WO2023245171A1 - Bacteria engineered to treat diseases associated with bile acid metabolism and methods of use thereof - Google Patents

Bacteria engineered to treat diseases associated with bile acid metabolism and methods of use thereof Download PDF

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WO2023245171A1
WO2023245171A1 PCT/US2023/068591 US2023068591W WO2023245171A1 WO 2023245171 A1 WO2023245171 A1 WO 2023245171A1 US 2023068591 W US2023068591 W US 2023068591W WO 2023245171 A1 WO2023245171 A1 WO 2023245171A1
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cells
nmol
bile acid
seq
recombinant bacterium
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PCT/US2023/068591
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French (fr)
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Jillian Marie MEANS
Douglas KENNY
Chengyi Jenny SHU
Afrand Kamali SARVESTANI
Michael James
Abhi SHAH
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Synlogic Operating Company, Inc.
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Publication of WO2023245171A1 publication Critical patent/WO2023245171A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01145—3-Beta-hydroxy-DELTA5-steroid dehydrogenase (1.1.1.145)
    • C—CHEMISTRY; METALLURGY
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01225—Chlordecone reductase (1.1.1.225)
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    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01003—DELTA4-3-oxosteroid 5-beta-reductase (1.3.1.3)
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    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01022—3-Oxo-5alpha-steroid 4-dehydrogenase (NADP+) (1.3.1.22), i.e. cortisone alpha-reductase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • IBDs Inflammatory bowel diseases
  • 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.
  • the recombinant bacteria are capable of catabolizing in low-oxygen environments, e.g., the gut of a subject.
  • 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).
  • the bacteria of the disclosure comprise one or more gene(s) encoding a bile acid catabolism enzyme(s) or a bile acid catabolism enzyme cassette(s).
  • the bile acid catabolism enzyme is a catabolizes a secondary bile acid, e.g., lithocholic acid (LCA).
  • the bile acid catabolism enzyme is a 3 -alpha-hydroxy steroid dehydrogenase, e.g., a bacterial 3-alpha-hydroxysteroid dehydrogenase.
  • the 3- alpha-hydroxysteroid dehydrogenase is from Eggerthella lenta.
  • the bile acid catabolism enzyme is a 5-alpha reductase, e.g., a bacterial 5-alpha reductase.
  • the 5-alpha reductase is from Bacteroides dorei DSM 17855.
  • the bile acid catabolism enzyme is a 5-beta reductase, e.g., a bacterial 5-beta reductase.
  • the 5-beta reductase is from Bacteroides dorei DSM 17855.
  • the bile acid catabolism enzyme is a 3-beta hydroxysteroid dehydrogenase, e.g. , a bacterial 3-beta hydroxysteroid dehydrogenase.
  • the 3- beta hydroxysteroid dehydrogenase is from Bacteroides dorei DSM 17855.
  • the bile acid catabolism enzyme cassette comprises 5-alpha reductase, the 5-beta reductase and 3-beta hydroxysteroid dehydrogenase.
  • the 5-alpha reductase, the 5-beta reductase and 3-beta hydroxysteroid dehydrogenase are all from Bacteroides dorei DSM 17855.
  • the bile acid catabolism enzyme catabolizes lithocholic acid (LCA). In some embodiments, the bile acid catabolism enzyme or bile acid catabolism enzyme cassette catabolizes 3-oxo lithocholic acid. In some embodiments, the bile acid catabolism enzyme increases levels of 3-oxo lithocholic acid. In some embodiments, the bile acid catabolism enzyme increases levels of isoallo lithocholic acid (isoalloLCA). In some embodiments, the bile acid catabolism enzyme increases levels of isolithocholic acid (isoLCA).
  • the disclosure provides for a recombinant bacterium comprising a gene encoding an bile acid catabolism enzyme, wherein the gene encoding the bile acid catabolism enzyme is operably linked to a that is not associated with the gene encoding the bile acid catabolism enzyme in nature.
  • the promoter is constitutive or is a directly or indirectly inducible promoter, which optionally is induced by exogenous environmental conditions.
  • the inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.
  • the inducible promoter is an FNR- inducible promoter.
  • the inducible promoter is induced by temperature.
  • the inducible promoter is a cI857 promoter.
  • 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.
  • 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.
  • the 3-alpha-hydroxysteroid dehydrogenase 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.
  • the gene sequence encoding an 3- alpha-hydroxysteroid dehydrogenase 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.
  • the 5-alpha reductase 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: 514.
  • the gene sequence encoding 5-alpha reductase 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: 515.
  • the 5-beta reductase 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.
  • the gene sequence encoding 5-beta reductase 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.
  • the a 3 -beta hydroxysteroid dehydrogenase 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: 518.
  • the gene sequence encoding a 3- beta hydroxysteroid dehydrogenase 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: 519.
  • 5-alpha reductase, the 5-beta reductase and 3 -beta hydroxysteroid dehydrogenase are present in a cassette and the 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: 514, 516, and 518, respectively.
  • the gene sequence encoding the 5-beta reductase and 3 -beta hydroxysteroid dehydrogenase encode 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: 515, 517, and 519, respectively.
  • the bacterium further comprises a heterologous gene encoding a bile acid importer or transporter.
  • importers or transporters include homologs of human ASBT, baiG or PanS.
  • the homolog of human ASBT is from Neisseria meningitidis (ASBTNM), from Yersinia frederiksenii (ASBTYf), from A. coli M34 or from E. coli VREC0334.
  • the bile acid importer or transporter is baiG from Eubacterium sp. strain VPI 12708.
  • the bile acid importer or transporter is PanS is from Escherichia coli EC23.
  • 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 or 508.
  • the gene sequence encoding an 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.
  • 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,
  • the gene sequence encoding an 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.
  • 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.
  • the gene sequence encoding an 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.
  • the bacterium further comprises an insertion, deletion or mutation of an endogenous phage gene.
  • the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.
  • the bacterium further comprises a modified endogenous colibactin island.
  • 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),
  • 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/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), c/ > 2 (SEQ ID NO:
  • the bacterium is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
  • the auxotrophy is in an enzyme diaminopimelic acid synthesis or an enzyme in the thymine biosynthetic pathway.
  • the present disclosure provides for a pharmaceutically acceptable composition
  • a pharmaceutically acceptable composition comprising the recombinant bacterium as provided herein; and a pharmaceutically acceptable carrier.
  • the composition is formulated for oral administration.
  • 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.
  • the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.
  • 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,
  • 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 a
  • CNS central nervous system inflammation
  • 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.
  • the subject is a human.
  • the present disclosure provides a recombinant bacterium comprising at least one heterologous 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.
  • the bile acid catabolism enzyme is a 3 -beta hydroxysteroid dehydrogenase (3[3-HSDH).
  • the bile acid catabolism enzyme is a bacterial 3J3-HSDH.
  • the 3[3-HSDH is a Bacteroides dorei DSM 17855 3[3-HSDH.
  • the gene encoding the 3J3-HSDH 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: 518.
  • the gene encoding the 3J3-HSDH 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: 519.
  • the recombinant bacterium further comprising a second heterologous gene encoding a second bile acid catabolism enzyme and a third heterologous gene encoding a third bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 5- alpha reductase, and wherein the third bile acid catabolism enzyme is a 5 -beta reductase.
  • the 5-alpha reductase is a bacterial 5-alpha reductase.
  • the bacterial 5-alpha reductase is a Bacteroides dorei DSM 17855 5-alpha reductase.
  • the gene encoding the 5-alpha reductase 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: 514.
  • the gene encoding the 5-alpha reductase 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: 515.
  • the 5 -beta reductase is a bacterial 5 -beta reductase.
  • the bacterial 5 -beta reductase is a Bacteroides dorei DSM 17855 5-beta reductase.
  • the gene encoding the 5-beta reductase 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.
  • the gene encoding the 5-beta reductase 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.
  • the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are present in a gene cassette operably linked to the promoter.
  • the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are each individually linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature.
  • each promoter is a different promoter, or wherein each promoter is a separate copy of the same promoter.
  • the recombinant bacterium further comprising a fourth heterologous gene encoding a fourth bile acid catabolism enzyme, wherein the fourth bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
  • the 3a-HSDH is a bacterial 3a-HSDH.
  • the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
  • the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is operably linked to a promoter that is not associated with the fourth gene encoding the fourth bile acid catabolism enzyme in nature.
  • the promoter that is operably linked to the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the fourth gene encoding the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme.
  • the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme, the second gene encoding the second bile acid catabolism enzyme, and the third gene encoding the third bile acid catabolism enzyme.
  • the recombinant bacterium further comprising a second heterologous gene encoding a second bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
  • the 3a-HSDH is a bacterial 3a-HSDH.
  • the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
  • the second heterologous gene encoding the second bile acid catabolism enzyme is operably linked to a promoter that is not associated with the second gene encoding the second bile acid catabolism enzyme in nature.
  • the promoter that is operably linked to the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the second gene encoding the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme.
  • the second heterologous gene encoding the second bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme.
  • the bile acid catabolism enzyme is a 3 -alpha-hydroxy steroid dehydrogenase (3a-HSDH).
  • the 3a-HSDH is a bacterial 3a-HSDH.
  • the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
  • the gene encoding the 3a-HSDH 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.
  • the gene encoding the 3a-HSDH 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.
  • the promoter is a constitutive or an inducible promoter.
  • the promotor is an inducible promoter induced by exogenous environmental conditions.
  • the inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.
  • the inducible promoter is an FNR-inducible promoter.
  • the inducible promoter is induced by temperature.
  • the inducible promoter is a cI857 promoter.
  • the inducible promoter is induced by a chemical inducer.
  • the promoter is induced by IPTG.
  • the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a plasmid in the recombinant bacterium.
  • the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a chromosome in the recombinant bacterium.
  • the recombinant bacterium is a non-pathogenic bacterium.
  • the recombinant bacterium is a probiotic or a commensal bacterium.
  • the recombinant bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
  • the recombinant bacterium is Escherichia coli strain Nissle.
  • the recombinant bacterium further comprising a heterologous gene encoding a bile acid importer or transporter.
  • the importer or transporter is a homolog of human ASBT, baiG or PanS.
  • the homologs of human ASBT is from Neisseria meningitidis (ASBTNM), from Yersinia frederiksenii (ASBTYf), from E. coli M34 or from E. coli VREC0334.
  • ASBTNM Neisseria meningitidis
  • ASBTYf Yersinia frederiksenii
  • the gene encoding the bile acid transporter 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.
  • the gene 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: 503, 505, 507, 509, 511 or 513.
  • the bile acid importer or transporter is baiG from Eubacterium sp. strain VPI 12708.
  • the gene encoding the bile acid transporter 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.
  • the gene 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: 513.
  • the PanS is from Escherichia coli EC23.
  • the gene encoding the bile acid transporter 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.
  • the gene 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: 511.
  • the recombinant bacterium further comprising an insertion, deletion or mutation of an endogenous phage gene.
  • the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.
  • the recombinant bacterium further comprising a modified endogenous colibactin island.
  • 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:
  • 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/ ⁇ (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:
  • the recombinant bacterium is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
  • the auxotrophy is in an enzyme diaminopimelic acid synthesis or an enzyme in the thymine biosynthetic pathway.
  • the bile acid catabolism enzyme catabolizes a secondary bile acid.
  • the bile acid catabolism enzyme catabolizes lithocholic acid (LCA).
  • the bile acid catabolism enzyme catabolizes 3-oxo lithocholic acid.
  • the bile acid catabolism enzyme is capable of producing 3-oxo lithocholic acid.
  • the bile acid catabolism enzyme is capable of producing isoallolithocholic acid (isoalloLCA).
  • the bile acid catabolism enzyme is capable of producing isolithocholic acid (isoLCA).
  • the bacterium produces isoalloLCA at a rate of at least about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4
  • the bacterium produces isoalloLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/
  • the bacterium produces isoalloLCA at a rate of about 0.025 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells.
  • the bacterium produces isoalloLCA at a rate of about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
  • the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 n
  • the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/
  • the bacterium produces 3-oxoLCA at a rate of about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
  • the present disclosure provides a pharmaceutically acceptable composition
  • a pharmaceutically acceptable composition comprising the recombinant bacterium as provided herein; and a pharmaceutically acceptable carrier.
  • the composition is formulated for oral administration.
  • the present disclosure provides 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.
  • the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.
  • 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,
  • the disease or disorder is 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 a
  • CNS central nervous system inflammation
  • 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.
  • the subject has a decreased level of a secondary bile acid in the gut after the composition is administered.
  • the bile acid is lithocholic acid (LCA).
  • the subject has an increased level of isoalloLCA in the gut after the composition is administered.
  • the present disclosure provides a method of decreasing a level of a secondary bile acid in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition as provided herein, thereby decreasing the level of the secondary bile acid in the gut of the subject.
  • the level of secondary bile acid in the gut is decreased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
  • the level of secondary bile acid in the gut is decreased by about 10% to about 200%, about 10% to about 190%, about 10% to about 180%, about 10% to about
  • the secondary bile acid is LCA.
  • the subject has an increased level of isoalloLCA, 3-oxoLCA and/or isoLCA in the gut after administration.
  • the present disclosure provides a method of increasing a level of isoalloLCA, 3-oxoLCA and/or isoLCA in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition as provided herein, thereby increasing the level of the isoalloLCA in the gut of the subject.
  • the level of isoalloLCA in the gut is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
  • the level of isoalloLCA in the gut is increased by at least about 10% to about 200%, about 10% to about 190%, about 10% to about 180%, about 10% to about 170%, about 10% to about 160%, about 10% to about 150%, about 10% to about 140%, about 10% to about 130%, about 10% to about 120%, about 10% to about 110%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 200%, about 20% to about 190%, about 20% to about 180%, about 20% to about 170%, about 20% to about 160%, about 20% to about 150%, about 20% to about 140%, about 20% to about 130%, about 20% to about 120%, about 20% to about 110%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to to about 20% to about 20% to about
  • the level of isoalloLCA in the gut is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%.
  • the subject is a human.
  • FIG. 1 provides a schematic showing a genetically engineered bacterium which is capable of converting a toxic bile acid into a non-toxic bile acid.
  • FIG. 2A depicts a schematic showing the catabolism of lithohcholic acid (LCA) to 3- oxo-LCA catalyzed by 3-alpha-hydroxysteroid dehydrogenase, e.g., by 3 -alpha-hydroxy steroid dehydrogenase from Eggerthella lenta.
  • LCA lithohcholic acid
  • 3- oxo-LCA catalyzed by 3-alpha-hydroxysteroid dehydrogenase, e.g., by 3 -alpha-hydroxy steroid dehydrogenase from Eggerthella lenta.
  • FIG. 2B depicts a schematic showing the catabolism of 3-oxo-LCA to isoalloLCA.
  • FIG. 2C depicts a schematic showing the genes encoding the enzymes involved in catabolism of 3-oxo-LCA to isoalloLCA, 5-alpha reductase, 5-beta reductase, and 3-beta hydroxysteroid dehydrogenase, which are derived from three bacterial strains, Parabacteroides merdae, B. dorei and Bacteroides vulgatus.
  • An exemplary engineered bacterium of the disclosure may comprise gene sequences encoding 3-alpha-hydroxysteroid dehydrogenase or a circuit for the catabolism of 3-oxo-LCA to isoalloLCA, or both.
  • FIG. 3 is a diagram showing 3-oxo-LCA transformation in EcN recombinant cells.
  • SYN8590 engineered to express a 3-gene pathway to convert 3-oxo-LCA to isoalloLCA.
  • FIG. 3 depicts a LC-MS/MS trace showing a peak consistent with isoalloLCA. The peak is seen in supernatant of the prototype SYN8590 after incubation with substrate 3-oxoLCA and is not seen from the WT EcN or Media only controls. Bacteria were incubated with 3-oxo-LCA in M9 media for 30 mins. The 3-oxoLCA and isoalloLCA were quantified by LCMS/MS. The peak at -1.77 min is isoalloLCA. Samples analyzed were media, control EcN, and engineered SYN8590.
  • FIGs. 4A, 4B, and 4C depict schematics of exemplary LCA and 3-oxoLCA converting prototype strains.
  • FIG. 4A depicts SYN8630, which utilizes a four enzyme pathway to convert LCA to isoalloLCA.
  • FIG. 4B depicts SYN8590, which utilizes the last three enzymes of the same pathway to convert 3-oxoLCA to isoalloLCA.
  • FIG. 4C depicts SYN8629, which utilizes only the first enzyme of the pathway to convert LCA to 3-oxoLCA.
  • FIG. 5 depicts a schematic showing the conversion pathways active in the exemplary prototype strains of the disclosure.
  • FIGs. 6A and 6B depict graphs showing activity of LCA catabolizing prototypes in vitro.
  • FIG. 6A SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively.
  • FIG. 6B SYN8629 produces 3-oxoLCA from LCA (it does not produce isoalloLCA as it is lacking the 3 enzyme operon involved).
  • FIGs. 6C and 6D depict graphs showing rate of activity of LCA catabolizing prototypes in vitro.
  • FIG. 6C rate at which SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively.
  • FIG. 6D rate at which SYN8629 produces 3-oxoLCA from LCA.
  • FIGs. 7A and 7B depict graphs showing the depletion of starting substrate by prototypes.
  • FIG. 7A Depletion of 3-oxoLCA by SYN8590, as it is converted to isoalloLCA, isoLCA and intermediates.
  • FIG. 7B Depletion of LCA by SYN8629 and SYN8630, as it is converted to 3- oxoLCA (SYN8629) or isoalloLCA, isoLCA and intermediates (SYN8630).
  • FIGs. 8A, 8B, and 8C depict graphs showing the accumulation of intermediates of LCA or 3-oxoLCA conversion to isoalloLCA in vitro.
  • FIG. 8A 3-oxo-A4-LCA accumulation by SYN8590 and SYN8630, starting in media containing either 3-oxoLCA or LCA, respectively.
  • FIG. 8B A4-isoLCA accumulation by SYN8590 and SYN8630, starting in media containing either 3- oxoLCA or LCA respectively.
  • FIG. 8C 3-oxoLCA accumulation by SYN8360, starting in media containing LCA.
  • FIGs. 9A, 9B, and 9C depict graphs and schematics of production of isoLCA in vitro.
  • FIG. 9A isoLCA is produced by SYN8590 from 3-oxoLCA and by SYN8360 from LCA.
  • FIG. 9B Potential enzyme pathway by which the strains could both produce isoLCA from 3-oxoLCA via the 3J3-HSDH.
  • FIG. 9C Potential enzyme pathway by which the strains could both produce isoLCA from A4-isoLCA via the 5[3-reductase.
  • BA disease-causing bile acids
  • 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.
  • Enterohepatic circulation of primary and secondary bile acids 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.
  • 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.
  • the present disclosure provides recombinant bacteria for catabolism of certain bile acids which cause inflammation and disease.
  • 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.
  • the recombinant bacteria are capable of catabolizing in low-oxygen environments, e.g., the gut.
  • the bile acid isoallolithocholic acid has been shown to enhance the differentiation of anti-inflammatory regulatory T cells (Treg cells) and its biosynthetic genes are significantly reduced in patients with inflammatory bowel diseases.
  • isoalloLCA bile acid isoallolithocholic acid
  • Treg cells anti-inflammatory regulatory T cells
  • increasing levels of isoalloLCA e.g., by administration of a bacterium disclosed herein, may promote gut homeostasis in a human subject in having an inflammatory bowel disease.
  • 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.
  • the present disclosure provides recombinant bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with inflammation.
  • the recombinant bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, a bile acid catabolism enzyme or a gene cassette of bile acid catabolism enzymes 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.
  • auxotrophies auxotrophies
  • a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid catabolism enzyme(s) or a gene cassette of bile acid catabolism enzymes and is capable of processing (e.g., metabolizing or catabolizing) and reducing levels of BA, e.g., lithocholic acid.
  • the bacteria are capable of producing isoalloLCA.
  • a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid catabolism enzyme(s) or a gene cassette of bile acid catabolism enzymes 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.
  • 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 more gut-beneficial metabolite, which can promote gut homeostasis, such as isoalloLCA, 3-oxoLCA, or isoLCA, in order to treat and/or prevent diseases associated with inflammation, such as IBD, Crohn’s disease or ulcerative colitis.
  • gut homeostasis such as isoalloLCA, 3-oxoLCA, or isoLCA
  • IBD, Crohn’s disease or ulcerative colitis e.g., IBD, Crohn’s disease or ulcerative colitis.
  • 3-oxoLCA and isoalloLCA regulate T cell effector functions; 3-oxoLCA binds to the transcription factor RORyt and suppresses Th 17 cell differentiation, while isoalloLCA promotes Treg cell differentiation (Hang et al.
  • IsoLCA and 3-oxoLCA both suppress differentiation of T helper 17 cells, which are associated with inflammation, and, moreover, both bile acids are significantly decreased in patients with IBD.
  • Paik et al. Human gut bacteria produce TH17-modulating bile acid metabolites. Nature. 2022 Mar; 603(7903): 907-912).
  • 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.
  • 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.
  • recombinant bacterial cells may comprise exogenous or heterologous nucleotide sequences stably incorporated into their chromosome.
  • 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.
  • 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.
  • 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.
  • heterologous gene refers to a nucleotide sequence that is not normally found in a given cell in nature.
  • 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.
  • 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.
  • 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.
  • the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.
  • 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.
  • bactericidal refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.
  • 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.
  • anti-toxin refers to a protein or enzyme which is capable of inhibiting the activity of a toxin.
  • 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.
  • coding region refers to a nucleotide sequence that codes for a specific amino acid sequence.
  • 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.
  • regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures.
  • the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.
  • 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.
  • operably linked refers to a nucleic acid sequence, e.g., a gene or gene cassette encoding at least one bile acid catabolism enzyme, 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.
  • a gene may be “directly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • 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.
  • 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.
  • 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.
  • inducible promoter Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.”
  • inducible promoters include, but are not limited to, an FNR promoter, a P ara c 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.
  • 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.
  • non-native genetic material e.g., a bile acid catabolism enzyme
  • 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.
  • 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.
  • 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.
  • 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
  • Plasmid 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.
  • 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.
  • genetic modification 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.
  • 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.
  • 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.
  • 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.
  • inactivated 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).
  • 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.
  • any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome.
  • 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.
  • 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.
  • 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.
  • 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.
  • the exogenous environmental condition is a tissuespecific or disease-specific metabolite or molecule(s).
  • the exogenous environmental condition is a low-pH environment.
  • the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter.
  • the exogenous environmental conditions of the disclosure refer to a specific temperature, for example, a temperature between 37 and 42 C.
  • exogenous 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.
  • 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.
  • the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter.
  • 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.
  • 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.
  • the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure.
  • 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).
  • the loss of exposure to an exogenous environmental condition 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).
  • 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.
  • oxygen level -dependent transcription factors include, but are not limited to, FNR, ANR, and DNR.
  • FNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et a/., 1998; Hoeren et al., 1993; Salmon et al., 2003).
  • Non-limiting examples are shown in Table 1.
  • a promoter was derived from the E. coll 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • Constant 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 o 32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli o 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E.
  • a constitutive Escherichia coli o s promoter e.g.,
  • 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 A promoter (e.g.
  • promoter veg (BBa_K143013), promoter 43 (BBa_K143013), Pi iaG (BBa_K823000), Pi epA (BBa_K823002), P veg (BBa_K823003)), a constitutive Bacillus subtilis 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; BB
  • “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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian gut, e.g.
  • 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.
  • mmHg O2 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).
  • 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.
  • 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.
  • 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.
  • O2 oxygen
  • Table 2 summarizes the amount of oxygen present in various organs and tissues.
  • DO dissolved oxygen
  • 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.
  • O2 oxygen
  • 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
  • oxygen producers or consumers are 100% air saturated.
  • 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%.
  • 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%,
  • 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.
  • 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%.
  • 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.
  • the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules or proteins of interest.
  • the microorganism is engineered to take up and catabolize certain metabolites or other compounds from its environment, e.g., the gut.
  • the microorganism is engineered to synthesize certain beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment.
  • the engineered microorganism is an engineered bacterium.
  • the engineered microorganism is an engineered virus.
  • Non-pathogenic bacteria refer to bacteria that are not capable of causing disease or harmful responses in a host.
  • non-pathogenic bacteria are Gram-negative bacteria.
  • non-pathogenic bacteria are Gram-positive bacteria.
  • non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut.
  • non-pathogenic bacteria examples 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
  • Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
  • 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.
  • the host organism is a mammal.
  • the host organism is a human.
  • Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic.
  • probiotic bacteria examples 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).
  • Bifidobacterium bifidum Enterococcus faecium
  • Escherichia coli Escherichia coli strain Nissle
  • Lactobacillus acidophilus Lactobacillus bulg
  • 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.
  • 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.
  • non-native genetic material e.g., amino acid metabolism gene
  • 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.
  • 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.
  • copy number affects the stability of expression of the non-native genetic material, e.g., an amino acid metabolism gene.
  • copy number affects the level of expression of the non-native genetic material, e.g., heterologous gene.
  • auxotroph 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.
  • 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).
  • module and “treat” and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof.
  • modulate and “treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient.
  • 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.
  • modulate” and “treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition.
  • 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.
  • 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)
  • CD Crohn’s disease
  • UC ulcerative colitis
  • 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.
  • 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
  • ADAM acute dissemin
  • 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.
  • 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)) 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.
  • a secondary bile acid such as lithocholic acid (LCA)
  • 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.
  • a phenylalanine transporter such as PheP imports phenylalanine into the microorganism.
  • payload refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as bacteria or a virus.
  • the payload is a therapeutic payload, e.g., an amino acid catabolic enzyme or an amino acid transporter polypeptide.
  • the payload is a regulatory molecule, e.g. , a transcriptional regulator such as FNR.
  • the payload comprises a regulatory element, such as a promoter or a repressor.
  • the payload comprises an inducible promoter, such as from FNRS.
  • the payload comprises a repressor element, such as a kill switch.
  • the payload is encoded by a gene or multiple genes or an operon.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism.
  • the genetically engineered microorganism comprises two or more payloads.
  • 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.
  • 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.
  • 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).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • 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.
  • dipeptide refers to a peptide of two linked amino acids.
  • tripeptide refers to a peptide of three linked amino acids.
  • 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.
  • 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.
  • 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.
  • Polypeptides also include fusion proteins.
  • the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the improvement of transcription and/or translation involves increasing the level of transcription and/or translation.
  • the improvement of transcription and/or translation involves decreasing the level of transcription and/or translation.
  • 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.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • phage and “bacteriophage” are used interchangeably herein. Both terms refer to a virus that infects and replicates within a bacterium.
  • phage or bacteriophage” collectively refers to prophage, lysogenic, dormant, temperate, intact, defective, cryptic, and satellite phage, phage tail bacteriocins, tailiocins, and gene transfer agents.
  • 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.
  • 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.
  • 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).
  • 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.
  • the inactivated phage or phage knockout refers to the inactivation of a temperate phage in its lysogenic state, i.e. , to a prophage.
  • 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.
  • 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.
  • 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.
  • 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.
  • a "pharmaceutical composition” refers to a preparation of bacterial cells disclosed herein with other components such as a physiologically suitable carrier and/or excipient.
  • 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.
  • 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.”
  • a heterologous gene encoding an bile acid transporter should be understood to mean “at least one heterologous gene encoding at least one bile acid transporter.”
  • 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.
  • “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.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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.
  • the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile acid catabolism enzyme.
  • the bacterial cell is a non-pathogenic bacterial cell.
  • the bacterial cell is a commensal bacterial cell.
  • the bacterial cell is a probiotic bacterial cell.
  • 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.
  • a Bacteroides fragilis Bacteroides thetaiotaomicron
  • Bacteroides subtilis Bacteroides subtilis
  • Bifidobacterium animalis Bifidobacterium bifidum
  • Bifidobacterium infantis Bifidobacterium lactis
  • 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.
  • 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 .
  • the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.
  • the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterobacteriaceae 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.
  • E. coli a-hemolysin, P-fimbrial adhesins) (Schultz, 2008)
  • 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).
  • Mutaflor medicinal capsules
  • 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).
  • the recombinant bacterial cell does not colonize the subject.
  • genes from one or more different species can be introduced into one another, e.g., an bile acid catabolism gene from Homo sapiens can be expressed in Escherichia coli.
  • 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.
  • the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.
  • the disclosure provides a recombinant bacterial culture which reduces levels of a bile acid, e.g., lithocholic acid, in the media of the culture.
  • the levels of an amino acid are reduced by about 50%, about 75%, or about 100% in the media of the cell culture.
  • the levels of an amino acid are reduced by about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold in the media of the cell culture.
  • the levels of an bile acid, e.g., lithocholic acid are reduced below the limit of detection in the media of the cell culture.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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.
  • the gene encoding a s bile acid catabolism enzyme or bile acid catabolism enzyme cassette is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.
  • 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.
  • 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.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is chemically induced.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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.
  • the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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.
  • the genetically engineered bacteria is an auxotroph.
  • 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.
  • the engineered bacteria have more than one auxotrophy, for example, they may be a tdhyA and dapA auxotroph.
  • 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.
  • 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.
  • Bile acid catabolism enzymes 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 (LCA) into isoalloLCA or 3-oxo-LCA.
  • the genetically engineered bacteria comprising at least one heterologous gene encoding a bile acid catabolism enzyme or a bile acid catabolism enzyme cassette can catabolize lithocholic acid to isoalloLCA or 3-oxo-LCA 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.
  • 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.
  • 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.
  • 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.
  • Bile acid catabolism enzymes are well-known in the art. For example 3-alpha- hydroxysteroid dehydrogenase a catabolizes lithohcholic acid (LCA) to 3-oxo-LCA. Hydroxysteroid dehydrogenases (HSDH) are expressed by both host tissues and host-associated microbiota that modify the hydroxyl groups on steroids such as bile acids. Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids through the generation of oxo-bile acid with implications for increased/decreased toxicity for the host.
  • HSDH Hydroxysteroid dehydrogenases
  • 5-beta reductase which can convert 3-oxoLCA to 3-oxo-A4- LCA
  • 3-beta hydroxysteroid dehydrogenase which can convert 3-oxo-A4-LCA to A4-isoLCA or 3-oxoalloLCA to isoalloLCA
  • 5-alpha reductase which can convert 3-oxo-A4- LCA to 3-oxoalloLCA or convert 3-oxo-A4-LCA to A4isoLCA to isoalloLCA.
  • the bacteria of the disclosure comprise gene sequence(s) or gene cassettes encoding 5-alpha reductase, 5-beta reductase, and 3-beta hydroxysteroid dehydrogenase.
  • the bacteria comprise gene sequences encoding Bacteroides dorei DSM 17855 5-alpha reductase, Bacteroides dorei DSM 17855 5-beta reductase and/or Bacteroides dorei DSM 17855 3-beta hydroxysteroid dehydrogenase.
  • the bacteria in addition to the three enzyme cassette, further comprise 3-alpha- hydroxysteroid dehydrogenase, e.g., from A. lenta.
  • 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.
  • the bile acid catabolism enzyme is a Hydroxysteroid dehydrogenases (HSDH).
  • HSDH Hydroxysteroid dehydrogenases
  • the HSDH is a 3-alpha-hydroxysteroid dehydrogenase .
  • the 3-alpha-hydroxysteroid dehydrogenase is from Eggerthella lenta.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming 3-oxoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming 3-oxoLCA, 3-oxo-A4-LCA, 3-oxoalloLCA and/or A4- isoLCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from 3-oxoLCA, 3- oxo-A4-LCA, 3-oxoalloLCA, and/or A4-isoLCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing A4-isoLCA, e.g., from 3-oxoLCA and/or 3-oxo-A4-LCA. .
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoalloLCA, e.g., from 3-oxoLCA and/or 3-oxo-A4-LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-A4-LCA, e.g., from 3-oxoLCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoLCA, e.g., from LCA or from 3-oxoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoLCA, e.g., from A4-isoLCA.
  • the genetically engineered bacterium is capable of producing isoalloLCA, e.g., from 3-oxoLCA at a rate of at least about 0.15 nmol/h/le9 cells. In one embodiment, the genetically engineered bacterium is capable of producing isoalloLCA, e.g., from 3- oxoLCA at a rate of at least about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells; 0.1 nmol/h/le cells to about 0.2 nmol/h/le9 cells; or about 0.125 nmol/h/le9 cells to about 1.175 nmol/h/le9 cells.
  • the genetically engineered bacterium comprises gene(s) encoding one or more of 5 -alpha reductase, 5 -beta reductase, and 3 -beta hydroxysteroid dehydrogenase from B. dorei.
  • the bacterium produces isoalloLCA at a rate of at least about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4
  • the bacterium produces isoalloLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/
  • the bacterium produces isoalloLCA at a rate of about 0.025 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
  • the bacterium produces isoalloLCA at a rate of about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoLCA, e.g., from LCA.
  • the genetically engineered bacterium produces 3-oxoLCA, e.g., from LCA at a rate of at least about 0.15 nmol/h/le9 cells.
  • the genetically engineered bacterium comprises gene(s) encoding 3 -alpha hydroxysteroid dehydrogenase from Eggerthella lenta.
  • the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 n
  • the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/
  • the bacterium produces 3-oxoLCA at a rate of about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA, 3-oxo-LCA, 3-oxo-A4-LCA, 3-oxoalloLCA and/or A4-isoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from LCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from 3-oxo-LCA, 3- oxo-A4-LCA, 3-oxoalloLCA and/or A4-isoLCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing A4-isoLCA, e.g., from LCA, 3-oxo-LCA and/or 3-oxo-A4-LCA.
  • the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoalloLCA, e.g., from LCA, 3-oxo-LCA and/or 3-oxo-A4- LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-A4-LCA, e.g., from LCA and/or 3-oxo-LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-LCA, e.g., from LCA.
  • the genetically engineered bacterium produces isoalloLCA, e.g. , from LCA at a rate of at least about 0.04 nmol/h/le9 cells.
  • the genetically engineered bacterium comprises gene(s) encoding one or more of 5-alpha reductase, 5-beta reductase, and 3 -beta hydroxy steroid dehydrogenase from B. dorei and 3 -alpha hydroxy steroid dehydrogenase from Eggerthella lenta.
  • the 3-alpha-hydroxysteroid dehydrogenase gene has at least about 80% identity with the sequence of SEQ ID NO: 500. Accordingly, in some embodiments, the 3-alpha-hydroxysteroid dehydrogenase gene has at least about 90% identity with the sequence of SEQ ID NO: 500. Accordingly, in some embodiments, the 5-alpha reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 500.
  • the 3-alpha- hydroxysteroid dehydrogenase 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.
  • the 3-alpha-hydroxysteroid dehydrogenase gene comprises the sequence of SEQ ID NO: 500.
  • the 3-alpha-hydroxysteroid dehydrogenase gene consists of the sequence of SEQ ID NO: 500.
  • the gene sequence encoding an 3-alpha-hydroxysteroid dehydrogenase 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.
  • the bile acid catabolism enzyme is a 5-alpha reductase.
  • the 5-alpha reductase is from Bacteroides dorei DSM 17855.
  • the 5-alpha reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 514. Accordingly, in some embodiments, the 5-alpha reductase gene has at least about 90% identity with the sequence of SEQ ID NO: 514. In some embodiments, the 5-alpha reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 514. Accordingly, in some embodiments, the 5-alpha reductase 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: 514. In another embodiment, the 5-alpha reductase gene comprises the sequence of SEQ ID NO: 514. In yet another embodiment the 5-alpha reductase gene consists of the sequence of SEQ ID NO: 514.
  • the gene sequence encoding a 5-alpha reductase 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: 515.
  • the bile acid catabolism enzyme is a 5-beta reductase.
  • the 5-beta reductase is from Bacteroides dorei DSM 17855.
  • the 5-beta reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 516.
  • the 5 -beta reductase gene has at least about 90% identity with the sequence of SEQ ID NO: 516.
  • the 5-beta reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 516.
  • the 5-beta reductase 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.
  • the 5-beta reductase gene comprises the sequence of SEQ ID NO: 516.
  • the 5-beta reductase gene consists of the sequence of SEQ ID NO: 516.
  • the gene sequence encoding a 5 beta reductase 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: 517.
  • the bile acid catabolism enzyme is a 3-beta hydroxysteroid dehydrogenase.
  • the 3-beta hydroxysteroid dehydrogenase is from Bacteroides dorei DSM 17855.
  • the 5-beta reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 518. Accordingly, In some embodiments, the 3-beta hydroxysteroid dehydrogenase gene has at least about 90% identity with the sequence of SEQ ID NO: 518. Accordingly, In some embodiments, the 3-beta hydroxysteroid dehydrogenase gene has at least about 95% identity with the sequence of SEQ ID NO: 518.
  • the 3-beta hydroxysteroid dehydrogenase 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: 518.
  • the 3-beta hydroxysteroid dehydrogenase gene comprises the sequence of SEQ ID NO: 518.
  • the 3-beta hydroxysteroid dehydrogenase gene consists of the sequence of SEQ ID NO: 518.
  • the gene sequence encoding a 3-beta hydroxysteroid dehydrogenase 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: 519.
  • the bile acid catabolism cassette comprises gene sequences encoding a 5 -alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase.
  • he bile acid catabolism cassette comprises gene sequences encoding a Bacteroides dorei DSM 17855 5-alpha reductase, a Bacteroides dorei DSM 17855 5-beta reductase, and a Bacteroides dorei DSM 17855 3-beta hydroxysteroid dehydrogenase.
  • the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 80% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively. Accordingly, in some embodiments, the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 90% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively.
  • the gene sequences in a cassete encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 95% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively.
  • the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively.
  • the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase comprise the sequence of SEQ ID NO: 514, 516 and 518, respectively.
  • the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase consist of the sequence of SEQ ID NO: 514, 516 and 518, respectively.
  • the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have encode an polypeptides that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 515, 517 and 519, respectively.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene(s) for producing a bile acid catabolism enzyme or bile acid catabolism enzyme cassette, such that the bile acid catabolism enzyme or cassette 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.
  • a bacterium may comprise multiple copies of the gene encoding the bile acid catabolism enzyme or cassette.
  • the gene(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette is expressed on a low-copy plasmid.
  • 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(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile acid catabolism enzyme or bile acid catabolism enzyme cassette. In some embodiments, the gene(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette are expressed on a chromosome.
  • 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.
  • MO As mechanisms of action
  • the genetically engineered bacteria may include four copies of the gene(s) encoding a particular bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at four different insertion sites.
  • the genetically engineered bacteria may include three copies of the gene(s) encoding a particular bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at three different insertion sites and three copies of the gene(s) encoding a different bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at three different insertion sites.
  • the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the bile acid catabolism enzyme or bile acid catabolism enzyme cassette, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.
  • qPCR quantitative PCR
  • Primers specific for bile acid catabolism enzyme or bile acid catabolism enzyme cassette gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • 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).
  • CT threshold cycle
  • qPCR quantitative PCR
  • Primers specific for bile acid catabolism enzyme or bile acid catabolism enzyme cassette gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • a fluorophore is added to a sample reaction mixture that may contain bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 bile acid catabolism enzyme or bile acid catabolism enzyme cassette gene(s).
  • CT threshold cycle
  • the bacterial cell comprises a heterologous gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette. In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile acid transporter and a heterologous gene encoding bile acid catabolism enzyme or bile acid catabolism enzyme cassette. 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.
  • the bacterial cell comprises a heterologous gene encoding a transporter of bile acids, a heterologous gene encoding bile acid catabolism enzyme or bile acid catabolism enzyme cassette, and a genetic modification that reduces export of bile acids.
  • Transporters and exporters are described in more detail in the subsections, below.
  • Bile acid transporters 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.
  • 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.
  • 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).
  • the bile acid transporter is an ASBT homolog.
  • ASBT homolog is from Neisseria meningitidis (ASBTNM).
  • ASBTNM Neisseria meningitidis
  • 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 502.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 502.
  • the gene sequence encoding an 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.
  • the bile acid transporter is an ASBT homolog from Yersinia frederiksenii (ASBTYf).
  • ASBTYf Yersinia frederiksenii
  • 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 504.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 504.
  • the gene sequence encoding an bile acid transporter e.g. , an ASBT analog, 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: 505.
  • 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 506.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 506.
  • the gene sequence encoding a bile acid transporter 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: 507.
  • the bile acid transporter is an ASBT homolog from Escherichia coli VREC0334.
  • 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, 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 508.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 508.
  • the gene sequence encoding a bile acid transporter 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: 509.
  • the bile acid transporter is 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, 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 510.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 510.
  • the gene sequence encoding a bile acid transporter 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: 511.
  • the bile acid transporter is BaiG or a homolog thereof.
  • the BaiG is from Eubacterium sp. strain VPI 12708.
  • 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, 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.
  • 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.
  • the gene encoding the bile acid transporter comprises the sequence of SEQ ID NO: 512.
  • the gene encoding the bile acid transporter consists of the sequence of SEQ ID NO: 512.
  • the gene sequence encoding an 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.
  • 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.
  • a bacterium may comprise multiple copies of the gene encoding the bile acid transporter.
  • 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.
  • the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions.
  • the gene encoding the bile acid transporter is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing expression of bile acid transporter.
  • the gene encoding the bile acid transporter is expressed on a chromosome.
  • 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.
  • the recombinant bacterial cells described herein comprise a genetic modification that reduces export of a bile acid, e.g., LCA
  • 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.
  • 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 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.
  • bile acid e.g., LCA
  • any bile acid catabolism enzyme expressed in the organism e.g., co-expressed bile acid catabolism enzyme
  • the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the polypeptides disclosed herein, e.g., bile acid catabolism enzyme(s) and/or bile acid 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.
  • a bacterial cell comprises two or more distinct bile acid catabolism enzyme(s), genes or operons, e.g., two or more genes or operons.
  • bacterial cell comprises three or more distinct bile acid catabolism enzyme gene(s) or operons, e.g., three or more bile acid catabolism enzyme(s) gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct bile acid catabolism enzyme gene(s) or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more bile acid catabolism enzyme(s) genes.
  • a bacterial cell comprises three or more distinct bile acid transporter gene(s) or operons, e.g., three or more bile acid transporter gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct bile acid transporter genes, or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more s bile acid transporter gene(s).
  • the genetically engineered bacteria comprise multiple copies of the same bile acid catabolism enzyme(s) and /or bile acid transporter gene(s).
  • the gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid transporter is present on a plasmid and operably linked to a directly or indirectly inducible promoter.
  • the gene encoding the bile acid catabolism enzyme(s), and/or bile acid transporter is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding the bile acid catabolism enzyme(s), and/or bile acid transporter is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the bile acid catabolism enzyme(s), and/or bile acid transporter is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding the bile acid catabolism enzyme(s) and/or bile acid transporter is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline, arabinose or Isopropyl B-D- 1 -thiogalactopyranoside (IPTG) .
  • the inducible promoter is an IPTG inducible promoter.
  • 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.
  • the recombinant bacterium further comprises a gene sequence encoding a repressor of the Lac promoter.
  • 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.
  • 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.
  • the promoter that is operably linked to the gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid transporter is directly induced by exogenous environmental conditions.
  • the promoter that is operably linked to the gene encoding bile acid catabolism enzyme(s) and/or bile acid transporter is indirectly induced by exogenous environmental conditions.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal.
  • the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal.
  • 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.
  • ATC anhydrotetracycline
  • the bacterial cell comprises a gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid transporter is expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter.
  • FNR fumarate and nitrate reductase regulator
  • 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 table below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
  • 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.
  • multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria.
  • the genetically engineered bacteria comprise a gene encoding a bile acid catabolism enzyme(s) and/or bile acid 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).
  • expression of the bile acid catabolism enzyme(s) and/or bile acid transporter gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability.
  • the mammalian gut is a human mammalian gut.
  • 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 bile acid catabolism enzyme(s) and/or bile acid transporter, in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions.
  • the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N.
  • 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 wildtype activity.
  • 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 bile acid catabolism enzyme(s) and/or bile acid transporter, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions.
  • 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 bile acid catabolism enzyme(s) and/or bile acid transporter, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions.
  • 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).
  • the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene.
  • the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the bile acid catabolism enzyme(s) and/or bile acid transporter are present on different plasmids.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter are present on different chromosomes.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter.
  • expression of the transcriptional regulator is controlled by the same promoter that controls expression of the bile acid catabolism enzyme(s) and/or bile acid transporter.
  • the transcriptional regulator and the bile acid catabolism enzyme(s) and/or bile acid transporter are divergently transcribed from a promoter region.
  • any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites.
  • one or more copies of one or more encoding a bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter and also permits fine-tuning of the level of expression.
  • 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.
  • 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.
  • a gene of interest cloned downstream of the X promoters can be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage X.
  • cI857 binds to the oL or oR regions of the pR promoter and inhibits transcription by RNA polymerase.
  • the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated.
  • 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.
  • the cultures may be grown aerobically. Alternatively, the cultures are grown anaerobically.
  • 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.
  • 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.
  • the gene sequence(s) are induced upon or during in vivo administration.
  • 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.
  • the genetically engineered bacteria further comprise gene sequence (s) encoding a transcription factor which is capable of binding to the temperature sensitive promoter.
  • the transcription factor is a repressor of transcription.
  • 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.
  • a second promoter e.g., a second constitutive or inducible promoter.
  • 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.
  • 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).
  • the first inducing conditions may be culture conditions, e.g., permissive temperature
  • the second inducing conditions may be in vivo conditions.
  • in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain.
  • 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).
  • 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.
  • 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, comprising or consisting of SEQ ID NO: 19.
  • 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% ;
  • 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% ;
  • 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.
  • 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, comprising or consisting of SEQ ID NO: 20.
  • 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, comprising or consisting of the polypeptide encoded by any of the sequences of SEQ ID NO: 21.
  • 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.
  • 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, comprising or consisting of SEQ ID NO: 23.
  • 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, comprising or consisting of the polypeptide encoded by SEQ ID NO: 24.
  • 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, comprising or consisting of the polypeptide encoded by SEQ ID NO: 25.
  • SEQ ID Nos: 19-25 are shown in Table 5.
  • the genetically engineered bacteria comprise one or more E. coli Nissle bacteriophage, e.g., Phage 1, Phage 2, and Phage 3.
  • 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.
  • the one or more insertions comprise an antibiotic cassette.
  • the mutation is a deletion.
  • 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, ECOLIN_10100, ECOLIN_10105, ECOLINJOl lO, ECOLIN_10115, ECOLIN_10120, ECOLIN 10125
  • 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.
  • 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.
  • the sequence of SEQ ID NO: 292 is deleted from the Phage 3 genome.
  • a sequence comprising SEQ ID NO: 292 is deleted from the Phage 3 genome.
  • the engineered bacterium further comprises a modified pks island (colibactin island).
  • a modified pks island colibactin island
  • 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.
  • 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.
  • 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.”
  • Colibactin undergoes a prodrug activation mechanism that incorporates an N-terminal structural motif, which is removed during the final stage of biosynthesis.
  • the engineered microorganism comprises a modified pks island (colibactin island).
  • 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.
  • a suitable control e.g., the native pks island in an unmodified bacterium of the same strain and/or subtype
  • the modified clb sequence is an insertion, a substitution, and/or a deletion as compared to the control.
  • 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.
  • 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, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, and clbR.
  • the clbS gene e.g., a deletion of clb A, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbl, clbJ, clbK, clbL, clbM, clbN, clbO, clbP
  • 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), clbA (SEQ ID NO
  • 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: 294)
  • 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).
  • 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.
  • 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.
  • any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth.
  • the essential gene is an oligonucleotide synthesis gene, for example, thyA.
  • the essential gene is a cell wall synthesis gene, for example, dapA.
  • 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.
  • 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).
  • 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.
  • 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).
  • Diaminopimelic acid 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).
  • 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.
  • 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).
  • the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene.
  • the uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et 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.
  • 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).
  • 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.
  • the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth.
  • 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, Ig
  • 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).
  • the SLiDE bacterial cell comprises a mutation in an essential gene.
  • the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk.
  • the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C.
  • the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C.
  • 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.
  • 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.
  • the genetically engineered bacterium is complemented by a ligand.
  • 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.
  • bacterial cells comprising mutations in metG 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 are complemented by benzothiazole, indole or 2- aminobenzothiazole.
  • Bacterial cells comprising mutations in pheS are complemented by benzothiazole or 2-aminobenzothiazole.
  • Bacterial cells comprising mutations in tyrS are complemented by benzothiazole or 2- aminobenzothiazole.
  • Bacterial cells comprising mutations in adk I4L, L5I and L6G are complemented by benzothiazole or indole.
  • the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand.
  • the bacterial cell comprises mutations in two essential genes.
  • the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C).
  • the bacterial cell comprises mutations in three essential genes.
  • 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).
  • the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.
  • 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.
  • 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).
  • 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).
  • the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a bile acid catabolism enzyme(s) and/or bile acid transporter operably linked to a first inducible promoter.
  • the disclosure provides an isolated plasmid comprising a second nucleic acid encoding at least one bile acid catabolism enzyme(s) and/or bile acid transporter.
  • the first nucleic acid and the second nucleic acid are operably linked to the first promoter.
  • the second nucleic acid is operably linked to a second inducible promoter.
  • 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.
  • FNR fumarate and nitrate reduction regulator
  • 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.
  • 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, an bile acid catabolism enzyme(s) and/or bile acid transporter gene
  • gene cassette for example, a gene cassette comprising a bile acid catabolism enzyme(s) and/or bile acid 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 other enzymes of the gene cassette, and also permits fine-tuning of the level of expression.
  • 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.
  • the bile acid catabolism enzyme(s) and/or bile acid transporter genes are integrated to facilitate bile acid import and metabolism.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the pharmaceutical composition is administered before the subject eats a meal.
  • the pharmaceutical composition is administered currently with a meal.
  • the pharmaceutical composition is administered after the subject eats a meal.
  • 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.
  • 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.
  • 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.
  • the genetically engineered microorganisms may be administered intravenously, e.g. , by infusion or injection.
  • 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.
  • 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.
  • 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.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.
  • the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product.
  • 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.
  • 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.
  • 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 carbo
  • 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); fdlers (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).
  • binding agents e.g., prege
  • 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-
  • 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).
  • the pH profile may be modified.
  • 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.
  • 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.
  • the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to adult subjects or pediatric subjects.
  • a composition suitable for administration to adult subjects may include easy-to- swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers.
  • a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.
  • 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.
  • the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life.
  • the gummy candy may also comprise sweeteners or flavors.
  • the composition suitable for administration to pediatric subjects may include a flavor.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product.
  • 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.
  • the food product is a fermented food, such as a fermented dairy product.
  • the fermented dairy product is yogurt.
  • the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir.
  • the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics.
  • the food product is a beverage.
  • the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts.
  • 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.
  • the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a single dosage form may be administered over a period of time, e.g., by infusion.
  • 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.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • 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).
  • 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.
  • 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.
  • 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.
  • Another aspect of the disclosure provides methods of treating diseases and disorders, e.g., autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • ADAM
  • 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.
  • 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).
  • 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; Fra
  • 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.
  • the subject to be treated is a human patient.
  • 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.
  • the recombinant bacteria are administered orally, e.g., in a liquid suspension.
  • the recombinant bacteria are lyophilized in a gel cap and administered orally.
  • the recombinant bacteria are administered via a feeding tube or gastric shunt.
  • the recombinant bacteria are administered rectally, e.g., by enema.
  • the recombinant bacteria are administered topically, intraintestinally, intrajej unally, intraduodenally, intraileally, and/or intracolically.
  • the recombinant bacteria described herein are administered to treat, manage, ameliorate, or prevent metabolic diseases in a subject.
  • 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.
  • the symptom e.g., obesity, insulin resistance
  • the subject is a human subject.
  • metabolic symptoms and manifestations may be measured in a biological sample, e.g., blood, serum, plasma, urine, fecal mater, peritoneal fluid, a sample collected from a tissue, such as liver, skeletal muscle, pancreas, epididymal fat, subcutaneous fat, and beige fat.
  • a biological sample e.g., blood, serum, plasma, urine, fecal mater, 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 faty acid levels, triglyceride levels, and nonesterified faty 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.
  • 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.
  • 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.
  • the recombinant bacteria may be administered alone or in combination with one or more additional therapeutic agents, e.g., insulin.
  • 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.
  • 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.
  • Colitis is characterized by inflammation of the inner lining of the colon, and is one form of IBD.
  • 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.
  • 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).
  • 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).
  • the bacteria of the disclosure may be evaluated in a CD45RBHi T cell transfer mouse model of IBD.
  • 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).
  • DSS dextran sodium sulfate
  • 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).
  • 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).
  • the bacteria of the disclosure may be evaluated in a DSS mouse model of IBD.
  • TNBS Trinitrobenzenesulfonic acid
  • 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.
  • 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.
  • 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.
  • 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
  • Example 1 Generation of E. coli Nissle strains expressing the three gene pathway from Bacteroides dorei, 5a-Reductase, -5 -Reducatase, and 3 -HSDH.
  • the 3-alpha hydroxysteroid dehydrogenase gene from Eggerthella lenta DSM 2243 was sourced from the NCBI genome database (CPOO 1726.1).
  • a ribosome binding site was designed using deNovo DNA software, the gene was codon optimized for expression in E. coli and was synthesized (Integrated DNA Technologies), fused to a pTac promoter, and cloned into a medium copy plasmid by Gibson assembly to generate Logic2758.
  • Each plasmid described above was transformed alone or in combination into E. coli Nissle to generate the strains SYN8590 (designed to convert 3-oxoLCA to isoalloLCA), SYN8629 (designed to convert LCA to 3-oxoLCA), and SYN8630 (designed to convert LCA to isoalloLCA) respectively (Table 6).
  • FIGs. 4A, 4B, and 4C depict schematics of the prototype strains of Table 5.
  • FIG. 4A depicts SYN8630, which utilizes a four enzyme pathway to convert LCA to isoalloLCA.
  • FIG. 4B depicts SYN8590, which utilizes the last three enzymes of the same pathway to convert 3-oxoLCA to isoalloLCA.
  • FIG. 4C depicts SYN8629, which utilizes only the first enzyme of the pathway to convert LCA to 3-oxoLCA.
  • FIG. 5 depicts proposed active conversion pathways.
  • Example 2 Functional Assays demonstrating the conversion of lithocholic acid (LCA) or 3-oxoLCA to isoalloLCA by recombinant bacteria
  • samples of each engineered strain and a control wild type E. coli Nissle strain are 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 are resuspended in phosphate buffer with glycerol and frozen for storage. They are then normalized by optical density at 600nm (ODeoo), added to assay buffer (M9 minimal salts with glucose) containing 15 pM or 100 pM of lithocholic acid (LCA) or 3-oxo-LCA.
  • LCA or 3-oxoLCA Transformation of LCA or 3-oxoLCA was assessed over 3h. At several time points up to 3h, a sample of the reaction was quenched with the addition of 400pl ice cold methanol. Samples were then centrifuged at 15000rpm for 5 min and supernatant collected for analysis. LCA, 3-oxo-LCA, isoLCA and isoalloLCA as well as putative intermediates, 3-oxo-A4-LCA, and A4-isoLCA, in the supernatant are quantified by LCMS using established methods.
  • SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively (FIG 6A).
  • SYN8629 produces 3-oxoLCA from LCA (FIG. 6B); SYN8629 does not produce isoalloLCA because it is lacking the 3 enzyme operon present in SYN8590 and SYN8630.
  • SYN8590 and SYN8630 produce isoalloLCA, at a rate of approximately 0.15 nmol/h/le9 cells and 0.04 nmol/h/le9 cells, respectively FIG. 6C).
  • SYN8629 produces 3-oxoLCA at a rate of approximately 0.15 nmol/h/le9 cells (FIG. 6D).
  • SYN8590 depletes 3-oxoLCA, converting it to isoalloLCA, isoLCA and intermediates (FIG. 7A).
  • SYN8629 and SYN8630 deplete LCA, converting it to 3-oxoLCA (SYN8629) or isoalloLCA, isoLCA and intermediates (SYN8630).
  • FIGs. 8A, 8B, and 8C show intermediates which accumulate as a result of the conversion of LCA or 3-oxoLCA to isoalloLCA.
  • SYN8590 and SYN8630 produce 3-oxo-A4-LCA, starting in media containing either 3-oxoLCA or LCA, respectively.
  • FIGs. 9A, 9B, and 9C show production of isoLCA in vitro.
  • SYN8590 produces isoLCA from 3-oxoLCA.
  • SYN8360 produces isoLCA from LCA.
  • a potential enzyme pathway by which the strains could both produce isoLCA from 3-oxoLCA via the 3J3-HSDH is shown in FIG. 9B.
  • a potential enzyme pathway by which the strains could both produce isoLCA from A4-isoLCA via the 5[3-reductase is shown in FIG. 9C.

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] The instant application claims priority to U.S. Serial No. 63/353,171, filed on June 17, 2022. The entire contents of the foregoing application are expressly incorporated by reference herein in their entirety.
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. 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). In some embodiments, the bacteria of the disclosure comprise one or more gene(s) encoding a bile acid catabolism enzyme(s) or a bile acid catabolism enzyme cassette(s).
[05] In some embodiments, the bile acid catabolism enzyme is a catabolizes a secondary bile acid, e.g., lithocholic acid (LCA). [06] In some embodiments, the bile acid catabolism enzyme is a 3 -alpha-hydroxy steroid dehydrogenase, e.g., a bacterial 3-alpha-hydroxysteroid dehydrogenase. In some embodiments, the 3- alpha-hydroxysteroid dehydrogenase is from Eggerthella lenta.
[07] In some embodiments, the bile acid catabolism enzyme is a 5-alpha reductase, e.g., a bacterial 5-alpha reductase. In some embodiments, the 5-alpha reductase is from Bacteroides dorei DSM 17855. In some embodiments, the bile acid catabolism enzyme is a 5-beta reductase, e.g., a bacterial 5-beta reductase. In some embodiments, the 5-beta reductase is from Bacteroides dorei DSM 17855. In some embodiments, the bile acid catabolism enzyme is a 3-beta hydroxysteroid dehydrogenase, e.g. , a bacterial 3-beta hydroxysteroid dehydrogenase. In some embodiments, the 3- beta hydroxysteroid dehydrogenase is from Bacteroides dorei DSM 17855.
[08] In some embodiments, the bile acid catabolism enzyme cassette comprises 5-alpha reductase, the 5-beta reductase and 3-beta hydroxysteroid dehydrogenase. In some embodiments, the 5-alpha reductase, the 5-beta reductase and 3-beta hydroxysteroid dehydrogenase are all from Bacteroides dorei DSM 17855.
[09] In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA). In some embodiments, the bile acid catabolism enzyme or bile acid catabolism enzyme cassette catabolizes 3-oxo lithocholic acid. In some embodiments, the bile acid catabolism enzyme increases levels of 3-oxo lithocholic acid. In some embodiments, the bile acid catabolism enzyme increases levels of isoallo lithocholic acid (isoalloLCA). In some embodiments, the bile acid catabolism enzyme increases levels of isolithocholic acid (isoLCA).
[010] In one aspect, the disclosure provides for a recombinant bacterium comprising a gene encoding an bile acid catabolism enzyme, wherein the gene encoding the bile acid catabolism enzyme is operably linked to a 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.
[Oi l] 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.
[012] 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.
[013] 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. [014] In some embodiments, the 3-alpha-hydroxysteroid dehydrogenase 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 an 3- alpha-hydroxysteroid dehydrogenase 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.
[015] In some embodiments, the 5-alpha reductase 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: 514. In some embodiments, the gene sequence encoding 5-alpha reductase 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: 515.
[016] In some embodiments, the 5-beta reductase 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 5-beta reductase 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.
[017] In some embodiments, the a 3 -beta hydroxysteroid dehydrogenase 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: 518. In some embodiments, the gene sequence encoding a 3- beta hydroxysteroid dehydrogenase 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: 519.
[018] In some embodiments, 5-alpha reductase, the 5-beta reductase and 3 -beta hydroxysteroid dehydrogenase are present in a cassette and the 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: 514, 516, and 518, respectively. In some embodiments, the gene sequence encoding the 5-beta reductase and 3 -beta hydroxysteroid dehydrogenase encode 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: 515, 517, and 519, respectively.
[019] 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 A. 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.
[020] 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 or 508. In some embodiments, the gene sequence encoding an 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.
[021] 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 an 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.
[022] 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 an 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.
[023] 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.
[024] 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).
[025] 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/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), c/ > 2 (SEQ ID NO: 310), and clbR (SEQ ID NO: 311).
[026] 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 an enzyme diaminopimelic acid synthesis or an enzyme in the thymine biosynthetic pathway.
[027] 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. [028] 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.
[029] 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.
[030] 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.
[031] 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.
[032] In some embodiments, the subject is a human.
[033] In another aspect, the present disclosure provides a recombinant bacterium comprising at least one heterologous 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. [034] In some embodiments, the bile acid catabolism enzyme is a 3 -beta hydroxysteroid dehydrogenase (3[3-HSDH).
[035] In some embodiments, the bile acid catabolism enzyme is a bacterial 3J3-HSDH.
[036] In some embodiments, the 3[3-HSDH is a Bacteroides dorei DSM 17855 3[3-HSDH.
[037] In some embodiments, the gene encoding the 3J3-HSDH 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: 518.
[038] In some embodiments, the gene encoding the 3J3-HSDH 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: 519.
[039] In some embodiments, the recombinant bacterium further comprising a second heterologous gene encoding a second bile acid catabolism enzyme and a third heterologous gene encoding a third bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 5- alpha reductase, and wherein the third bile acid catabolism enzyme is a 5 -beta reductase.
[040] In some embodiments, the 5-alpha reductase is a bacterial 5-alpha reductase.
[041] In some embodiments, the bacterial 5-alpha reductase is a Bacteroides dorei DSM 17855 5-alpha reductase.
[042] In some embodiments, the gene encoding the 5-alpha reductase 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: 514.
[043] In some embodiments, the gene encoding the 5-alpha reductase 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: 515.
[044] In some embodiments, the 5 -beta reductase is a bacterial 5 -beta reductase.
[045] In some embodiments, the bacterial 5 -beta reductase is a Bacteroides dorei DSM 17855 5-beta reductase.
[046] In some embodiments, the gene encoding the 5-beta reductase 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.
[047] In some embodiments, the gene encoding the 5-beta reductase 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.
[048] In some embodiments, the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are present in a gene cassette operably linked to the promoter. [049] In some embodiments, the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are each individually linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature.
[050] In some embodiments, each promoter is a different promoter, or wherein each promoter is a separate copy of the same promoter.
[051] In some embodiments, the recombinant bacterium further comprising a fourth heterologous gene encoding a fourth bile acid catabolism enzyme, wherein the fourth bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
[052] In some embodiments, the 3a-HSDH is a bacterial 3a-HSDH.
[053] In some embodiments, the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
[054] In some embodiments, the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is operably linked to a promoter that is not associated with the fourth gene encoding the fourth bile acid catabolism enzyme in nature.
[055] In some embodiments, the promoter that is operably linked to the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the fourth gene encoding the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme.
[056] In some embodiments, the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme, the second gene encoding the second bile acid catabolism enzyme, and the third gene encoding the third bile acid catabolism enzyme.
[057] In some embodiments, the recombinant bacterium further comprising a second heterologous gene encoding a second bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
[058] In some embodiments, the 3a-HSDH is a bacterial 3a-HSDH.
[059] In some embodiments, the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
[060] In some embodiments, the second heterologous gene encoding the second bile acid catabolism enzyme is operably linked to a promoter that is not associated with the second gene encoding the second bile acid catabolism enzyme in nature.
[061] In some embodiments, the promoter that is operably linked to the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the second gene encoding the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme. [062] In some embodiments, the second heterologous gene encoding the second bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme.
[063] In some embodiments, the bile acid catabolism enzyme is a 3 -alpha-hydroxy steroid dehydrogenase (3a-HSDH).
[064] In some embodiments, the 3a-HSDH is a bacterial 3a-HSDH.
[065] In some embodiments, the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
[066] In some embodiments, the gene encoding the 3a-HSDH 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.
[067] In some embodiments, the gene encoding the 3a-HSDH 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.
[068] In some embodiments, the promoter is a constitutive or an inducible promoter.
[069] In some embodiments, the promotor is an inducible promoter induced by exogenous environmental conditions.
[070] In some embodiments, the inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.
[071] In some embodiments, the inducible promoter is an FNR-inducible promoter.
[072] In some embodiments, the inducible promoter is induced by temperature.
[073] In some embodiments, the inducible promoter is a cI857 promoter.
[074] In some embodiments, the inducible promoter is induced by a chemical inducer.
[075] In some embodiments, the promoter is induced by IPTG.
[076] In some embodiments, the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a plasmid in the recombinant bacterium.
[077] In some embodiments, the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a chromosome in the recombinant bacterium.
[078] In some embodiments, the recombinant bacterium is a non-pathogenic bacterium.
[079] In some embodiments, the recombinant bacterium is a probiotic or a commensal bacterium.
[080] In some embodiments, the recombinant bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
[081] In some embodiments, the recombinant bacterium is Escherichia coli strain Nissle.
[082] In some embodiments, the recombinant bacterium further comprising a heterologous gene encoding a bile acid importer or transporter. [083] In some embodiments, the importer or transporter is a homolog of human ASBT, baiG or PanS.
[084] In some embodiments, the homologs of human ASBT is from Neisseria meningitidis (ASBTNM), from Yersinia frederiksenii (ASBTYf), from E. coli M34 or from E. coli VREC0334.
[085] In some embodiments, the gene encoding the bile acid transporter 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.
[086] In some embodiments, the gene 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: 503, 505, 507, 509, 511 or 513.
[087] In some embodiments, the bile acid importer or transporter is baiG from Eubacterium sp. strain VPI 12708.
[088] In some embodiments, the gene encoding the bile acid transporter 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.
[089] In some embodiments, the gene 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: 513.
[090] In some embodiments, the PanS is from Escherichia coli EC23.
[091] In some embodiments, the gene encoding the bile acid transporter 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.
[092] In some embodiments, the gene 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: 511.
[093] In some embodiments, the recombinant bacterium further comprising an insertion, deletion or mutation of an endogenous phage gene.
[094] In some embodiments, the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.
[095] In some embodiments, the recombinant bacterium further comprising a modified endogenous colibactin island.
[096] 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).
[097] 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/^ (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).
[098] 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.
[099] In some embodiments, the auxotrophy is in an enzyme diaminopimelic acid synthesis or an enzyme in the thymine biosynthetic pathway.
[0100] In some embodiments, the bile acid catabolism enzyme catabolizes a secondary bile acid.
[0101] In some embodiments, the bile acid catabolism enzyme catabolizes lithocholic acid (LCA).
[0102] In some embodiments, the bile acid catabolism enzyme catabolizes 3-oxo lithocholic acid.
[0103] In some embodiments, the bile acid catabolism enzyme is capable of producing 3-oxo lithocholic acid.
[0104] In some embodiments, the bile acid catabolism enzyme is capable of producing isoallolithocholic acid (isoalloLCA).
[0105] In some embodiments, the bile acid catabolism enzyme is capable of producing isolithocholic acid (isoLCA).
[0106] In some embodiments, the bacterium produces isoalloLCA at a rate of at least about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.45 nmol/h/le9 cells, or at least about 0.5 nmol/h/le9 cells.
[0107] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.02 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.1 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 0.45 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 0.35 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 0.25 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.25 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 0.45 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, or about 0.45 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells in vitro.
[0108] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.025 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells.
[0109] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
[0110] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.45 nmol/h/le9 cells, or at least about 0.5 nmol/h/le9 cells.
[0111] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.02 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.1 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 0.45 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 0.35 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 0.25 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.25 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 0.45 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, or about 0.45 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells in vitro.
[0112] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
[0113] In another aspect, the present disclosure provides a pharmaceutically acceptable composition comprising the recombinant bacterium as provided herein; and a pharmaceutically acceptable carrier.
[0114] In some embodiments, the composition is formulated for oral administration.
[0115] In another aspect, the present disclosure provides 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.
[0116] In some embodiments, the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.
[0117] 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.
[0118] In some embodiments, the disease or disorder is 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-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 disease. [0119] In some embodiments, the disease or disorder is ulcerative colitis or Crohn’s disease.
[0120] 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.
[0121] In some embodiments, the subject has a decreased level of a secondary bile acid in the gut after the composition is administered.
[0122] In some embodiments, the bile acid is lithocholic acid (LCA).
[0123] In some embodiments, the subject has an increased level of isoalloLCA in the gut after the composition is administered.
[0124] In another aspect, the present disclosure provides a method of decreasing a level of a secondary bile acid in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition as provided herein, thereby decreasing the level of the secondary bile acid in the gut of the subject.
[0125] In some embodiments, the level of secondary bile acid in the gut is decreased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
[0126] In some embodiments, the level of secondary bile acid in the gut is decreased by about 10% to about 200%, about 10% to about 190%, about 10% to about 180%, about 10% to about
170%, about 10% to about 160%, about 10% to about 150%, about 10% to about 140%, about 10% to about 130%, about 10% to about 120%, about 10% to about 110%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 200%, about 20% to about 190%, about 20% to about 180%, about 20% to about 170%, about 20% to about 160%, about 20% to about 150%, about 20% to about 140%, about 20% to about 130%, about 20% to about 120%, about 20% to about 110%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 200%, about 30% to about 190%, about 30% to about 180%, about 30% to about 170%, about 30% to about 160%, about 30% to about 150%, about 30% to about 140%, about 30% to about 130%, about 30% to about 120%, about 30% to about 110%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 200%, about 40% to about 190%, about 40% to about 180%, about 40% to about 170%, about 40% to about 160%, about 40% to about 150%, about 40% to about 140%, about 40% to about 130%, about 40% to about 120%, about 40% to about 110%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 200%, about 50% to about 190%, about 50% to about 180%, about 50% to about 170%, about 50% to about 160%, about 50% to about 150%, about 50% to about 140%, about 50% to about 130%, about 50% to about 120%, about 50% to about 110%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 200%, about 60% to about 190%, about 60% to about 180%, about 60% to about 170%, about 60% to about 160%, about 60% to about 150%, about 60% to about 140%, about 60% to about 130%, about 60% to about 120%, about 60% to about 110%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 200%, about 70% to about 190%, about 70% to about 180%, about 70% to about 170%, about 70% to about 160%, about 70% to about 150%, about 70% to about 140%, about 70% to about 130%, about 70% to about 120%, about 70% to about 110%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 200%, about 80% to about 190%, about 80% to about 180%, about 80% to about 170%, about 80% to about 160%, about 80% to about 150%, about 80% to about 140%, about 80% to about 130%, about 80% to about 120%, about 80% to about 110%, about 80% to about 100%, about 80% to about 90%, about 90% to about 200%, about 90% to about 190%, about 90% to about 180%, about 90% to about 170%, about 90% to about 160%, about 90% to about 150%, about 90% to about 140%, about 90% to about 130%, about 90% to about 120%, about 90% to about 110%, about 90% to about 100%, about 100% to about 200%, about 100% to about 190%, about 100% to about 180%, about 100% to about 170%, about 100% to about 160%, about 100% to about 150%, about 100% to about 140%, about 100% to about 130%, about 100% to about 120%, about 100% to about 110%, about 110% to about 200%, about 110% to about 190%, about 110% to about 180%, about 110% to about 170%, about 110% to about 160%, about 110% to about 150%, about 110% to about 140%, about 110% to about 130%, about 110% to about 120%, about 120% to about 200%, about 120% to about 190%, about 120% to about 180%, about 120% to about 170%, about 120% to about 160%, about 120% to about 150%, about 120% to about 140%, about 120% to about 130%, about 130% to about 200%, about 130% to about 190%, about 130% to about 180%, about 130% to about 170%, about 130% to about 160%, about 130% to about 150%, about 130% to about 140%, about 140% to about 200%, about 140% to about 190%, about 140% to about 180%, about 140% to about 170%, about 140% to about 160%, about 140% to about 150%, about 150% to about 200%, about 150% to about 190%, about 150% to about 180%, about 150% to about 170%, about 150% to about 160%, about 160% to about 200%, about 160% to about 190%, about 160% to about 180%, about 160% to about 170%, about 170% to about 200%, about 170% to about 190%, about 170% to about 180%, about 180% to about 200%, about 180% to about 190%, or about 190% to about 200%.
[0127] In some embodiments, the secondary bile acid is LCA. [0128] In some embodiments, the subject has an increased level of isoalloLCA, 3-oxoLCA and/or isoLCA in the gut after administration.
[0129] In another aspect, the present disclosure provides a method of increasing a level of isoalloLCA, 3-oxoLCA and/or isoLCA in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition as provided herein, thereby increasing the level of the isoalloLCA in the gut of the subject.
[0130] In some embodiments, the level of isoalloLCA in the gut is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
[0131] In some embodiments, the level of isoalloLCA in the gut is increased by at least about 10% to about 200%, about 10% to about 190%, about 10% to about 180%, about 10% to about 170%, about 10% to about 160%, about 10% to about 150%, about 10% to about 140%, about 10% to about 130%, about 10% to about 120%, about 10% to about 110%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 200%, about 20% to about 190%, about 20% to about 180%, about 20% to about 170%, about 20% to about 160%, about 20% to about 150%, about 20% to about 140%, about 20% to about 130%, about 20% to about 120%, about 20% to about 110%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 200%, about
30% to about 190%, about 30% to about 180%, about 30% to about 170%, about 30% to about 160%, about 30% to about 150%, about 30% to about 140%, about 30% to about 130%, about 30% to about 120%, about 30% to about 110%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 200%, about 40% to about 190%, about 40% to about 180%, about 40% to about 170%, about 40% to about 160%, about 40% to about 150%, about 40% to about 140%, about 40% to about 130%, about 40% to about 120%, about 40% to about 110%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 200%, about 50% to about 190%, about 50% to about 180%, about 50% to about 170%, about 50% to about 160%, about 50% to about 150%, about 50% to about 140%, about 50% to about 130%, about 50% to about 120%, about 50% to about 110%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 200%, about 60% to about 190%, about 60% to about 180%, about 60% to about 170%, about 60% to about 160%, about 60% to about 150%, about 60% to about 140%, about 60% to about 130%, about 60% to about 120%, about 60% to about
110%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 200%, about 70% to about 190%, about 70% to about 180%, about 70% to about 170%, about 70% to about 160%, about 70% to about 150%, about 70% to about 140%, about 70% to about 130%, about 70% to about 120%, about 70% to about 110%, about 70% to about
100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 200%, about 80% to about 190%, about 80% to about 180%, about 80% to about 170%, about 80% to about 160%, about 80% to about 150%, about 80% to about 140%, about 80% to about 130%, about 80% to about 120%, about 80% to about 110%, about 80% to about 100%, about 80% to about 90%, about 90% to about 200%, about 90% to about 190%, about 90% to about 180%, about 90% to about 170%, about 90% to about 160%, about 90% to about 150%, about 90% to about 140%, about 90% to about 130%, about 90% to about 120%, about 90% to about 110%, about 90% to about 100%, about 100% to about 200%, about 100% to about 190%, about 100% to about 180%, about 100% to about 170%, about 100% to about 160%, about 100% to about 150%, about 100% to about 140%, about 100% to about 130%, about 100% to about 120%, about 100% to about 110%, about 110% to about 200%, about 110% to about 190%, about 110% to about 180%, about 110% to about 170%, about 110% to about 160%, about 110% to about 150%, about 110% to about 140%, about 110% to about 130%, about 110% to about 120%, about 120% to about 200%, about 120% to about 190%, about 120% to about 180%, about 120% to about 170%, about 120% to about 160%, about 120% to about 150%, about 120% to about 140%, about 120% to about 130%, about 130% to about 200%, about 130% to about 190%, about 130% to about 180%, about 130% to about 170%, about 130% to about 160%, about 130% to about 150%, about 130% to about 140%, about 140% to about 200%, about 140% to about 190%, about 140% to about 180%, about 140% to about 170%, about 140% to about 160%, about 140% to about 150%, about 150% to about 200%, about 150% to about 190%, about 150% to about 180%, about 150% to about 170%, about 150% to about 160%, about 160% to about 200%, about 160% to about 190%, about 160% to about 180%, about 160% to about 170%, about 170% to about 200%, about 170% to about 190%, about 170% to about 180%, about 180% to about 200%, about 180% to about 190%, or about 190% to about 200%.
[0132] In some embodiments, the level of isoalloLCA in the gut is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%.
[0133] In some embodiments, the subject is a human.
Brief Description of the Drawings
[0134] FIG. 1 provides a schematic showing a genetically engineered bacterium which is capable of converting a toxic bile acid into a non-toxic bile acid. [0135] FIG. 2A depicts a schematic showing the catabolism of lithohcholic acid (LCA) to 3- oxo-LCA catalyzed by 3-alpha-hydroxysteroid dehydrogenase, e.g., by 3 -alpha-hydroxy steroid dehydrogenase from Eggerthella lenta.
[0136] FIG. 2B depicts a schematic showing the catabolism of 3-oxo-LCA to isoalloLCA.
[0137] FIG. 2C depicts a schematic showing the genes encoding the enzymes involved in catabolism of 3-oxo-LCA to isoalloLCA, 5-alpha reductase, 5-beta reductase, and 3-beta hydroxysteroid dehydrogenase, which are derived from three bacterial strains, Parabacteroides merdae, B. dorei and Bacteroides vulgatus. An exemplary engineered bacterium of the disclosure may comprise gene sequences encoding 3-alpha-hydroxysteroid dehydrogenase or a circuit for the catabolism of 3-oxo-LCA to isoalloLCA, or both.
[0138] FIG. 3 is a diagram showing 3-oxo-LCA transformation in EcN recombinant cells. SYN8590, engineered to express a 3-gene pathway to convert 3-oxo-LCA to isoalloLCA. FIG. 3 depicts a LC-MS/MS trace showing a peak consistent with isoalloLCA. The peak is seen in supernatant of the prototype SYN8590 after incubation with substrate 3-oxoLCA and is not seen from the WT EcN or Media only controls. Bacteria were incubated with 3-oxo-LCA in M9 media for 30 mins. The 3-oxoLCA and isoalloLCA were quantified by LCMS/MS. The peak at -1.77 min is isoalloLCA. Samples analyzed were media, control EcN, and engineered SYN8590.
[0139] FIGs. 4A, 4B, and 4C depict schematics of exemplary LCA and 3-oxoLCA converting prototype strains. FIG. 4A depicts SYN8630, which utilizes a four enzyme pathway to convert LCA to isoalloLCA. FIG. 4B depicts SYN8590, which utilizes the last three enzymes of the same pathway to convert 3-oxoLCA to isoalloLCA. FIG. 4C depicts SYN8629, which utilizes only the first enzyme of the pathway to convert LCA to 3-oxoLCA.
[0140] FIG. 5 depicts a schematic showing the conversion pathways active in the exemplary prototype strains of the disclosure.
[0141] FIGs. 6A and 6B depict graphs showing activity of LCA catabolizing prototypes in vitro. FIG. 6A: SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively. FIG. 6B: SYN8629 produces 3-oxoLCA from LCA (it does not produce isoalloLCA as it is lacking the 3 enzyme operon involved).
[0142] FIGs. 6C and 6D depict graphs showing rate of activity of LCA catabolizing prototypes in vitro. FIG. 6C: rate at which SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively. FIG. 6D: rate at which SYN8629 produces 3-oxoLCA from LCA.
[0143] FIGs. 7A and 7B depict graphs showing the depletion of starting substrate by prototypes. FIG. 7A: Depletion of 3-oxoLCA by SYN8590, as it is converted to isoalloLCA, isoLCA and intermediates. FIG. 7B: Depletion of LCA by SYN8629 and SYN8630, as it is converted to 3- oxoLCA (SYN8629) or isoalloLCA, isoLCA and intermediates (SYN8630).
[0144] FIGs. 8A, 8B, and 8C depict graphs showing the accumulation of intermediates of LCA or 3-oxoLCA conversion to isoalloLCA in vitro. FIG. 8A: 3-oxo-A4-LCA accumulation by SYN8590 and SYN8630, starting in media containing either 3-oxoLCA or LCA, respectively. FIG. 8B: A4-isoLCA accumulation by SYN8590 and SYN8630, starting in media containing either 3- oxoLCA or LCA respectively. FIG. 8C: 3-oxoLCA accumulation by SYN8360, starting in media containing LCA.
[0145] FIGs. 9A, 9B, and 9C depict graphs and schematics of production of isoLCA in vitro. FIG. 9A: isoLCA is produced by SYN8590 from 3-oxoLCA and by SYN8360 from LCA. FIG. 9B: Potential enzyme pathway by which the strains could both produce isoLCA from 3-oxoLCA via the 3J3-HSDH. FIG. 9C: Potential enzyme pathway by which the strains could both produce isoLCA from A4-isoLCA via the 5[3-reductase.
Detailed Description
[0146] 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.
[0147] 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.
[0148] 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. [0149] 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.
[0150] 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. The recombinant bacteria are capable of catabolizing in low-oxygen environments, e.g., the gut.
[0151] The bile acid isoallolithocholic acid (isoalloLCA) has been shown to enhance the differentiation of anti-inflammatory regulatory T cells (Treg cells) and its biosynthetic genes are significantly reduced in patients with inflammatory bowel diseases. Without wishing to be bound by theory, increasing levels of isoalloLCA, e.g., by administration of a bacterium disclosed herein, may promote gut homeostasis in a human subject in having an inflammatory bowel disease.
[0152] 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.
[0153] 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 catabolism enzyme or a gene cassette of bile acid catabolism enzymes 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.
[0154] In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid catabolism enzyme(s) or a gene cassette of bile acid catabolism enzymes and is capable of processing (e.g., metabolizing or catabolizing) and reducing levels of BA, e.g., lithocholic acid. Alternatively, or in addition, in some embodiments, the bacteria are capable of producing isoalloLCA. In some embodiments, a bacterial cell disclosed herein has been genetically engineered to comprise a heterologous gene sequence encoding one or more bile acid catabolism enzyme(s) or a gene cassette of bile acid catabolism enzymes 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 more gut-beneficial metabolite, which can promote gut homeostasis, such as isoalloLCA, 3-oxoLCA, or isoLCA, in order to treat and/or prevent diseases associated with inflammation, such as IBD, Crohn’s disease or ulcerative colitis. For example, in the lamina propria, 3-oxoLCA and isoalloLCA regulate T cell effector functions; 3-oxoLCA binds to the transcription factor RORyt and suppresses Th 17 cell differentiation, while isoalloLCA promotes Treg cell differentiation (Hang et al. Bile acid metabolites control TH17 and Treg cell differentiation. Nature. 2019;576(7785): 143-148. doi: 10.1038/s41586- 019-1785-z). IsoLCA and 3-oxoLCA both suppress differentiation of T helper 17 cells, which are associated with inflammation, and, moreover, both bile acids are significantly decreased in patients with IBD. (Paik et al., Human gut bacteria produce TH17-modulating bile acid metabolites. Nature. 2022 Mar; 603(7903): 907-912).
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] As used herein, the term “bactericidal” refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.
[0161] 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. [0162] 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.
[0163] “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 or gene cassette encoding at least one bile acid catabolism enzyme, 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.
[0164] 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.
[0165] 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 Parac 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.
[0166] 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.
[0167] 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
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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., 23TA-T, 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)).
[0173] 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.
[0174] “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 and 42 C.
[0175] 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.
[0176] 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).
[0177] 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. [0178] Examples of oxygen level -dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et a/., 1998; Hoeren et al., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1.
[0179] In a non -limiting example, a promoter (PfhrS) was derived from the E. coll 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
Figure imgf000034_0001
[0180] 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.
[0181] “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 os 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 oA promoter (e.g. , promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PiiaG (BBa_K823000), PiepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis oBpromoter (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.
[0182] “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.
[0183] 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.
[0184] 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% CL.etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
Table 2
Compartment
Figure imgf000037_0002
Oxygen Tension
Figure imgf000037_0001
Figure imgf000038_0001
[0185] “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.
[0186] “Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria 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.
[0187] “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.
[0188] 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.
[0189] 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).
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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)) 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.
[0196] 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. [0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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 an bile acid transporter” should be understood to mean “at least one heterologous gene encoding at least one bile acid transporter.”
[0210] 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.
[0211] 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.
[0212] 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
[0213] 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.
[0214] 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 .
[0215] In one embodiment, the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.
[0216] In some embodiments, the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterobacteriaceae 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).
[0217] In one embodiment, the recombinant bacterial cell does not colonize the subject.
[0218] 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., an bile acid catabolism gene from Homo sapiens can be expressed in Escherichia coli.
[0219] 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.
[0220] 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, 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, nine-fold, or ten-fold in the media of the cell culture. In one embodiment, the levels of an bile acid, e.g., lithocholic acid, are reduced below the limit of detection in the media of the cell culture.
[0221] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 or bile acid catabolism enzyme cassette 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.
[0222] In some embodiments of the above described genetically engineered bacteria, the gene encoding a s bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.
[0223] 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. [0224] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette 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 catabolism enzyme or bile acid catabolism enzyme cassette is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is chemically induced.
[0225] In some embodiments of the above described genetically engineered bacteria, the gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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.
[0226] 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 tdhyA and dapA auxotroph.
[0227] 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
[0228] Bile acid catabolism enzymes 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 (LCA) into isoalloLCA or 3-oxo-LCA. For example, the genetically engineered bacteria comprising at least one heterologous gene encoding a bile acid catabolism enzyme or a bile acid catabolism enzyme cassette can catabolize lithocholic acid to isoalloLCA or 3-oxo-LCA 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.
[0229] 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.
[0230] Bile acid catabolism enzymes are well-known in the art. For example 3-alpha- hydroxysteroid dehydrogenase a catabolizes lithohcholic acid (LCA) to 3-oxo-LCA. Hydroxysteroid dehydrogenases (HSDH) are expressed by both host tissues and host-associated microbiota that modify the hydroxyl groups on steroids such as bile acids. Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids through the generation of oxo-bile acid with implications for increased/decreased toxicity for the host.
[0231] Other examples include 5-beta reductase, which can convert 3-oxoLCA to 3-oxo-A4- LCA, and 3-beta hydroxysteroid dehydrogenase, which can convert 3-oxo-A4-LCA to A4-isoLCA or 3-oxoalloLCA to isoalloLCA. Another example is 5-alpha reductase, which can convert 3-oxo-A4- LCA to 3-oxoalloLCA or convert 3-oxo-A4-LCA to A4isoLCA to isoalloLCA.
[0232] Taken together, these three bile acid catabolism enzymes can together catabolize 3- oxo-LCA to isoalloLCA (i.e., 5-alpha reductase, 5-beta reductase, and 3-beta hydroxysteroid dehydrogenase). A gene cluster was identified in the gut bacterial phylum Bacteroidetes containing these three enzymes, which are responsible for responsible for isoalloLCA production from the gut metabolite 3-oxoLCA (Li et al., Cell Host & Microbe 29, 1366-1377; September 8, 2021). The cluster was found in many prevalent Bacteroidetes strains, suggesting that this pathway may play an important role in the gut environment. Accordingly, in some embodiments, the bacteria of the disclosure comprise gene sequence(s) or gene cassettes encoding 5-alpha reductase, 5-beta reductase, and 3-beta hydroxysteroid dehydrogenase. In some embodiments, the bacteria comprise gene sequences encoding Bacteroides dorei DSM 17855 5-alpha reductase, Bacteroides dorei DSM 17855 5-beta reductase and/or Bacteroides dorei DSM 17855 3-beta hydroxysteroid dehydrogenase. In some embodiments, the bacteria, in addition to the three enzyme cassette, further comprise 3-alpha- hydroxysteroid dehydrogenase, e.g., from A. lenta.
[0233] 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.
[0234] In some embodiments, the bile acid catabolism enzyme is a Hydroxysteroid dehydrogenases (HSDH). In some embodiments, the HSDH is a 3-alpha-hydroxysteroid dehydrogenase . In one embodiments, the 3-alpha-hydroxysteroid dehydrogenase is from Eggerthella lenta.
[0235] In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming 3-oxoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming 3-oxoLCA, 3-oxo-A4-LCA, 3-oxoalloLCA and/or A4- isoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from 3-oxoLCA, 3- oxo-A4-LCA, 3-oxoalloLCA, and/or A4-isoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing A4-isoLCA, e.g., from 3-oxoLCA and/or 3-oxo-A4-LCA. . In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoalloLCA, e.g., from 3-oxoLCA and/or 3-oxo-A4-LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-A4-LCA, e.g., from 3-oxoLCA.
[0236] In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoLCA, e.g., from LCA or from 3-oxoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoLCA, e.g., from A4-isoLCA.
[0237] In one embodiment, the genetically engineered bacterium is capable of producing isoalloLCA, e.g., from 3-oxoLCA at a rate of at least about 0.15 nmol/h/le9 cells. In one embodiment, the genetically engineered bacterium is capable of producing isoalloLCA, e.g., from 3- oxoLCA at a rate of at least about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells; 0.1 nmol/h/le cells to about 0.2 nmol/h/le9 cells; or about 0.125 nmol/h/le9 cells to about 1.175 nmol/h/le9 cells. In one embodiment, the genetically engineered bacterium comprises gene(s) encoding one or more of 5 -alpha reductase, 5 -beta reductase, and 3 -beta hydroxysteroid dehydrogenase from B. dorei.
[0238] In some embodiments, the bacterium produces isoalloLCA at a rate of at least about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.45 nmol/h/le9 cells, or at least about 0.5 nmol/h/le9 cells.
[0239] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.02 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.1 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 0.45 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 0.35 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 0.25 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.25 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 0.45 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, or about 0.45 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells in vitro..
[0240] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.025 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
[0241] In some embodiments, the bacterium produces isoalloLCA at a rate of about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
[0242] In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoLCA, e.g., from LCA.
[0243] In one embodiment, the genetically engineered bacterium produces 3-oxoLCA, e.g., from LCA at a rate of at least about 0.15 nmol/h/le9 cells. In one embodiment, the genetically engineered bacterium comprises gene(s) encoding 3 -alpha hydroxysteroid dehydrogenase from Eggerthella lenta.
[0244] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells, at least about 0.02 nmol/h/le9 cells, at least about 0.03 nmol/h/le9 cells, at least about 0.04 nmol/h/le9 cells, at least about 0.05 nmol/h/le9 cells, at least about 0.06 nmol/h/le9 cells, at least about 0.07 nmol/h/le9 cells, at least about 0.08 nmol/h/le9 cells, at least about 0.09 nmol/h/le9 cells, at least about 0.1 nmol/h/le9 cells, at least about 0.15 nmol/h/le9 cells, at least about 0.2 nmol/h/le9 cells, at least about 0.25 nmol/h/le9 cells, at least about 0.3 nmol/h/le9 cells, at least about 0.35 nmol/h/le9 cells, at least about 0.4 nmol/h/le9 cells, at least about 0.45 nmol/h/le9 cells, or at least about 0.5 nmol/h/le9 cells.
[0245] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.01 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.01 nmol/h/le9 cells to about 0.02 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.02 nmol/h/le9 cells to about 0.03 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.03 nmol/h/le9 cells to about 0.04 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.04 nmol/h/le9 cells to about 0.05 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.05 nmol/h/le9 cells to about 0.06 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.06 nmol/h/le9 cells to about 0.07 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.07 nmol/h/le9 cells to about 0.08 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.1 nmol/h/le9 cells, about 0.08 nmol/h/le9 cells to about 0.09 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.09 nmol/h/le9 cells to about 0.1 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 0.45 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 0.35 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 0.25 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.1 nmol/h/le9 cells to about 0.15 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.25 nmol/h/le9 cells, about 0.15 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.25 nmol/h/le9 cells to about 0.25 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 0.45 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.3 nmol/h/le9 cells to about 0.3 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.4 nmol/h/le9 cells, about 0.35 nmol/h/le9 cells to about 0.35 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells, about 0.4 nmol/h/le9 cells to about 0.45 nmol/h/le9 cells, or about 0.45 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells in vitro.
[0246] In some embodiments, the bacterium produces 3-oxoLCA at a rate of about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
[0247] In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of consuming LCA, 3-oxo-LCA, 3-oxo-A4-LCA, 3-oxoalloLCA and/or A4-isoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing isoalloLCA, e.g., from 3-oxo-LCA, 3- oxo-A4-LCA, 3-oxoalloLCA and/or A4-isoLCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing A4-isoLCA, e.g., from LCA, 3-oxo-LCA and/or 3-oxo-A4-LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxoalloLCA, e.g., from LCA, 3-oxo-LCA and/or 3-oxo-A4- LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-A4-LCA, e.g., from LCA and/or 3-oxo-LCA. In one embodiment, the genetically engineered bacterium comprising gene(s) encoding one or more bile acid catabolism enzyme(s) is capable of producing 3-oxo-LCA, e.g., from LCA.
[0248] In one embodiment, the genetically engineered bacterium produces isoalloLCA, e.g. , from LCA at a rate of at least about 0.04 nmol/h/le9 cells. In one embodiment, the genetically engineered bacterium comprises gene(s) encoding one or more of 5-alpha reductase, 5-beta reductase, and 3 -beta hydroxy steroid dehydrogenase from B. dorei and 3 -alpha hydroxy steroid dehydrogenase from Eggerthella lenta.
[0249] In some embodiments, the 3-alpha-hydroxysteroid dehydrogenase gene has at least about 80% identity with the sequence of SEQ ID NO: 500. Accordingly, In some embodiments, the 3-alpha-hydroxysteroid dehydrogenase gene has at least about 90% identity with the sequence of SEQ ID NO: 500. Accordingly, in some embodiments, the 5-alpha reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 500. Accordingly, in some embodiments, the 3-alpha- hydroxysteroid dehydrogenase 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 3-alpha-hydroxysteroid dehydrogenase gene comprises the sequence of SEQ ID NO: 500. In yet another embodiment the 3-alpha-hydroxysteroid dehydrogenase gene consists of the sequence of SEQ ID NO: 500.
[0250] In some embodiments, the gene sequence encoding an 3-alpha-hydroxysteroid dehydrogenase 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.
[0251] In some embodiments, the bile acid catabolism enzyme is a 5-alpha reductase. In some embodiments, the 5-alpha reductase is from Bacteroides dorei DSM 17855.
[0252] In some embodiments, the 5-alpha reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 514. Accordingly, in some embodiments, the 5-alpha reductase gene has at least about 90% identity with the sequence of SEQ ID NO: 514. In some embodiments, the 5-alpha reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 514. Accordingly, in some embodiments, the 5-alpha reductase 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: 514. In another embodiment, the 5-alpha reductase gene comprises the sequence of SEQ ID NO: 514. In yet another embodiment the 5-alpha reductase gene consists of the sequence of SEQ ID NO: 514.
[0253] In some embodiments, the gene sequence encoding a 5-alpha reductase 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: 515.
[0254] In some embodiments, the bile acid catabolism enzyme is a 5-beta reductase. In some embodiments, the 5-beta reductase is from Bacteroides dorei DSM 17855. In some embodiments, the 5-beta reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 516. In some embodiments, the 5 -beta reductase gene has at least about 90% identity with the sequence of SEQ ID NO: 516. Accordingly, In some embodiments, the 5-beta reductase gene has at least about 95% identity with the sequence of SEQ ID NO: 516. In some embodiments, the 5-beta reductase 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 5-beta reductase gene comprises the sequence of SEQ ID NO: 516. In yet another embodiment the 5-beta reductase gene consists of the sequence of SEQ ID NO: 516.
[0255] In some embodiments, the gene sequence encoding a 5 beta reductase 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: 517.
[0256] In some embodiments, the bile acid catabolism enzyme is a 3-beta hydroxysteroid dehydrogenase. In some embodiments, the 3-beta hydroxysteroid dehydrogenaseis from Bacteroides dorei DSM 17855. In some embodiments, the 5-beta reductase gene has at least about 80% identity with the sequence of SEQ ID NO: 518. Accordingly, In some embodiments, the 3-beta hydroxysteroid dehydrogenase gene has at least about 90% identity with the sequence of SEQ ID NO: 518. Accordingly, In some embodiments, the 3-beta hydroxysteroid dehydrogenase gene has at least about 95% identity with the sequence of SEQ ID NO: 518. Accordingly, In some embodiments, the 3-beta hydroxysteroid dehydrogenase 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: 518. In another embodiment, the 3-beta hydroxysteroid dehydrogenase gene comprises the sequence of SEQ ID NO: 518. In yet another embodiment the 3-beta hydroxysteroid dehydrogenase gene consists of the sequence of SEQ ID NO: 518.
[0257] In some embodiments, the gene sequence encoding a 3-beta hydroxysteroid dehydrogenase 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: 519.
[0258] In some embodiments, the bile acid catabolism cassette comprises gene sequences encoding a 5 -alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase. In some embodiments, he bile acid catabolism cassette comprises gene sequences encoding a Bacteroides dorei DSM 17855 5-alpha reductase, a Bacteroides dorei DSM 17855 5-beta reductase, and a Bacteroides dorei DSM 17855 3-beta hydroxysteroid dehydrogenase.
[0259] In some embodiments, the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 80% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively. Accordingly, in some embodiments, the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 90% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively. Accordingly, in some embodiments, the gene sequences in a cassete encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 95% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively. Accordingly, In some embodiments, the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences of SEQ ID NO: 514, 516 and 518, respectively. In another embodiment the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase comprise the sequence of SEQ ID NO: 514, 516 and 518, respectively. In yet another embodiment the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase consist of the sequence of SEQ ID NO: 514, 516 and 518, respectively.
[0260] In some embodiments, the gene sequences in a cassette encoding a 5-alpha reductase, a 5-beta reductase, and a 3-beta hydroxysteroid dehydrogenase have encode an polypeptides that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to, comprises, or consists of SEQ ID NO: 515, 517 and 519, respectively.
[0261] Assays for testing the activity of a bile acid catabolism enzyme, are known, to one of ordinary skill in the art.
[0262] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene(s) for producing a bile acid catabolism enzyme or bile acid catabolism enzyme cassette, such that the bile acid catabolism enzyme or cassette 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 catabolism enzyme or cassette. In some embodiments, the gene(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile acid catabolism enzyme or bile acid catabolism enzyme cassette. In some embodiments, the gene(s) encoding the bile acid catabolism enzyme or bile acid catabolism enzyme cassette are expressed on a chromosome.
[0263] 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(s) encoding a particular bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at four different insertion sites. Altematively, the genetically engineered bacteria may include three copies of the gene(s) encoding a particular bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at three different insertion sites and three copies of the gene(s) encoding a different bile acid catabolism enzyme or bile acid catabolism enzyme cassette inserted at three different insertion sites.
[0264] In some embodiments, under conditions where the bile acid catabolism enzyme or bile acid catabolism enzyme cassette is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the bile acid catabolism enzyme or bile acid catabolism enzyme cassette, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.
[0265] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the bile acid catabolism enzyme or bile acid catabolism enzyme cassette gene(s). Primers specific for bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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).
[0266] 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 bile acid catabolism enzyme or bile acid catabolism enzyme cassette 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 or bile acid catabolism enzyme cassette 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 bile acid catabolism enzyme or bile acid catabolism enzyme cassette gene(s).
[0267] In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile acid catabolism enzyme or bile acid catabolism enzyme cassette. In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile acid transporter and a heterologous gene encoding bile acid catabolism enzyme or bile acid catabolism enzyme cassette. 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 bile acid catabolism enzyme or bile acid catabolism enzyme cassette, and a genetic modification that reduces export of bile acids. Transporters and exporters are described in more detail in the subsections, below.
B, Transporters of Bile acids
[0268] 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.
[0269] 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). [0270] 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)).
[0271] 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.
[0272] In some embodiments, the gene sequence encoding an 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.
[0273] 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.
[0274] In some embodiments, the gene sequence encoding an bile acid transporter, e.g. , an ASBT analog, 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: 505.
[0275] 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.
[0276] In some embodiments, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, 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: 507.
[0277] 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, 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: 506. 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.
[0278] In some embodiments, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, 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: 509.
[0279] In some embodiment, the bile acid transporter is 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, 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.
[0280] In some embodiments, the gene sequence encoding a bile acid transporter, e.g. , an ASBT analog, 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: 511.
[0281] In one embodiment, the bile acid transporter is BaiG or a homolog thereof. In some embodiments, the BaiG is from Eubacterium 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, 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.
[0282] In some embodiments, the gene sequence encoding an 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.
[0283] 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
[0284] 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, 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 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
[0285] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the polypeptides disclosed herein, e.g., bile acid catabolism enzyme(s) and/or bile acid 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, a bacterial cell comprises two or more distinct bile acid catabolism enzyme(s), genes or operons, e.g., two or more genes or operons. In some embodiments, bacterial cell comprises three or more distinct bile acid catabolism enzyme gene(s) or operons, e.g., three or more bile acid catabolism enzyme(s) gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct bile acid catabolism enzyme gene(s) or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more bile acid catabolism enzyme(s) genes.
[0286] In some embodiments, a bacterial cell comprises three or more distinct bile acid transporter gene(s) or operons, e.g., three or more bile acid transporter gene(s). In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct bile acid transporter genes, or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more s bile acid transporter gene(s).
[0287] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same bile acid catabolism enzyme(s) and /or bile acid transporter gene(s). In some embodiments, the gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid transporter, is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the bile acid catabolism enzyme(s), and/or bile acid 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 bile acid catabolism enzyme(s), and/or bile acid transporter is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the bile acid catabolism enzyme(s), and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline, arabinose or Isopropyl B-D- 1 -thiogalactopyranoside (IPTG) .
[0288] In some embodiments, the inducible promoter is an 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
Figure imgf000067_0001
Figure imgf000068_0001
[0289] In some embodiments, the promoter that is operably linked to the gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid transporter is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding bile acid catabolism enzyme(s) and/or bile acid 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.
[0290] In certain embodiments, the bacterial cell comprises a gene encoding a polypeptide described herein, e.g., bile acid catabolism enzyme(s) and/or bile acid 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 table below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
Table 4
Figure imgf000068_0002
Figure imgf000069_0001
[0291] 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.
[0292] 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 bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid 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.
[0293] 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 bile acid catabolism enzyme(s) and/or bile acid 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 wildtype activity.
[0294] 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 bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid 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).
[0295] 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 bile acid catabolism enzyme(s) and/or bile acid transporter are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid transporter. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the bile acid catabolism enzyme(s) and/or bile acid transporter. In some embodiments, the transcriptional regulator and the bile acid catabolism enzyme(s) and/or bile acid transporter are divergently transcribed from a promoter region.
[0296] 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 bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid 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
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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).
[0301] 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.
[0302] 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, comprising or consisting 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, comprising or consisting 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, comprising or consisting 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, comprising or consisting 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, comprising or consisting of the polypeptide encoded by any of the sequences 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, comprising or consisting 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, comprising or consisting 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, comprising or consisting of the polypeptide encoded by SEQ ID NO: 25.
[0303] SEQ ID NOs: 19-25 are shown in Table 5.
Table 5: Inducible promoter construct sequences and related elements
Figure imgf000073_0001
Figure imgf000074_0001
F, Phage Deletion
[0304] 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, ECOLIN_10100, 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)
[0305] 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.
[0306] 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, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, and clbR.
[0307] 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
[0308] 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).
[0309] 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.
[0310] 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).
[0311] 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).
[0312] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et 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).
[0313] 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.
[0314] 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.
-TI- [0315] 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).
[0316] 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.
[0317] 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.
[0318] 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).
[0319] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.
[0320] 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
[0321] In other embodiments, the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a bile acid catabolism enzyme(s) and/or bile acid 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 bile acid catabolism enzyme(s) and/or bile acid 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.
[0322] 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
[0323] 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, an bile acid catabolism enzyme(s) and/or bile acid transporter gene) or gene cassette (for example, a gene cassette comprising a bile acid catabolism enzyme(s) and/or bile acid 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 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.
[0324] In one non-limiting example, the bile acid catabolism enzyme(s) and/or bile acid transporter genes are integrated to facilitate bile acid import and metabolism.
Pharmaceutical Compositions and Formulations
[0325] 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.
[0326] 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.
[0327] ‘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.
[0328] 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.
[0329] 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.
[0330] 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.
[0331] The genetically engineered microorganisms may be administered intravenously, e.g. , by infusion or injection.
[0332] 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.
[0333] 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.
[0334] 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); fdlers (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.
[0335] 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.
[0336] 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.
[0337] 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- 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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).
[0345] 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. [0346] 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.
[0347] 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.
[0348] 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. [0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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
[0354] Another aspect of the disclosure provides methods of treating diseases and disorders, e.g., 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 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.
[0355] 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).
[0356] 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. In some embodiments, the subject to be treated is a human patient.
[0357] 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.
[0358] 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.
[0359] 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 mater, 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 faty acid levels, triglyceride levels, and nonesterified faty 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.
[0360] 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
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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 infdtration 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).
[0365] 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.
[0366] 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
[0367] 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 expressing the three gene pathway from Bacteroides dorei, 5a-Reductase, -5 -Reducatase, and 3 -HSDH.
[0368] The sequence of a 3 gene operon encoding 5 -alpha reductase, 5 -beta reductase, and 3- beta hydroxysteroid dehydrogenase from Bacteroides dorei DSM 17855 was sourced from NCBI genome database (DS995567. 1) and codon optimized for expression in E. coli. A ribosome binding site for each gene in the operon was designed using deNovo DNA synthesis software (www.denovodna.com) and is located immediately upstream of each coding sequence respectively. A synthetic construct was generated (IDT) in which expression of the operon is controlled by the pTac, IPTG-inducible promoter on a low copy plasmid with the pSClOl origin to generate Logic2759.
[0369] The 3-alpha hydroxysteroid dehydrogenase gene from Eggerthella lenta DSM 2243 was sourced from the NCBI genome database (CPOO 1726.1). A ribosome binding site was designed using deNovo DNA software, the gene was codon optimized for expression in E. coli and was synthesized (Integrated DNA Technologies), fused to a pTac promoter, and cloned into a medium copy plasmid by Gibson assembly to generate Logic2758.
[0370] Each plasmid described above was transformed alone or in combination into E. coli Nissle to generate the strains SYN8590 (designed to convert 3-oxoLCA to isoalloLCA), SYN8629 (designed to convert LCA to 3-oxoLCA), and SYN8630 (designed to convert LCA to isoalloLCA) respectively (Table 6).
Table 6. Strains
Figure imgf000094_0001
[0371] FIGs. 4A, 4B, and 4C depict schematics of the prototype strains of Table 5. FIG.
4A depicts SYN8630, which utilizes a four enzyme pathway to convert LCA to isoalloLCA. FIG. 4B depicts SYN8590, which utilizes the last three enzymes of the same pathway to convert 3-oxoLCA to isoalloLCA. FIG. 4C depicts SYN8629, which utilizes only the first enzyme of the pathway to convert LCA to 3-oxoLCA. FIG. 5 depicts proposed active conversion pathways.
Example 2 Functional Assays demonstrating the conversion of lithocholic acid (LCA) or 3-oxoLCA to isoalloLCA by recombinant bacteria
[0372] For in vitro studies, samples of each engineered strain and a control wild type E. coli Nissle strain are 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 are resuspended in phosphate buffer with glycerol and frozen for storage. They are then normalized by optical density at 600nm (ODeoo), added to assay buffer (M9 minimal salts with glucose) containing 15 pM or 100 pM of lithocholic acid (LCA) or 3-oxo-LCA. Transformation of LCA or 3-oxoLCA was assessed over 3h. At several time points up to 3h, a sample of the reaction was quenched with the addition of 400pl ice cold methanol. Samples were then centrifuged at 15000rpm for 5 min and supernatant collected for analysis. LCA, 3-oxo-LCA, isoLCA and isoalloLCA as well as putative intermediates, 3-oxo-A4-LCA, and A4-isoLCA, in the supernatant are quantified by LCMS using established methods.
Table 7. Summary of compounds produced
Figure imgf000095_0001
*quantities are estimated based on isoalloLCA standard curve [0373] Results are shown in FIG. 6- FIG. 9.
[0374] SYN8590 and SYN8630 produce isoalloLCA from either 3-oxoLCA or LCA respectively (FIG 6A). SYN8629 produces 3-oxoLCA from LCA (FIG. 6B); SYN8629 does not produce isoalloLCA because it is lacking the 3 enzyme operon present in SYN8590 and SYN8630. SYN8590 and SYN8630 produce isoalloLCA, at a rate of approximately 0.15 nmol/h/le9 cells and 0.04 nmol/h/le9 cells, respectively FIG. 6C). SYN8629 produces 3-oxoLCA at a rate of approximately 0.15 nmol/h/le9 cells (FIG. 6D).
[0375] SYN8590 depletes 3-oxoLCA, converting it to isoalloLCA, isoLCA and intermediates (FIG. 7A). SYN8629 and SYN8630 deplete LCA, converting it to 3-oxoLCA (SYN8629) or isoalloLCA, isoLCA and intermediates (SYN8630). FIGs. 8A, 8B, and 8C show intermediates which accumulate as a result of the conversion of LCA or 3-oxoLCA to isoalloLCA. is SYN8590 and SYN8630 produce 3-oxo-A4-LCA, starting in media containing either 3-oxoLCA or LCA, respectively. SYN8590 and SYN8630 produce A4-isoLCA, starting in media containing either 3-oxoLCA or LCA, respectively. SYN8360 produces 3-oxoLCA, starting in media containing LCA. [0376] FIGs. 9A, 9B, and 9C show production of isoLCA in vitro. SYN8590 produces isoLCA from 3-oxoLCA. SYN8360 produces isoLCA from LCA. A potential enzyme pathway by which the strains could both produce isoLCA from 3-oxoLCA via the 3J3-HSDH is shown in FIG. 9B. A potential enzyme pathway by which the strains could both produce isoLCA from A4-isoLCA via the 5[3-reductase is shown in FIG. 9C.
Table 8. Bile Acid Catabolism Enzymes
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Table 9. Bile Acid Catabolism Enzymes
Figure imgf000100_0002
Figure imgf000101_0001
Table 10. Bile salt transporter polypeptide sequences
Figure imgf000101_0002
Figure imgf000102_0001
Table 11. Bile acid transporter polynucleotide sequence
Figure imgf000102_0002
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
-Ill-
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001

Claims

Claims
1. A recombinant bacterium comprising at least one heterologous 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.
2. The recombinant bacterium of claim 1, wherein the bile acid catabolism enzyme is a 3 -beta hydroxysteroid dehydrogenase (3P-HSDH).
3. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme is a bacterial 3P-HSDH.
4. The recombinant bacterium of any one of the previous claims, wherein the 3P-HSDH is a Bacteroides dorei DSM 17855 3P-HSDH.
5. The recombinant bacterium any one of claims 2-4, wherein the gene encoding the 3P-HSDH 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: 518.
6. The recombinant bacterium of any one of claims 2-5, wherein the gene encoding the 3p-
HSDH 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: 519.
7. The recombinant bacterium of any one of claims 2-6, further comprising a second heterologous gene encoding a second bile acid catabolism enzyme and a third heterologous gene encoding a third bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 5- alpha reductase, and wherein the third bile acid catabolism enzyme is a 5 -beta reductase.
8. The recombinant bacterium of claim 7, wherein the 5 -alpha reductase is a bacterial 5 -alpha reductase.
9. The recombinant bacterium of claim 8, wherein the bacterial 5-alpha reductase is a Bacteroides dorei DSM 17855 5-alpha reductase.
10. The recombinant bacterium any one of claims 7-9, wherein the gene encoding the 5 -alpha reductase 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: 514.
11. The recombinant bacterium of any one of claims 7-10, wherein the gene encoding the 5-alpha reductase 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: 515.
12. The recombinant bacterium of any one of claims 7-11, wherein the 5-beta reductase is a bacterial 5-beta reductase.
13. The recombinant bacterium of claim 12, wherein the bacterial 5 -beta reductase is a Bacteroides dorei DSM 17855 5-beta reductase.
14. The recombinant bacterium any one of claims 7-13, wherein the gene encoding the 5-beta reductase 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.
15. The recombinant bacterium of any one of claims 7-14, wherein the gene encoding the 5-beta reductase 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.
16. The recombinant bacterium of any one of claims 7-15, wherein the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are present in a gene cassette operably linked to the promoter.
17. The recombinant bacterium of any one of claims 7-15, wherein the at least one gene encoding the bile acid catabolism enzyme, the second heterologous gene encoding the second bile acid catabolism enzyme, and the third heterologous gene encoding the third bile acid catabolism enzyme are each individually linked to a promoter that is not associated with the gene encoding the bile acid catabolism enzyme in nature.
18. The recombinant bacterium of claim 17, wherein each promoter is a different promoter, or wherein each promoter is a separate copy of the same promoter.
19. The recombinant bacterium of any one of claims 7-18, further comprising a fourth heterologous gene encoding a fourth bile acid catabolism enzyme, wherein the fourth bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
20. The recombinant bacterium of claim 19, wherein the 3a-HSDH is a bacterial 3a-HSDH.
21. The recombinant bacterium of claim 20, wherein the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
22. The recombinant bacterium of any one of claims 19-21, wherein the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is operably linked to a promoter that is not associated with the fourth gene encoding the fourth bile acid catabolism enzyme in nature.
23. The recombinant bacteria of claim 22, wherein the promoter that is operably linked to the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the fourth gene encoding the fourth bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme.
24. The method of any one of claims 19-22, wherein the fourth heterologous gene encoding the fourth bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme, the second gene encoding the second bile acid catabolism enzyme, and the third gene encoding the third bile acid catabolism enzyme.
25. The recombinant bacterium of any one of claims 2-6, further comprising a second heterologous gene encoding a second bile acid catabolism enzyme, wherein the second bile acid catabolism enzyme is a 3-alpha-hydroxysteroid dehydrogenase (3a-HSDH).
26. The recombinant bacterium of claim 25, wherein the 3a-HSDH is a bacterial 3a-HSDH.
27. The recombinant bacterium of claim 26, wherein the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
28. The recombinant bacterium of any one of claims 25-27, wherein the second heterologous gene encoding the second bile acid catabolism enzyme is operably linked to a promoter that is not associated with the second gene encoding the second bile acid catabolism enzyme in nature.
29. The recombinant bacteria of claim 28, wherein the promoter that is operably linked to the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme, or wherein the promoter that is operably linked to the second gene encoding the second bile acid catabolism enzyme is a different promoter than the promoter that is operably linked to the bile acid catabolism enzyme.
30. The method of any one of claims 25-29, wherein the second heterologous gene encoding the second bile acid catabolism enzyme is present in a gene cassette with the at least one gene encoding the bile acid catabolism enzyme.
31. The recombinant bacterium of claim 1, wherein the bile acid catabolism enzyme is a 3-alpha- hydroxysteroid dehydrogenase (3a-HSDH).
32. The recombinant bacterium of claim 31, wherein the 3a-HSDH is a bacterial 3a-HSDH.
33. The recombinant bacterium of claim 32, wherein the bacterial 3a-HSDH is an Eggerthella lenta 3a-HSDH.
34. The recombinant bacterium any one of claims 19-33, wherein the gene encoding the 3a- HSDH 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.
35. The recombinant bacterium of any one of claims 19-34, wherein the gene encoding the 3a- HSDH 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.
36. The recombinant bacterium of any one of the previous claims, wherein the promoter is a constitutive or an inducible promoter.
37. The recombinant bacterium of any one of the previous claims, wherein the promotor is an inducible promoter induced by exogenous environmental conditions.
38. The recombinant bacterium of claim 36 or claim 37, wherein the inducible promoter is directly or indirectly induced by low-oxygen or anaerobic conditions.
39. The recombinant bacterium of claim 38, wherein the inducible promoter is an FNR-inducible promoter.
40. The recombinant bacterium of claim 36 or claim 37, wherein the inducible promoter is induced by temperature.
41. The recombinant bacterium of claim 40, wherein the inducible promoter is a cI857 promoter.
42. The recombinant bacterium of claim 36, wherein the inducible promoter is induced by a chemical inducer.
43. The recombinant bacterium of claim 42, wherein the promoter is induced by IPTG.
44. The recombinant bacterium of any one of the previous claims, wherein the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a plasmid in the recombinant bacterium.
45. The recombinant bacterium of any one of the previous claims, wherein the at least one heterologous gene encoding the bile acid catabolism enzyme is present on a chromosome in the recombinant bacterium.
46. The recombinant bacterium of any one of the previous claims, wherein the recombinant bacterium is a non-pathogenic bacterium.
47. The recombinant bacterium of any one of the previous claims, wherein the recombinant bacterium is a probiotic or a commensal bacterium.
48. 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 .
49. The recombinant bacterium of claim 48, wherein the recombinant bacterium is Escherichia coli strain Nissle.
50. The recombinant bacterium of any one of the previous claims, further comprising a heterologous gene encoding a bile acid importer or transporter.
51. The recombinant bacterium of claim 50, wherein the importer or transporter is a homolog of human ASBT, baiG or PanS.
52. The recombinant bacterium of claim 51, wherein the homologs of human ASBT is from Neisseria meningitidis (ASBTNM), from Yersinia frederiksenii (ASBTYf), from E. coli M34 or from E. coli VREC0334.
53. The recombinant bacterium of any one of claims 50-52, wherein the gene encoding the bile acid transporter 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.
54. The recombinant bacterium of any one of claims 50-53, wherein the gene 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: 503, 505, 507, 509, 511 or 513.
55. The recombinant bacterium of any one of claims 50 or 51, wherein the bile acid importer or transporter is baiG from Eubacterium sp. strain VPI 12708.
56. The recombinant bacterium of any one of claims 50, 51, or 55, wherein the gene encoding the bile acid transporter 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.
57. The recombinant bacterium of any claim 50, 51, 55, or 56, wherein the gene 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: 513.
58. The recombinant bacterium of claim 51, wherein the PanS is from Escherichia coli EC23.
59. The recombinant bacterium of any one of claims 50, 51, or 58, wherein the gene encoding the bile acid transporter 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.
60. The recombinant bacterium of any one of claims 50, 51, 58, or 59, wherein the gene 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: 511.
61. The recombinant bacterium of any one of the previous claims, further comprising an insertion, deletion or mutation of an endogenous phage gene.
62. The recombinant bacterium of claim 61, wherein the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence SEQ ID NO: 292.
63. The recombinant bacterium of any one of the previous claims, further comprising a modified endogenous colibactin island.
64. The recombinant bacterium of claim 63, 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), 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).
65. The recombinant bacterium of claim 63 or claim 64, 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), clbE (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).
66. 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.
67. The recombinant bacterium of claim 66, wherein the auxotrophy is in an enzyme diaminopimelic acid synthesis or an enzyme in the thymine biosynthetic pathway.
68. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme catabolizes a secondary bile acid.
69. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme catabolizes lithocholic acid (LCA).
70. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme catabolizes 3-oxo lithocholic acid.
71. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme is capable of producing 3-oxo lithocholic acid.
72. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme is capable of producing isoallolithocholic acid (isoalloLCA).
73. The recombinant bacterium of any one of the previous claims, wherein the bile acid catabolism enzyme is capable of producing isolithocholic acid (isoLCA).
74. The recombinant bacterium of any one of the previous claims, wherein the bacterium produces isoalloLCA at a rate of about 0.025 nmol/h/le9 cells to about 0.2 nmol/h/le9 cells.
75. The recombinant bacterium of any one of the previous claims, wherein the bacterium produces isoalloLCA at a rate of about 0. 15 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
76. The recombinant bacterium of any one of the previous claims, wherein the bacterium produces 3-oxoLCA at a rate of about 0.1 nmol/h/le9 cells to about 0.5 nmol/h/le9 cells.
77. A pharmaceutically acceptable composition comprising the recombinant bacterium of any one of the previous claims; and a pharmaceutically acceptable carrier.
78. The pharmaceutically acceptable composition of claim 77, wherein the composition is formulated for oral administration.
79. 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 77 or claim 78, thereby treating the disease or disorder.
80. The method of claim 79, wherein the disease or disorder is an autoimmune disease or an inflammatory disease or disorder.
81. The method of claim 79, 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.
82. The method of claim 80, wherein the disease or disorder is 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.
83. The method of claim 79, wherein the disease or disorder is ulcerative colitis or Crohn’s disease.
84. 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 77 or claim 78, wherein the symptom of the disease or disorder is inflammation.
85. The method of any one of claims 77-84, wherein the subject has a decreased level of a secondary bile acid in the gut after the composition is administered.
86. The method of claim 85, wherein the bile acid is lithocholic acid (LCA).
87. The method of any one of claims 77-86, wherein the subject has an increased level of a isoalloLCA in the gut after the composition is administered.
88. A method of decreasing a level of a secondary bile acid in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition of claim 77 or claim 78, thereby decreasing the level of the secondary bile acid in the gut of the subject.
89. The method of claim 88, wherein the secondary bile acid is LCA.
90. The method of claim 88 or claim 89, wherein the subject has an increased level of isoalloLCA, 3-oxoLCA and/or isoLCA in the gut after administration.
91. A method of increasing a level of isoalloLCA in the gut of a subject, the method comprising administering to the subject the pharmaceutical composition of claim 77 or claim 78, thereby increasing the level of the isoalloLCA, 3-oxoLCA and/or isoLCA in the gut of the subject.
92. The method of any one of claims 77-91, wherein the subject is a human.
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