assessed over 3h. At several time points up to 3h, a sample of the reaction was quenched with the addition of 400 pL ice cold methanol. Samples are centrifuged at 15000 rpm for 5 min and supernatant collected for analysis. CA, CA-7 -sulfate, CDCA, and CDCA -7 -sulfate were quantified by LCMS using established methods. Results are shown in FIG. 6B, and FIGs. 12A and 12B.

Example 4: In vivo D4-LCA sulfonation activity by EcN-SULT2Al prototype strains

[0346] Next, in vivo target engagement assessment of EcN-SULT2Al strains, including SYN9056 and SYN8978 (with an ASBT transporter and without respectively), specifically their in vivo sulfonation activity on lithocholic acid (LCA), was assessed in a mouse model. The study utilized ten weeks old male C57BL/6NTac mice as subjects. SYN9056 and SYN8978 were used for this investigation. [0347] To evaluate the target engagement, mice were administered a single dose of deuterium -labeled LCA (D4-LCA) mixed in equal proportion with unlabeled LCA (50 mg per kg of body weight each; D4-LCA DLM-9560, Cambridge Isotope Laboratories, MA; LCA, ULM-9559, Cambridge Isotope Laboratories, MA) through oral gavage. After 1 hour, the mice received a single dose of lelO cells of a EcN-SULT2Al strain, either SYN9056 or SYN8978.The control groups were D4-LCA-only treated mice and EcN-SULT2Al-only treated mice. The experimental design for the both D4-LCA/LCA and SYN9056 or SYN8978 dosed animals included three separate groups (n=5) of mice, which were euthanized at 1.5 hours (T1.5), 2 hours (T2), and 2.5 hours (T2.5) after administration of the D4-LCA/LCA dose.. Ileal luminal contents were collected in bead beating tubes and subsequently homogenized using mechanical disruption. LCMS-based quantification was performed on the samples to determine the levels of D4-LCA and D4-labeled lithocholic acid-3 - sulfate (D4-LCA-3S) as well as LCA and LCA-3S using established methodologies.

[0348] The results of the study demonstrated that EcN-SULT2Al administration mediated the sulfonation of orally administered D4-LCA into D4-LCA-3S, and LCA into LCA-3S, as evidenced by the detection of D4-LCA-3S and LCA-3S at all timepoints.

[0349] When comparing SYN9056 and SYN8978, a trend of greater D4-LCA-3S and LCA- 3S production was observed for SYN9056 relative to SYN8978. This indicates that SYN9056, having ASBT expression, and a more favorable viability profile, may display improved target engagement over SYN8978 in vivo. Results are shown in FIG. 11.

Table 7. Bile Acid Catabolism Enzymes

Table 8. Bile salt transporter polypeptide sequences

Table 9. Bile acid transporter polynucleotide sequence

-Ill-

Claims

1. A recombinant bacterium comprising:

(i) a heterologous gene encoding a bile acid catabolism enzyme, wherein the bile acid catabolism enzyme is a human SULT2A1 sulfotransferase or a human SULT2A8 sulfotransferase, and wherein the gene encoding the bile acid catabolism enzyme is operably linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature;

(ii) a heterologous gene encoding a bile acid transporter, wherein the bile acid transporter is an ASBT transporter from Yersinia frederiksenii, and wherein the gene encoding the bile acid transporter is operably linked to a promoter that is not associated with the gene encoding the bile acid transporter in nature; and

(iii) a knock-out of an endogenous cysH gene.

2. The recombinant bacterium of claim 1, wherein the bile acid catabolism enzyme catabolizes lithocholic acid (LCA) and/or cholic acid (CA).

3. The recombinant bacterium of any one of the previous claims, wherein the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are operably linked to different promoters, wherein the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are operably linked to different copies of the same promoter, or wherein the heterologous gene encoding the bile acid catabolism enzyme and the heterologous gene encoding the bile acid transporter are present in a gene cassette linked to the same promoter.

4. The recombinant bacterium of any one of the previous claims, wherein the promoter operably linked to the bile acid catabolism enzyme is an inducible promoter or a constitutive promoter; and/or wherein the promoter operably linked to the bile acid transporter is an inducible promoter or a constitutive promoter.

5. The recombinant bacterium of any one of the previous claims, wherein the promoter operably linked to the bile acid catabolism enzyme is induced by a chemical inducer; and/or wherein the promoter operably linked to the bile acid transporter is induced by a chemical inducer.

6. The recombinant bacterium of claim 5, wherein the chemical inducer is isopropylthio-beta- galactoside (IPTG).

7. The recombinant bacterium of any one of claims 1-4, wherein the promoter operably linked to the bile acid catabolism enzyme is induced by exogenous environmental conditions; and/or wherein the promoter operably linked to the bile acid transporter is induced by exogenous environmental conditions.

8. The recombinant bacterium of claim 7, wherein the promoter operably linked to the bile acid catabolism enzyme is induced by low- oxygen or anaerobic conditions; and/or wherein the promoter operably linked to the bile acid transporter is induced by low-oxygen or anaerobic conditions.

9. The recombinant bacterium of claim 8, wherein the promoter operably linked to the bile acid catabolism enzyme is an FNR-inducible promoter; and/or wherein the promoter operably linked to the bile acid transporter is an FNR-inducible promoter.

10. The recombinant bacterium of any one of claims 1-4, wherein the promoter operably linked to the bile acid catabolism enzyme is induced by temperature; and/or wherein the promoter operably linked to the bile acid transporter is induced by temperature.

11. The recombinant bacterium of claim 10, wherein the promoter operably linked to the bile acid catabolism enzyme is a cI857 promoter; and/or wherein the promoter operably linked to the bile acid transporter is a cI857 promoter.

12. The recombinant bacterium any one of the previous claims, wherein the human SULT2A1 sulfotransferase gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 500.

13. The recombinant bacterium of any one of the previous claims, wherein the gene sequence encoding the human SULT2A1 sulfotransferase encodes an polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 501.

14. The recombinant bacterium of any one of the previous claims, wherein the bile acid transporter gene comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 504.

15. The recombinant bacterium of any one of the previous claims, wherein the gene sequence encoding the bile acid transporter encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 505.

16. The recombinant bacterium of any one of the previous claims, wherein the gene encoding the bile acid catabolism enzyme is present on a plasmid in the recombinant bacterium.

17. The recombinant bacterium of any one of claims 1-16, wherein the gene encoding the bile acid catabolism enzyme is present on a chromosome in the recombinant bacterium.

18. The recombinant bacterium of any one of the previous claims, wherein the recombinant bacterium is a non-pathogenic bacterium.

19. The recombinant bacterium of any one of the previous claims, wherein the recombinant bacterium is a probiotic or a commensal bacterium.

20. The recombinant bacterium any one of the previous claims, wherein the recombinant bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus .

21. The recombinant bacterium of claim 20, wherein the recombinant bacterium is Escherichia coli strain Nissle.

22. The recombinant bacterium of any one of the previous claims, further comprising an insertion, deletion or mutation of an endogenous phage gene.

23. The recombinant bacterium of claim 22, wherein the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.

24. The recombinant bacterium of any one the previous claims, further comprising a modified endogenous colibactin island.

25. The recombinant bacterium of claim 24, wherein the modified endogenous colibactin island comprises one or more modified clb sequences selected from the group consisting of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), clbl (SEQ ID NO: 302), c//?./ (SEQ ID NO: 303), c//rA (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), c/WV(SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), clbR (SEQ ID NO: 311), and clbS (SEQ ID NO: 312).

26. The recombinant bacterium of claim 24 or claim 25, wherein the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 294), clbB (SEQ ID NO: 295), clbC (SEQ ID NO: 296), clbD (SEQ ID NO: 297), clbE (SEQ ID NO: 298), clbF (SEQ ID NO: 299), clbG (SEQ ID NO: 300), clbH (SEQ ID NO: 301), c/W (SEQ ID NO: 302), clbJ (SEQ ID NO: 303), clbK (SEQ ID NO: 304), clbL (SEQ ID NO: 305), c/ (SEQ ID NO: 306), clbN (SEQ ID NO: 307), clbO (SEQ ID NO: 308), clbP (SEQ ID NO: 309), clbQ (SEQ ID NO: 310), and clbR (SEQ ID NO: 311).

27. The recombinant bacterium of any one of the previous claims, wherein the recombinant bacterium is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.

28. The recombinant bacterium of claim 27, wherein the auxotrophy is a in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.

29. The recombinant bacterium of any one of the previous claims, wherein the bacterium has at least about 65% viability, at least about 70% viability, at least about 75% viability, at least about 80% viability, at least about 85% viability, at least about 90% viability, or at least about 95% viability.

30. The recombinant bacterium of any one of the previous claims, wherein the bacterium is capable of sulfonating chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), tauro-litocholic acid (TLCA), glycol-litocholic acid (GLCA), and/or lithocholic acid (LCA).

31. The recombinant bacterium of claim 30, wherein the bacterium is further capable sulfonating cholic acid (CA).

32. The recombinant bacterium of any one of the previous claims, wherein the bacterium has an lithocholic acid (LCA) sulfonation rate of at least about 0.5 nmol/h/le9 cells in vitro.

33. The recombinant bacterium of claim 32, wherein the bacterium sulfonates LCA at a rate of about 0.5 nmol/h/le9 cells to about 1.5 nmol/h/le9 cells.

34. The recombinant bacterium of any one of the previous claims, wherein the bacterium sulfonates CA at a rate of about 0.001 to about 0.004 nmol/hr/le9 cells.

35. The recombinant bacterium of claim 34, wherein the bacterium sulfonates CA at a rate of about 0.003 nmol/hr/le9 cells.

36. The recombinant bacterium of any one of the previous claims, wherein the bacterium sulfonates CDCA at a rate of about 0.004 to about 0.006 nmol/hr/le9 cells.

37. The recombinant bacterium of claim 36, wherein the bacterium sulfonates CDCA at a rate of about 0.005 nmol/hr/le9 cells.

38. A pharmaceutically acceptable composition comprising the recombinant bacterium of any one of the previous claims, and a pharmaceutically acceptable carrier.

39. The pharmaceutically acceptable composition of claim 38, wherein the composition is formulated for oral administration.

40. A method for decreasing a level of a bile acid in the gut of a subject, the method comprising a step of administering to the subject the pharmaceutical composition of claim 38 or claim 39, thereby decreasing the level of the bile acid in the gut of the subject.

41. A method of treating a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition of claim 38 or claim 39, thereby treating the disease or disorder.

42. The method of claim 41, wherein the disease or disorder is an autoimmune disease or an inflammatory disease or disorder

43. The method of claim 41, wherein the disease or disorder is a metabolic disease selected from the group consisting of liver disease; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); liver cirrhosis; obesity; type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjcson-Forsmann-Lchmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome.

44. The method of claim 42, wherein the disease or disorder selected an autoimmune disease selected from the group consisting of multiple sclerosis, central nervous system inflammation (CNS) inflammation, 2,4,6-trinitrobenzene sulfonic acid (TNBS) -induced colitis, T cell-induced colitis, T cell-induced small bowel inflammation, chronic colitis, rheumatoid arthritis, celiac disease, myasthenia gravis, and B-cell-mediated T-cell-dependent autoimmune disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal & neuronal neuropathies, Balo disease, Behcet’s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage -Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener’s disease.

45. The method of claim 42, wherein the disease or disorder is ulcerative colitis or Crohn’s disease.

46. A method of treating, reducing, or ameliorating symptoms of a disease or disorder in a subject in need thereof comprising the step of administering to the subject the pharmaceutical composition of claim 38 or claim 39, wherein the symptom of the disease or disorder is inflammation.

47. The method of any one of claims 40-46, wherein the subject has a decreased level of a secondary bile acid in the gut after the composition is administered.

48. The method of claim 47, wherein the secondary bile acid is lithocholic acid (LCA).

49. The method of any one of claims 40-46, wherein the subject has an decreased level of a primary bile acid in the gut after the composition is administered.

50. The method of claim 49, wherein the primary bile acid is cholic acid (CA).

51. The method of any one of claims 40-50, wherein the subject is a human.