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WO2017123610A2 - Bactéries modifiées pour détoxifier les molécules délétères - Google Patents

Bactéries modifiées pour détoxifier les molécules délétères Download PDF

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Publication number
WO2017123610A2
WO2017123610A2 PCT/US2017/012982 US2017012982W WO2017123610A2 WO 2017123610 A2 WO2017123610 A2 WO 2017123610A2 US 2017012982 W US2017012982 W US 2017012982W WO 2017123610 A2 WO2017123610 A2 WO 2017123610A2
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WIPO (PCT)
Prior art keywords
gene
promoter
genetically engineered
bacterium
payload
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2017/012982
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English (en)
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WO2017123610A3 (fr
Inventor
Dean Falb
Jonathan W. Kotula
Vincent M. Isabella
Paul F. Miller
Adam B. FISHER
Yves Millet
Suman MACHINANI
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Synlogic Inc
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Synlogic Inc
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Publication date
Priority claimed from PCT/US2016/020530 external-priority patent/WO2016141108A1/fr
Priority claimed from PCT/US2016/032565 external-priority patent/WO2016183532A1/fr
Priority claimed from PCT/US2016/039444 external-priority patent/WO2016210384A2/fr
Priority claimed from PCT/US2016/050836 external-priority patent/WO2017074566A1/fr
Priority claimed from PCT/US2016/069052 external-priority patent/WO2017123418A1/fr
Application filed by Synlogic Inc filed Critical Synlogic Inc
Publication of WO2017123610A2 publication Critical patent/WO2017123610A2/fr
Publication of WO2017123610A3 publication Critical patent/WO2017123610A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)

Definitions

  • compositions and therapeutic methods for detoxifying deleterious molecules relate to compositions and therapeutic methods for detoxifying deleterious molecules.
  • the disclosure relates to genetically engineered bacteria that are capable of inhibiting and/or metabolizing toxic molecules, metabolites, or other deleterious molecules.
  • the compositions and methods disclosed herein may be used to detoxify chemotherapeutic drugs or metabolites or byproducts thereof (e.g., 5-fluorouracil, irinotecan), nonsteroidal ant i- inflammatory drugs or metabolites or byproducts thereof (e.g., naproxen, indomethacin), or exogenous toxic molecules (e.g. , lead, dioxin).
  • chemotherapeutic drugs or metabolites or byproducts thereof e.g., 5-fluorouracil, irinotecan
  • nonsteroidal ant i- inflammatory drugs or metabolites or byproducts thereof e.g., naproxen, indomethacin
  • exogenous toxic molecules
  • PCT/US2016/34200 detoxifying excess ammonia
  • PCT/US2016/32562 PCT/US2016/32562
  • PCT/US2016/062369 detoxifying excess phenylalanine
  • PCT/US2016/37098 detoxifying excess branch chain amino acids
  • PCT/US2016/044922 detoxifying excess propionate
  • PCT/US2016/049781 detoxifying excess oxalate
  • Naturally occurring hepatic enzymes are capable of detoxifying ammonia, a toxic substance that can be endogenously produced during amino acid biosynthesis, and alcohol, a toxic substance that can be exogenously ingested.
  • many toxic substances cannot be detoxified naturally by the body, particularly when they are present at high levels. These toxic substances can cause disastrous physiological effects, including death.
  • Heavy metal poisoning can cause damage to the brain, lungs, kidneys, liver, and blood (Jaishankar et al., 2014).
  • copper poisoning is capable of causing gastrointestinal hemorrhaging, basal ganglia neurodegeneration, stroke, and death.
  • Lead poisoning is capable of causing renal dysfunction, mental retardation, and death.
  • Heavy metal poisoning can occur by exposure through the skin, ingestion, and/or inhalation, and young children are particularly vulnerable.
  • Current therapies for heavy metal poisoning include natural and synthetic chelating agents such as metal binding proteins, metallothioneins, and small organic molecules. However, only certain heavy metals can be chelated by particular chelating agents, and the treatment window can be limited (Smith 2013 ; Sears 2013).
  • Chemotherapeutic drugs and nonsteroidal ant i- inflammatory drugs are administered for therapeutically beneficial effects, but those drugs and/or their metabolites or byproducts are also capable of causing deleterious effects.
  • chemotherapy is commonly associated with dose-limiting diarrhea, and chemotherapy-induced diarrhea is a notable cause of morbidity and mortality (Andreyev et al., 2014; Stein et al., 2010).
  • Diarrhea occurs in about 50-80% of chemotherapy patients, particularly those treated with irinotecan, fluoropyrimidines, and/or 5-fluorouracil (Stein et al., 2010).
  • Grade 3 diarrhea is severe enough to affect daily life, grade 4 diarrhea is life-threatening, and grade 5 diarrhea can cause death.
  • Some medications are capable of reducing the general toxic effects on healthy cells caused by chemotherapeutic drugs.
  • No pharmacological strategies effectively prevent radiotherapy-induced diarrhea (Andreyev et al, 2014).
  • Suggested therapies to ameliorate diarrhea include activated charcoal, glutamine, celecoxib, racecadotril, and probiotics, but "evidence of efficacy is lacking for all" (Andreyev et aL., 2014).
  • Glutamine, sucralfate, sulfasalazine, and octreotide have all been tested in trials, but none decreased or prevented diarrhea (Andreyev et al., 2014).
  • the invention provides genetically engineered bacteria that are capable of detoxifying, inhibiting, and/or metabolizing deleterious molecules.
  • the genetically engineered bacteria are capable of detoxifying toxic molecules, metabolites, or other deleterious molecules selectively in low-oxygen environments, e.g., the mammalian gut.
  • the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxicity.
  • the toxic molecule also exerts therapeutically beneficial effects, e.g., cytotoxicity to cancer cells, and the genetically engineered bacteria detoxify the toxic molecule or its metabolite(s) or byproduct(s) after the therapeutically beneficial effects have been exerted.
  • the invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders and conditions caused by toxic molecules, metabolites, and other deleterious molecules, e.g., chemotherapy- induced diarrhea and heavy metal poisoning.
  • the genetically engineered bacteria are capable of inhibiting, metabolizing, and/or detoxifying chemotherapeutic drugs or metabolites or byproducts thereof.
  • the genetically engineered bacteria detoxify the drug or metabolite or byproduct after the chemotherapeutic drug exerts its therapeutically beneficial effects, e.g., cytotoxicity in cancerous cells.
  • the genetically engineered bacteria are administered before, together with, and/or after administration of the chemotherapeutic drug.
  • the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the drug or metabolite or byproduct, thereby reducing chemotherapy-induced diarrhea, reducing chemotherapy-induced toxicity, increasing chemotherapy dosage amount, increasing chemotherapy dosage frequency, and/or increasing chemotherapy efficacy.
  • the molecule to be detoxified is a
  • chemotherapeutic drug selected from irinotecan, methotrexate, an antimetabolite, gemcitabine, cytosine arabinoside, a fluoropyrimidine, fluoro uracil, capecitabine, tegafur- uracil, a multitargeted folinic acid antagonist, pemetrexed, raltitrexed, gemcitabine, a plant alkaloid, a vinca alkaloid, vincristine, vinorelbine, a epipodophyllotoxin, etoposide, a taxane, paclitaxel, docetaxel, a topoisomerase I inhibitor, a cytotoxic antibiotic, an anthracycline, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, an alkylating agent, cyclophosphamide, a platinum, cisplatin, carboplatin, ox
  • the engineered bacteria comprise gene sequence encoding one or more enzyme(s) capable of metabolizing or otherwise detoxifying a toxic or deleterious molecule. In some embodiments, the genetically engineered bacteria further comprises gene sequence encoding one or more enzyme(s) for the production of one or more ant i- inflammatory molecule(s). In some embodiments, the genetically engineered bacteria further comprise gene sequence for the production of one or more gut barrier enhancer molecule(s).
  • the genetically engineered bacteria comprise gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s).
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more antiinflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is an auxotroph.
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s )is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate.
  • the payload is a small molecule that is capable of inhibiting methotrexate.
  • the payload is an enzyme that is capable of metabolizing methotrexate into non-toxic metabolites.
  • the enzyme capable of metabolizing methotrexate is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • the genetically engineered bacteria comprise a gene encoding carboxypeptidase Gi (CPD Gi) and are capable of detoxifying methotrexate.
  • the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi (Chabner et ah, 1972).
  • the genetically engineered bacteria comprise a gene encoding carboxypeptidase G 2 (CPD G 2 ) and are capable of detoxifying methotrexate.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and/or SN-38.
  • the payload is a small molecule that inhibits ⁇ -glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38.
  • the small molecule that inhibits ⁇ -glucuronidase is D-saccharic acid 1.4-lactone (SAL).
  • the payload is a small molecule that is capable of inhibiting SN-38.
  • the payload is an enzyme that is capable of metabolizing SN-38 into non-toxic metabolites.
  • the payload is an enzyme that is capable of glucuroniding SN-38, thereby converting it into non-toxic SN-38G.
  • the enzyme is from a non- human species, e.g., a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • the payload is a molecule that is capable of inhibiting or killing the commensal bacteria that produce ⁇ -glucuronidase.
  • the payload is a molecule that promotes changes to the intestinal microflora, e.g., enhancing the survival and/or proliferation of the genetically engineered bacteria, thereby outcompeting commensal bacteria that produce ⁇ - glucuronidase.
  • the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase, which is capable of adding glucuronic acid to SN-38, and are capable of detoxifying SN-38.
  • the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying NSAIDs or metabolites or byproducts thereof.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying one or more NSAIDs, e.g., naproxen.
  • the payload is a small molecule that is capable of inhibiting one or more NSAIDs, e.g., naproxen.
  • the payload is a proton pump inhibitor, and the genetically engineered bacteria are capable of ameliorating NSAID-induced intestinal damage.
  • the payload is an enzyme that is capable of metabolizing the NSAID into non-toxic metabolites.
  • the enzyme capable of metabolizing the NSAID is from a non-human species, e.g. , a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • B -glucuronidase inhibition can alleviate NSAID-induced enteropathy.
  • the genetically engineered bacteria comprise a gene encoding
  • glucuronosyltransferase and are capable of glucuronidating and detoxifying naproxen.
  • the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more heavy metals, thereby ameliorating one or more symptoms of heavy metal poisoning. In some embodiments, the genetically engineered bacteria of the invention are capable of ameliorating acute heavy metal poisoning and/or chronic heavy metal poisoning.
  • genetically engineered bacteria of the invention are capable of ameliorating one or more symptoms of aluminum poisoning, antimony poisoning, arsenic poisoning, barium poisoning, bismuth poisoning, cadmium poisoning, chromium poisoning, cobalt poisoning, copper poisoning, gold poisoning, iron poisoning, lead poisoning, lithium poisoning, manganese poisoning, mercury poisoning, nickel poisoning, phosphorous poisoning, platinum poisoning, selenium poisoning, silver poisoning, thallium poisoning, tin poisoning, and/or zinc poisoning.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering a heavy metal.
  • the payload is a heavy metal chelator.
  • the payload is a plant phytochelatin.
  • the payload is from a non-human species, e.g. , a plant, bacterial, or other mammalian molecule.
  • the payload is a synthetic or modified molecule.
  • the genetically engineered bacteria are capable of expressing plant phytochelatins, particularly on the surface of the bacteria.
  • the genetically engineered bacteria of the invention are capable of binding to cadmium, thereby ameliorating one or more symptoms of cadmium poisoning.
  • the payload is carboxypeptidase Gi (CPD Gi) or carboxypeptidase G 2 (CPD G 2 ).
  • the payload is D-saccharic acid 1, 4- lactone (SAL).
  • the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38).
  • the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen..
  • the payload is a proton pump inhibitor.
  • the payload is a heavy metal chelator. In some embodiments, the payload is a plant phytochelatin. In some embodiments, the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate. In some embodiments, the payload is the enzyme Pseudomonas. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying a toxic or deleterious molecule and a gene or gene cassette for producing a short-chained fatty acid, e.g. butyrate, propionate, or acetate.
  • FIG. 1A and IB depict schematics of the gene organization of exemplary bacteria of the invention.
  • FIG. 1A depicts the gene organization of an exemplary recombinant bacterium of the invention comprising a butyrate synthetic cassette and a carboxypeptidase G2 cassette, wherein the butyrate synthetic cassette and the carboxypeptidase G2 cassette comprise a FNR-responsive promoter, and wherein FNR ⁇ e.g., a FNR dimer) binding to the FNR- responsive promoter induces the expression of the butyrate synthetic cassette and/or the carboxypeptidase G2 cassette, which leads to the production of butyrate and/or the production of carboxypeptidase G2 (CPD G2).
  • FNR ⁇ e.g., a FNR dimer binding to the FNR- responsive promoter induces the expression of the butyrate synthetic cassette and/or the carboxypeptidase G2 cassette, which leads to the production of butyrate and/or the production of carboxypeptidase G2
  • the bacteria may optionally include an auxo trophy, e.g., deletion or mutation of thyA ( ⁇ thyA; thymidine dependence) in the E. coli Nissle genome, such that thymidine must be supplied in the culture medium to support growth.
  • auxo trophy e.g., deletion or mutation of thyA ( ⁇ thyA; thymidine dependence) in the E. coli Nissle genome, such that thymidine must be supplied in the culture medium to support growth.
  • IB depicts the gene organization of an exemplary recombinant bacterium of the invention comprising a butyrate synthetic cassette and a glucuronosyl transferase cassette, wherein the butyrate synthetic cassette and the glucuronosyl transferase cassette comprise a FNR- responsive promoter, and wherein FNR ⁇ e.g., a FNR dimer) binding to the FNR-responsive promoter induces the expression of the butyrate synthetic cassette and/or the glucuronosyl transferase cassette, which leads to the production of butyrate and/or the production of glucuronosyl transferase (GT).
  • a FNR-responsive promoter e.g., a FNR dimer
  • the bacteria may optionally include an auxotrophy, e.g., deletion or mutation of thyA ( ⁇ thyA; thymidine dependence) in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth
  • auxotrophy e.g., deletion or mutation of thyA ( ⁇ thyA; thymidine dependence) in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth
  • FIG. 1C and FIG. ID depict the state of a non- limiting embodiment certain constructs of the invention under non-inducing (FIG. 1C) and inducing (FIG.1D) conditions.
  • FIG. 1C depicts relatively low carboxypeptidase G2 (CPD G2) and glucuronosyl transferase (GT) production under aerobic conditions due to oxygen (0 2 ) preventing FNR from dimerizing and activating carboxypeptidase G2 (CPD G2) and/or glucuronosyl transferase (GT) gene expression.
  • CPD G2 carboxypeptidase G2
  • GT glucuronosyl transferase
  • CPD G2 carboxypeptidase G2
  • GT glucuronosyl transferase
  • FIG. 2A, FIG. 2B, FIG. 2C, and FIG.2D depict schematics of a butyrate production pathway and schematics of different butyrate producing circuits.
  • FIG. 2A depicts a metabolic pathway for butyrate production.
  • FIG. 2B and FIG. 2C depict schematics of two different exemplary butyrate producing circuits, both under the control of a tetracycline inducible promoter.
  • FIG. 2B depicts a bdc2 butyrate cassette under control of a tet promoter on a plasmid.
  • a "bdc2 cassette” or “bdc2 butyrate cassette” refers to a butyrate producing cassette that comprises at least the following genes: bcd2, etfB3, etfA3, hbd, crt2, pbt, and buk genes.
  • FIG. 2C depicts a ter butyrate cassette (ter gene replaces the bcd2, etfB3, and etfA3 genes) under control of a tet promoter on a plasmid.
  • a “ter cassette” or “ter butyrate cassette” refers to a butyrate producing cassete that comprises at least the following genes: ter, thiAl, hbd, crt2, pbt, and buk genes.
  • 2D depicts a schematic of a third exemplary butyrate gene cassette under the control of a tetracycline inducible promoter, specifically, a tesB butyrate cassette (ter gene is present and tesB gene replaces the pbt gene and the buk gene) under control of a tet promoter on a plasmid.
  • a "tes or tesB cassette or "tes or tesB butyrate cassette” refers to a butyrate producing cassette that comprises at least ter, thiAl, hbd, crt2, and tesB genes.
  • An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiAl, hbd, crt2, and tesB genes.
  • the tes or tesB cassette is under the control of an inducible promoter other than tetracycline.
  • Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.
  • oxygen level-dependent promoters e.g., FNR- inducible promoter
  • RNS inflammatory response
  • ROS ROS promoters
  • promoters induced by a metabolite that may or may not be naturally present e.g., can be exogenously added
  • FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F depict schematics of the gene organization of exemplary bacteria of the disclosure.
  • FIG. 3A and FIG. 3B depict the gene organization of an exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions.
  • FIG. 3A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter").
  • FIG. 3B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • FIG. 3C and FIG. 3D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO).
  • NO nitric oxide
  • NsrR NsrR transcription factor
  • the NsrR transcription factor in the absence of NO, binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) are expressed.
  • the NsrR transcription factor in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 3E and FIG. 3F depict the gene organization of an exemplary
  • the OxyR transcription factor (circle, "OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) are expressed.
  • the OxyR transcription factor in the presence of H 2 O 2 , the OxyR transcription factor interacts with H 2 O 2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 4F depict schematics of the gene organization of exemplary bacteria of the invention.
  • FIG. 4A and FIG. 4B depict the gene organization of another exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions using a different butyrate circuit from that shown in FIG. 3.
  • FIG. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter").
  • FIG. 4B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • FIG. 4C and FIG. 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO).
  • NO nitric oxide
  • FIG. 4C in the absence of NO, the NsrR transcription factor (circle, "NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk) are expressed.
  • the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence.
  • FIG. 4E and FIG. 4F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H 2 O 2 .
  • the OxyR transcription factor (circle, "OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes ⁇ ter, thiAl, hbd, crt2, pbt, buk) are expressed.
  • the OxyR transcription factor interacts with H 2 O 2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F depict schematics of the gene organization of exemplary bacteria of the invention.
  • FIG. 5A and FIG. 5B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.
  • FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes ⁇ ter, thiAl, hbd, crt2, and tesB) are expressed.
  • FIG. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"
  • FIG. 5B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • FIG. 5C and FIG. 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO).
  • NO nitric oxide
  • FIG. 5E and FIG. 5F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H 2 O 2 .
  • the OxyR transcription factor (circle, "OxyR") binds to, but does not induce, the oxyS promoter.
  • FIG. 6 depicts a bar graph showing butyrate production of butyrate producing strains of the invention.
  • FIG. 6 shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen (designated as "02"), in which there is no significant difference in butyrate production.
  • Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous E. Coli tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl Co A).
  • FIG. 7 depicts a bar graph showing butyrate production of butyrate producing strains of the invention.
  • FIG. 7 shows butyrate production in strains comprising a tet-butyrate cassette having ter substitution (pLOGIC046) or the tesB substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production.
  • FIG. 8 depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion.
  • Strains depicted are BW25113 comprising a bcd- butyrate cassette, with or without a nuoB deletion, and BW25113 comprising a ter-butyrate cassette, with or without a nuoB deletion. Strains with deletion are labeled with nuoB.
  • the nuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains.
  • NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.
  • FIG. 9A, FIG. 9B, and FIG.9C depict schematics and graphs showing butyrate production of a butyrate producing circuit under the control of an FNR promoter.
  • FIG. 9A depicts a schematic showing a butyrate producing circuit under the control of an FNR promoter.
  • FIG. 9B depicts a bar graph of anaerobic induction of butyrate production.
  • FNR- responsive promoters were fused to butyrate cassettes containing either the bed or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes.
  • FIG. 9C depicts SYN-501 in the presence and absence of glucose and oxygen in vitro.
  • SYN-501 comprises pSClOl PydfZ-ter butyrate plasmid;
  • SYN-500 comprises pSClOl PydfZ-bcd butyrate plasmid;
  • SYN-506 comprises pSClOl nirB-bcd butyrate plasmid.
  • FIG. 10 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H 2 0, 100 mM butyrate in H 2 0, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid.
  • Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H 2 0 (+) 200 mM butyrate.
  • FIG. 11 depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints.
  • the Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.
  • FIG. 12 depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid copies.
  • Integrated butyrate strains, SYN1001 and SYN1002 both integrated at the agal/rsml locus) gave comparable butyrate production to the plasmid strain SYN501.
  • FIG. 13A and FIG. 13B depicts the construction and gene organization of an exemplary plasmids.
  • FIG. 13A depicts the construction and gene organization of an exemplary plasmids comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct).
  • FIG. 13B depicts the construction and gene organization of another exemplary plasmid comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic046- nsrR- norB -butyrogenic gene cassette).
  • FIG. 14 depicts butyrate production using SYN001 + tet (control wild-type Nissle comprising no plasmid), SYN067 + tet (Nissle comprising the pLOGIC031 ATC- inducible butyrate plasmid), and SYN080 + tet (Nissle comprising the pLOGIC046 ATC- inducible butyrate plasmid).
  • SYN001 + tet control wild-type Nissle comprising no plasmid
  • SYN067 + tet Non-type Nissle comprising the pLOGIC031 ATC- inducible butyrate plasmid
  • SYN080 + tet Non-type Nissle comprising the pLOGIC046 ATC- inducible butyrate plasmid
  • Nissle 15 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB -butyrate construct (SYN133) or the pLogic046-nsrR- norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001).
  • Fig. 16 depicts ⁇ -galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown Table 3 (Pfnrl-5).
  • FNR-responsive promoters were used to create a library of anaerobic/low oxygen conditions inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites.
  • Bacterial cultures were grown in either aerobic (+0 2 ) or anaerobic conditions (- 0 2 ). Samples were removed at 4 hrs and the promoter activity based on ⁇ -galactosidase levels was analyzed by performing standard ⁇ -galactosidase colorimetric assays.
  • FIG. 17A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P fnr s)- LacZ encodes the ⁇ -galactosidase enzyme and is a common reporter gene in bacteria.
  • Fig. 17B depicts FNR promoter activity as a function of ⁇ - galactosidase activity in SYN-PKU904.
  • SYN-PKU904 an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard ⁇ -galactosidase colorimetric assays are expressed in Miller units (Miller, 1972).
  • Fig. 17C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
  • Fig. 18 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC oriented in the reverse direction.
  • FIG. 19A depicts an "Oxygen bypass switch" useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more propionate catabolism enzyme(s) (POI1) and /or one or more transporter(s)/importer(s) and/or exporter(s) (POI2) under the control of a low oxygen FNR promoter in vitro in a culture vessel ⁇ e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture).
  • a culture vessel e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.
  • strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest.
  • FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ).
  • the 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety).
  • the 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A.
  • FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions.
  • strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and preloaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs.
  • This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.
  • a Lacl promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction).
  • a rhamnose inducible promoter is used in this system.
  • a temperature sensitive promoter is used to drive expression of FNRS24Y.
  • FIG 19B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y.
  • the levels of Fnr expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bio informatics tools for optimization of RBS are known in the art.
  • FIG. 19C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy.
  • Bio informatics tools for optimization of RBS are known in the art.
  • arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of Pfnrs-POI constructs are maintained to allow for strong in vivo induction.
  • Fig. 20 depicts the gene organization of an exemplary construct, comprising a cloned cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.
  • POI cloned cloned protein of interest
  • Fig. 21 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI).
  • this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG.
  • inducer e.g., IPTG.
  • this construct is used alone.
  • the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs.
  • the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. PheP expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is used in combination with a LAAD expression construct. In some
  • the PAL3 sequences are codon optimized for expression in E coli.
  • the construct is located on a plasmid, e.g., a low or high copy plasmid.
  • the construct is employed in a biosafety system, such as the system shown in Fig. 36A, Fig. 36B, Fig. 36C, and Fig. 36D.
  • the construct is integrated into the genome at one or more locations described herein.
  • Figs. 22A-C depict schematics of non-limiting examples of constructs expressing a protein of interest (POI).
  • Fig 22A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter.
  • the construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI.
  • the temperature sensitive CI repressor mutant, CI857 binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone.
  • the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs.
  • the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration.
  • the construct provides in vivo activity.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid.
  • the construct is located on a plasmid component of a biosafety system.
  • the construct is integrated into the bacterial chromosome at one or more locations.
  • the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • a temperature sensitive system can be used to set up a conditional auxotrophy.
  • a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).
  • Fig. 22B depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter.
  • a rhamnose inducible promoter For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only
  • the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration. In a non- limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations.
  • Fig. 22C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter.
  • the arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre- induction and pre-loading of one or more POI(s) prior to in vivo administration.
  • the construct is useful for pre-induction and is combined with low-oxygen inducible constructs.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid.
  • the construct is located on a plasmid component of a biosafety system.
  • the construct is integrated into the bacterial chromosome at one or more locations.
  • Fig. 23A depicts a schematic of the gene organization of a PssB promoter.
  • the ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair.
  • the PssB promoter was cloned in front of a LacZ reporter and beta- galactosidase activity was measured.
  • Fig. 23B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1: 100 and split into two different tubes.
  • the Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.
  • This promoter can be used to express a gene of interest under aerobic conditions.
  • This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions.
  • the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest.
  • the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions.
  • This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control.
  • this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine.
  • dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine.
  • Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).
  • Fig. 24 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites.
  • Fig. 25 depicts three bacterial strains which constitutively express red fluorescent protein (RFP).
  • RFP red fluorescent protein
  • strains 1-3 the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light.
  • Unmodified E. coli Nissle strain 4 is non-fluorescent.
  • Fig. 26 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post- administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.
  • Fig. 27 depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage.
  • Fig. 28 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (Mo As).
  • Figs. 29A-29C depict other non- limiting embodiments of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • Fig. 29A depicts an embodiment of heterologous gene expression in which, in the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P araBAD ), which induces expression of the Tet repressor (TetR) and an anti-toxin.
  • P araBAD ParaBAD promoter
  • TetR Tet repressor
  • Fig. 29A also depicts another non- limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a
  • Fig. 29B depicts a non-limiting embodiment of the disclosure, where an antitoxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin.
  • TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell.
  • the constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin.
  • the araC gene is under the control of a constitutive promoter in this circuit.
  • Fig. 29C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin.
  • Tet repressor Tet repressor
  • the anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site).
  • araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.
  • Fig. 30 depicts one non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.
  • Fig. 31 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated
  • the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
  • Fig. 32 depicts another non- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips at least one excision enzyme into an activated conformation.
  • the at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death.
  • the natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days.
  • the presence of multiple nested recombinases can be used to further control the timing of cell death.
  • Fig. 33 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a second recombinase from an inverted orientation to an active conformation.
  • the activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.
  • Fig. 34 depicts a one non- limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin.
  • the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin.
  • the cell produces equal amounts of toxin and anti-toxin and is stable.
  • the cell loses the plasmid and anti-toxin begins to decay.
  • the anti-toxin decays completely, and the cell dies.
  • Fig. 35 depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al, 2015.
  • Figs. 36A-36D depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 36A and Fig. 36B), which also contains a chromosomal component (shown in Fig. 36C and Fig. 36D).
  • the Biosafety Plasmid System Vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 36A) and thyA (Fig. 36B) and promoter elements driving expression of these components.
  • bla is knocked out and replaced with one or more constructs described herein, in which PAL3 and/or PheP and/or LAAD are expressed from an inducible or constitutive promoter.
  • Fig. 36C and Fig. 36D depict schematics of the gene organization of the chromosomal component of a biosafety system.
  • Fig. 36C depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter.
  • Fig. 36D depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter.
  • the plasmid containing the functional DapA is used (as shown in Fig. 36A)
  • the chromosomal constructs shown in Fig. 36C and Fig. 36D are knocked into the DapA locus.
  • the plasmid containing the functional ThyA is used (as shown in Fig. 36B)
  • the chromosomal constructs shown in Fig. 36C and Fig. 36D are knocked into the ThyA locus.
  • the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.
  • Fig. 37 depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.
  • Fig. 38 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N- terminal secretion signal, a linker and the beta-domain of an autotransporter.
  • the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence.
  • the beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure.
  • the therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence.
  • the therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.
  • Fig. 39 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP- binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes.
  • HlyB an ATP- binding cassette transporter
  • HlyD a membrane fusion protein
  • TolC an outer membrane protein
  • Fig. 40 depicts a schematic of the outer and inner membranes of a gram- negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the
  • extracellular space e.g. , therapeutic polypeptides of eukaryotic origin containing disulphide bonds.
  • Fig. 41 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen.
  • An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell.
  • An inducible promoter small arrow, bottom
  • a FNR-inducible promoter drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).
  • Fig. 42A, Fig. 42B, and Fig. 42C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted using components of the flagellar type III secretion system.
  • a therapeutic polypeptide of interest such as, PAL and/or LAAD, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (Fig. 42A and Fig. 42B) or a Tet-inducible promoter (Fig. 42C).
  • an inducible promoter such as oxygen level-dependent promoters (e.g., FNR- inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used.
  • the therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD).
  • an N terminal part of FliC is included in the construct, as shown in Fig. 42B and Fig. 42C.
  • Fig. 43A and Fig. 43B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system.
  • the therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space.
  • Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA).
  • the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpl.
  • periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm.
  • a FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a Tet promoter (Fig. 43A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, Fig. 43B), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose.
  • Fig. 44A depicts a schematic diagram of a wild-type clbA construct.
  • Fig. 44B depicts a schematic diagram of a clbA knockout construct.
  • Fig. 45 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7.
  • Figs. 46A, B, C, D, and E depict a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.
  • Fig. 46A depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm.
  • Fig. 46B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SCI, duration 1.5 hours, temperature 37° C, shaking at 250 rpm.
  • SCI starter culture 1
  • SC2 starter culture 2
  • 46C depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300- 1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours.
  • Fig. 46D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS.
  • Fig. 46E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
  • FIG. 47 A and FIG. 47B depict diagrams of bacterial tryptophan metabolism pathways.
  • FIG. 47A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g. , in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase
  • Trp Tryptophan
  • TrA Tryptamine
  • IAAld Indole-3- acetaldehyde
  • IAA Indole- 3 -acetic acid
  • FICZ 6-formylindolo(3,2-b)carbazole
  • IPyA Indole- 3-pyruvic acid
  • IAM Indole- 3 -acetamine
  • IAOx Indole-3-acetaldoxime
  • IAN Indole-3- acetonitrile
  • N-formyl Kyn N-formylkynurenine
  • Kyn Kynurenine
  • KynA Kynurenic acid
  • I3C Indole-3-carbinol
  • IAld Indole- 3 -aldehyde
  • DIM 3,3'-Diindolylmethane
  • ICZ 6,3'-Diindolylmethane
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIGs. 47A and 47B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 47A and 47B.
  • the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low- oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • FIG. 48 shows a schematic depicting an exemplary Tryptophan circuit.
  • Tryptophan is produced from the Chorismate precursor through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes.
  • trpE trpG-D
  • trpC-F also referred to as trpC
  • trpB also depicted.
  • Chorismate precursor through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. All of these genes are optionally expressed from an inducible promoter, e.g. , a FNR- inducible promoter.
  • the bacteria may also include an auxotrophy, e.g. , deletion of thyA ( ⁇ thyA; thymidine dependence).
  • the bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan.
  • a bacterial strain is listed.
  • FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG. 49E, FIG. 49F, FIG. 49G, and FIG. 49H depict schematics of non- limiting examples of embodiments of the disclosure.
  • optionally gene(s) which encode exporters may also be included.
  • FIG. 49A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan.
  • the optional circuits for tryptophan production are as depicted and described in FIG. 48.
  • FIG. 49B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan.
  • FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG. 49E, FIG. 49F, FIG. 49G, and FIG. 49H depict schematics of non- limiting examples of embodiments of the disclosure.
  • optionally gene(s) which encode exporters may also be included.
  • FIG. 49C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan.
  • FIG. 49D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan.
  • FIG. 49E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan.
  • FIG. 49F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan.
  • FIG. 49G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan.
  • FIG. 49D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan.
  • FIG. 49E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce
  • 49H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet.
  • the genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g. , from Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter.
  • the engineered bacterium shown in any of FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG.
  • FIG. 49E, FIG. 49F, FIG. 49G and FIG. 49H may also have an auxo trophy, e.g., in one example, the thy A gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • auxo trophy e.g., in one example, the thy A gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • FIG. 50A, FIG. 50B, FIG. 50C, FIG. 50D, and FIG. 50E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole- 3 -acetic acid.
  • FIG. 50A the optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 50B the optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 50C the optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 50D the optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 50E the optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 51A and FIG 51B depict schematics of cicuits for the production of indole metabolites.
  • FIG. 51A depicts a schematic of an indole-3-propionic acid (IP A) synthesis circuit.
  • IP A indole-3-propionic acid
  • FIG. 51A depicts a schematic of an indole-3-propionic acid (IP A) synthesis circuit.
  • IP A indole-3-propionic acid
  • IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from Rubrivivax benzoatilyticus) and indole- 3 -aery late reductase (e.g., from Clostridum botulinum). Tryptophan ammonia lyase converts tryptophan to indole-3- acrylic acid, and indole- 3 -aery late reductase converts indole- 3 -acrylic acid into IPA.
  • WAL Tryptophan ammonia lyase
  • indole- 3 -aery late reductase e.g., from Clostridum botulinum
  • strains further comprise optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 51B depicts a schematic of another indole-3-propionic acid (IPA) synthesis circuit.
  • Enzymes are as follows: 1. TrpDH: tryptophan dehydrogenase, e.g., from from Nostoc punctiforme NIES-2108; FldHl/FldH2: indole- 3 -lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole- 3 -lactate dehydratase, e.g., from
  • Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate, NH 3 , NAD(P)H and H + .
  • Indole- 3 -lactate dehydrogenase ((EC 1.1.1.110, e.g.
  • Clostridium sporogenes or Lactobacillus casei converts (indol-3yl)pyruvate and NADH and H+ to indole-3-lactate and NAD+.
  • Indole- 3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole- 3 -lactate and indol- 3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA.
  • Indole-3-acrylyl-CoA reductase (FldD) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3- propionyl-CoA.
  • Indole- 3 -lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole- 3-acrylyl-CoA.
  • the strains further comprise optional circuits for tryptophan production are as depicted and described in FIG. 41.
  • FIG. 52A, FIG. 52B, FIG. 52C, FIG. 52D, and FIG. 52E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid.
  • Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter.
  • Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g.
  • FNR- inducible promoter FNR- inducible promoter
  • promoters induced by inflammation or an inflammatory response RNS, ROS promoters
  • promoters induced by a metabolite that may or may not be naturally present e.g., can be exogenously added
  • the bacteria may also include an auxotrophy, e.g. , deletion of thyA ( ⁇ thyA; thymidine dependence).
  • FIG. 52A depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes.
  • AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production.
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 41 and/or described in the description of FIG. 41 and/or FIG. 52B.
  • Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • the bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan.
  • FIG. 52B depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production.
  • the strain further comprises either a wild type or a feedback resistant SerA gene.
  • Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD 1 to NADH.
  • E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved.
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 41 and/or described in the description of FIG. 41.
  • Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • the bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan.
  • FIG. 52C depicts non-limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41. Additionally, the strain comprises tdc
  • FIG. 52D depicts a non-limiting example of an indole- 3 -acetate producing strain. Tryptophan optionally is produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from
  • FIG. 52E depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises additional circuits as depicted and/or described in FIG. 52A and/or FIG. 52B and/or FIG. 41.
  • FIG. 53 depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways.
  • Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al, CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016)).
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 53.
  • the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 53, including but not limited to, kynurenine, indole-3- aldehyde, indole- 3 -acetic acid, and/or indole-3 acetaldehyde.
  • the invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating or treating disorders or conditions that are caused by toxic molecules, metabolites, or other deleterious molecules, e.g. , chemotherapy- induced diarrhea.
  • the genetically engineered bacteria are capable of reducing and/or metabolizing the toxic molecule, metabolite, or deleterious molecule, particularly in low- oxygen conditions, such as in the mammalian gut.
  • the engineered bacteria are further capable of producing one or more anti- inflammation and/or gut barrier function enhancer molecule(s).
  • the engineered bacteria are further capable of producing one or more anti- inflammation and/or gut barrier function enhancer molecule(s) in inducing environments, e.g., in the gut.
  • the term "toxin" in the context of a kill switch being expressed in a recombinant bacterium of the disclosure 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" in the context of a kill switch being expressed in a recombinant bacterium of the disclosure is intended to include bacteriostatic proteins and bactericidal proteins.
  • 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 or antiitoxin 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 in the context of a kill switch being expressed in a recombinant bacterium of the disclosure are known in the art and described in more detail infra.
  • Non- limiting examples of such toxic molecules include heavy metals, chemotherapeutic drugs, nonsteroidal ant i- inflammatory drugs (NSAIDs), natural toxic molecules such as botulinum and tetanus toxic molecules, and synthetic toxic molecules such as dioxin and sarin.
  • NSAIDs nonsteroidal ant i- inflammatory drugs
  • NSAIDs nonsteroidal ant i- inflammatory drugs
  • a toxic molecule may be a substance that has predominantly deleterious effects, e.g., lead poisoning and sarin poisoning.
  • a toxic molecule may be a substance that has some therapeutically beneficial effects, e.g., chemotherapeutic drugs and NSAIDs.
  • a toxic molecule has therapeutically beneficial effects and its metabolites or byproducts have deleterious effects; for example, irinotecan is an effective antineoplastic agent, and its metabolite SN-38 causes gastrointestinal toxicity and dose-limiting diarrhea (Stein et al., 2010).
  • a toxic molecule has therapeutically beneficial effects as well as deleterious effects; for example, methotrexate is an effective antineoplastic agent, but can cause bone marrow and gastrointestinal toxicity (Chabner et al, 1972).
  • a toxic molecule is therapeutically beneficial or innocuous at particular doses, e.g., low doses, but toxic at other doses, e.g., high doses.
  • Non-limiting examples of toxic chemotherapeutic drugs include antimetabolites, methotrexate, gemcitabine, cytosine arabinoside, fluoropyrimidines, fluorouracil, capecitabine, tegafur-uracil, multitargeted folinic acid antagonists, pemetrexed, raltitrexed, gemcitabine, plant alkaloids, vinca alkaloids, vincristine, vinorelbine, epipodophyllotoxins, etoposide, taxanes, paclitaxel, docetaxel, topoisomerase I inhibitors, irinotecan, cytotoxic antibiotics, anthracyclines, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, alkylating agents, cyclophosphamide, platinums, cisplatin, carboplatin, oxaliplatin, nedaplatin, antibodies
  • Non-limiting examples of toxic NSAIDs include etoricoxib, etodolac, rofecoxib, meloxicam, celecoxib, piroxicam, naproxen, indomethacin, ketoprofen, piroxicam, ibuprofen, diclofenac, and COX-2 inhibitors. These toxic molecules are capable of causing clinically important diarrhea that can dose-limit, delay, and/or interfere with or detract from treatment.
  • Non-limiting examples of toxic heavy metals include aluminum, antimony, arsenic, barium, bismuth, cadmium, chromium, cobalt, copper, gold, iron, lead, lithium, manganese, mercury, nickel, phosphorous, platinum, selenium, silver, thallium, tin, and zinc. These toxic molecules are capable of causing heavy metal poisoning.
  • Deleterious molecules is used to refer to the toxic molecules, heavy metals, chemotherapeutic drugs, NSAIDs, and their metabolites and byproducts as described above, as well as other compounds that adversely affect health, e.g. , radioactive substances and pesticides.
  • a "deleterious" molecule may have therapeutically beneficial effects, e.g. , antineoplastic/cytotoxic effects on cancerous cells.
  • “Detoxify” is used to refer to the process or processes, natural or synthetic, by which the above-referenced deleterious molecules are removed, inhibited, metabolized, and/or converted into one or more non-toxic molecules.
  • the deleterious molecule may be detoxified directly or indirectly.
  • detoxification may refer to the inhibition of a toxic substance, e.g. , a small molecule that inhibits ⁇ -glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38 (Wallace et al., 2010).
  • detoxification may refer to the conversion of a toxic substance into a non-toxic substance, e.g., an enzyme that metabolizes SN-38 into one or more non-toxic molecules, or an enzyme that reattaches glucuronide to SN-38, thereby converting it into non-toxic SN-38G.
  • detoxification refers to the modification of the intestinal microflora such that toxic molecule- promoting bacteria are reduced, e.g. , commensal bacteria that convert SN-38G into toxic SN- 38.
  • the toxic molecule e.g., a chemotherapeutic drug or NSAID
  • the toxic molecule is capable of exerting both therapeutically beneficial effects and deleterious effects, and the genetically engineered bacteria detoxify the deleterious molecule after the therapeutically beneficial effects have been exerted.
  • the payload refers to one or more molecules of interest to be produced by a genetically engineered bacterium.
  • the payload is a therapeutic payload, which refers to a molecule, substance, or drug that is useful for modulating or treating a disorder or condition, e.g., chemotherapy- induced diarrhea.
  • a disorder or condition e.g., chemotherapy- induced diarrhea
  • the payload is carboxypeptidase Gi (CPD Gi) (Chabner et al., 1972) or CPD G 2 .
  • the payload is a therapeutic molecule encoded by a gene.
  • the payload is a therapeutic molecule produced by a biosynthetic pathway, e.g.
  • the genetically engineered bacterium comprises two or more payloads; for example, the condition is methotrexate-induced diarrhea, and the first payload is carboxypeptidase G 2 encoded by a CPD G 2 gene, and the second payload is butyrate produced by a gene cassette encoding the genes of the butyrate biosynthesis pathway.
  • the payload is a therapeutic payload capable of detoxifying a toxic or deletorious molecule. These therapeutic or payload molecules are also referred to herein as "detoxification molecules".
  • Exemplary detoxifcation molecules include, but are not limited to, carboxypeptidase Gi (CPD Gi) or carboxypeptidase G 2 (CPD G 2 ), D- saccharic acid 1, 4-lactone (SAL), a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen, a proton pump inhibitor (e.g. , omeprazole, aspirin, lansoprazole,
  • NSAID non-steroidal ant i- inflammatory drug
  • dexlansoprazole rabeprazole, pantoprazole, esomeprazole, esomeproazole
  • the payload is an antiinflammatory or gut barrier enhancer molecule, e.g. butyrate, acetate, propionate, GLP-2, IL- 10, IL-22, IL-2, other interleukins, and/or tryptophan and/or one or more of its metabolites.
  • 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. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments the payload comprises an antibiotic resistance gene or genes. In some embodiments, the payload is encoded by a gene, multiple genes, gene cassette, or an operon. In alternate embodiments, the payload is produced by a bio synthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads.
  • gut inflammation and/or compromised gut barrier function include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases.
  • Inflammatory bowel diseases and “IBD” are used interchangeably herein to refer to a group of diseases associated with gut
  • inflammation which include, but are not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
  • diarrheal diseases include, but are not limited to, acute watery diarrhea, e.g., cholera; acute bloody diarrhea, e.g. , dysentery; and persistent diarrhea.
  • related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis.
  • Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of 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, inflammation of the skin, inflammation of the eyes, inflammation of the joints, inflammation of the liver, and inflammation of the bile ducts.
  • These symptoms relating to the disruption of gut health and other symptoms relating to an inflammatory state may arise as a result of a subject's exposure to one or more toxic or deleterious molecules.
  • grade 1 diarrhea, grade 2 diarrhea, grade 3 diarrhea, grade 4 diarrhea, and grade 5 diarrhea are all encompassed by the term "diarrhea.”
  • grade 1 diarrhea is characterized by an increase of ⁇ 4 bowel movements per day or a mild increase in stoma output.
  • Grade 2 diarrhea is characterized by an increase of 4-6 bowel movements per day, a moderate increase in stoma output, and moderate cramping or nocturnal stools.
  • Grade 3 diarrhea is characterized by an increase of 7-9 bowel movements per day, incontinence, or severe increase in stoma output, and severe cramping or nocturnal stools.
  • Grade 4 diarrhea is generally life-threatening and is characterized by an increase of more than 10 bowel movements per day, grossly bloody stool, severe dehydration, extremely low blood pressure, and/or need for parenteral support. Grade 5 diarrhea generally results in death. See, e.g., Andreyev et al., 2014; National Cancer Institute Common Toxicity Criteria for Diarrhea.
  • Diarrhea may optionally be co-morbid with steatorrhea (excess fat in the stool due to fat malabsorption in the gut).
  • anti-inflammation molecules and/or “gut barrier function enhancer molecules” include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP- 1, IL- 10, IL-27, TGF- ⁇ , TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein.
  • Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g. , a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN- ⁇ , IL- ⁇ , IL-6, IL-8, IL- 17, and/or chemokines, e.g. , CXCL-8 and CCL2.
  • pro-inflammatory molecules e.g. a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN- ⁇ , IL- ⁇ , IL-6, IL-8, IL- 17, and/or chemokines, e.g. , CXCL-8 and CCL2.
  • Such molecules also include AHR agonists ⁇ e.g. , which result in IL-22 production, e.g. , indole acetic acid, indole-3-aldehyde, and indole) and and
  • GPR109A e.g. , butyrate
  • inhibitors of NF-kappaB signaling e.g. , butyrate
  • modulators of PPARgamma e.g. , butyrate
  • activators of AMPK signaling e.g. , acetate
  • modulators of GLP- 1 secretion Such molecules also include hydro xyl radical scavengers and antioxidants (e.g., IP A).
  • a molecule may be primarily ant i- inflammatory, e.g., IL- 10, or primarily gut barrier function enhancing, e.g. , GLP-2.
  • a molecule may be both anti- inflammatory and gut barrier function enhancing.
  • An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g. , elafin is encoded by the PI3 gene.
  • an anti- inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g. , butyrate. These molecules may also be referred to as therapeutic molecules.
  • the "anti- inflammation molecules” and/or “gut barrier function enhancer molecules” are referred to herein as "effector molecules” or “therapeutic molecules” or “therapeutic polypeptides”.
  • a disease or condition associated with gut inflammation and/or compromised gut barrier function may be an autoimmune disorder.
  • a disease or condition associated with gut inflammation and/or compromised gut barrier function may be co-morbid with an autoimmune disorder.
  • autoimmune disorders 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, antiphospho lipid 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 polyneur
  • 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,
  • 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.
  • polypeptides include 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, and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post- expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology.
  • 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.
  • 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 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. No n- 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.
  • 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 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.
  • variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention.
  • Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones.
  • 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
  • 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.
  • amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.
  • An antibody generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond.
  • the recognized immunoglobulin genes include the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either ⁇ or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.
  • antibody or “antibodies” is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities.
  • antibody or “antibodies” is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies.
  • Antibodies can have more than one binding specificity, e.g. , be bispecific.
  • antibody is also meant to include so-called antibody mimetics.
  • Antibody mimetics refers to small molecules, e.g. , 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure.
  • Antibody mimetics include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus acidocaldarius), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif),
  • Fynomers SH3 domain of Fyn
  • Kunitz domain peptides Kunitz domains of various protease inhibitors
  • Ecallantide Kalbitor
  • Monobodies Monobodies.
  • antibody or “antibodies” is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional anti cancer antibodies can for example be found in Scott et al, Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety.
  • a “single-chain antibody” or “single-chain antibodies” typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or bond, such as a disulfide bond.
  • the single-chain antibody lacks the constant Fc region found in traditional antibodies.
  • the single-chain antibody is a naturally occurring single-chain antibody, e.g., a camelid antibody.
  • the single-chain antibody is a synthetic, engineered, or modified single-chain antibody.
  • the single-chain antibody is capable of retaining substantially the same antigen specificity as compared to the original immunoglobulin despite the addition of a linker and the removal of the constant regions.
  • the single chain antibody can be a "scFv antibody", which refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins (without any constant regions), optionally connected with a short linker peptide of ten to about 25 amino acids, as described, for example, in U.S. Patent No. 4,946,778, the contents of which is herein incorporated by reference in its entirety.
  • the Fv fragment is the smallest fragment that holds a binding site of an antibody, which binding site may, in some aspects, maintain the specificity of the original antibody. Techniques for the production of single chain antibodies are described in U.S. Patent No.
  • Vh and VL sequences of the scFv can be connected via the N-terminus of the VH connecting to the C-terminus of the VL or via the C-terminus of the VH connecting to the N-terminus of the VL.
  • ScFv fragments are independent folding entities that can be fused indistinctively on either end to other epitope tags or protein domains.
  • Linkers of varying length can be used to link the Vh and VL sequences, which the linkers can be glycine rich (provides flexibility) and serine or threonine rich (increases solubility). Short linkers may prevent association of the two domains and can result in multimers (diabodies, tribodies, etc.).
  • Linkers may result in proteolysis or weak domain association (described in Voelkel et al el., 2011). Linkers of length between 15 and 20 amino acids or 18 and 20 amino acids are most often used. Additional non- limiting examples of linkers, including other flexible linkers are described in Chen et al, 2013 (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and Functionality), the contents of which is herein incorporated by reference in its entirety. Flexible linkers are also rich in small or polar amino acids such as Glycine and Serine, but can contain additional amino acids such as Threonine and Alanine to maintain flexibility, as well as polar amino acids such as Lysine and Glutamate to improve solubility.
  • Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-Gly-Ser)n, KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8, and Gly and Ser rich flexible linker, GSAGSAAGSGEF.
  • Single chain antibodies as used herein also include single- domain antibodies, which include camelid antibodies and other heavy chain antibodies, light chain antibodies, including nanobodies and single domains VH or VL domains derived from human, mouse or other species. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.
  • Single domain antibodies include domain antigen-binding units which have a camelid scaffold, derived from camels, llamas, or alpacas.
  • Camelids produce functional antibodies devoid of light chains.
  • the heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding molecules (Fabs) or single chain variable fragments (scFvs).
  • VH heavy chain variable
  • Fabs classical antigen-binding molecules
  • scFvs single chain variable fragments
  • Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies.
  • Camelid scaffold-based antibodies can be produced using methods well known in the art.
  • Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained.
  • VNAR fragments single-domain antibodies
  • the dimeric variable domains from IgG from humans or mice can be split into monomers.
  • Nanobodies are single chain antibodies derived from light chains. The term “single chain antibody” also refers to antibody mimetic s.
  • linker 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.
  • 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.
  • the antibodies expressed by the engineered microorganisms are bispecfic.
  • a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.
  • Antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies.
  • scDb Monomeric single-chain diabodies
  • 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.
  • 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.
  • the term "gene” or “gene sequence” is meant to refer to a nucleic acid sequence encoding a payload capable of detoxifying a deleterious molecule and/or any of the anti- inflammatory and gut barrier function enhancing molecules described herein, e.g. , IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP- 1, IL- 10, IL-27, TGF- ⁇ , TGF- ⁇ 2, N-acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as others.
  • IL-2 IL-22
  • SOD superoxide dismutase
  • kynurenine e.g., IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP- 1, IL- 10, IL-27, TGF- ⁇ , TGF- ⁇ 2, N-acylphosphati
  • the nucleic acid sequence may comprise the entire gene sequence or a partial gene sequence encoding a functional molecule.
  • the nucleic acid sequence may be a natural sequence or a synthetic sequence.
  • the nucleic acid sequence may comprise a native or wild-type sequence or may comprise a modified sequence having one or more insertions, deletions, substitutions, or other modifications, for example, the nucleic acid sequence may be codon-optimized.
  • heterologous gene or heterologous sequence 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 and can be a native sequence (naturally found or expressed in the cell) or non- native sequence (not naturally found or expressed in the cell) and can be a natural or wild-type sequence or a variant, non-natural, or synthetic sequence.
  • “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 to include non-native regulatory regions that is reintroduced into the host cell.
  • a heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell.
  • 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.
  • a "gene cassette” or “operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce a molecule of interest, e.g. , a gut barrier function enhancer molecule such as butyrate.
  • the gene cassette may also comprise additional transcription and translation elements, e.g. , a ribosome binding site.
  • a "butyrogenic gene cassette,” “butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway.
  • Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema.
  • the genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • a butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C- acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate
  • butyryltransferase and butyrate kinase, respectively (Aboulnaga et al., 2013).
  • One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g. , codon optimized.
  • Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk.
  • a butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiAl from
  • Peptoclostridium difficile strain 630 and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.
  • a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile.
  • a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from
  • the butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.
  • the pbt and buk genes are replaced with tesB (e.g., from E coli).
  • a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.
  • a "propionate gene cassette” or “propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway.
  • Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola.
  • the genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and
  • Dehydration of (K)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC).
  • Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC).
  • the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC are replaced by the acul gene from R. sphaeroides.
  • This gene product catalyzes the NADPH-dependent acrylyl- CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013).
  • the propionate cassette comprises pet, IcdA, IcdB, IcdC, and acul.
  • the homo log of Acul in E coli, YhdH is used (see. e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004).
  • This the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH.
  • the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively.
  • the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.
  • a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH). Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g.
  • the SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA.
  • Sbm methylmalonyl-CoA mutase
  • YgfD is a Sbm- interacting protein kinase with GTPase activity
  • ygfG methylmalonylCoA decarboxylase
  • ygfH propionyl-CoA/succinylCoA transferase
  • propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009).
  • the propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.
  • One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • An "acetate gene cassette” or “acetate operon” refers to a set of genes capable of producing acetate in a biosynthetic pathway.
  • Bacteria “synthesize acetate from a number of carbon and energy sources,” including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008).
  • the genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994).
  • Several bacteria such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or C0 2 + H 2 into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012).
  • the acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate.
  • One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • Each gene or gene cassette may be present on a plasmid or bacterial chromosome.
  • multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein.
  • the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.
  • each gene or gene cassette for producing a payload may be present on a plasmid or bacterial chromosome.
  • 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.
  • 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 on, inter alia, 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
  • Reduced levels of a toxic molecule, metabolite, or other deleterious molecule is used to refer to a reduction in the amount of said toxic molecule, metabolite, or molecule after treatment with the genetically engineered bacteria of the invention, as compared to amount after treatment with unmodified bacteria of the same subtype under the same conditions. In some embodiments, reduction is measured by comparing the level of the toxic molecule, metabolite, or other deleterious molecule before and after administering the genetically engineered bacteria of the invention.
  • levels of the toxic molecule, metabolite, or other deleterious molecule is about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% as compared to levels in an untreated or control condition.
  • Each gene or gene cassette may be operably linked to a promoter that is induced by exogenous environmental conditions, for example low-oxygen conditions, as is found in a mammalian gut.
  • "Operably linked” refers a nucleic acid sequence, e.g., a carboxypeptidase G 2 (CPD G 2 ) gene, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis.
  • a regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • a regulatory region "operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding an anti- inflammatory or gut barrier enhancer molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the anti- inflammatory or gut barrier enhancer molecule. In other words, the regulatory sequence acts in cis.
  • 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.
  • oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR.
  • FNR-responsive promoters include, but are not limited to, FNR, ANR, and DNR.
  • ANR-responsive promoters include, but are not limited to, ANR, and DNR.
  • DNR-responsive promoters are known in the art ⁇ see, e.g.,
  • a promoter was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010).
  • the PfnrS promoter is activated under anaerobic conditions by the global
  • a "tunable regulatory region” refers to a nucleic acid sequence under direct or indirect control of a transcription factor and which is capable of activating, repressing, derepressing, or otherwise controlling gene expression relative to levels of an inducer.
  • the tunable regulatory region comprises a promoter sequence.
  • the inducer may be RNS, or other inducer described herein, and the tunable regulatory region may be a RNS -responsive regulatory region or other responsive regulatory region described herein.
  • the tunable regulatory region may be operatively linked to a gene sequence(s) or gene cassette for the production of one or more payloads, e.g. , a butyrogenic or other gene cassette or gene sequence(s).
  • the tunable regulatory region is a RNS-derepressible regulatory region, and when RNS is present, a RNS- sensing transcription factor no longer binds to and/or represses the regulatory region, thereby permitting expression of the operatively linked gene or gene cassette.
  • the tunable regulatory region derepresses gene or gene cassette expression relative to RNS levels.
  • Each gene or gene cassette may be operatively linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one RNS.
  • the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS).
  • exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut.
  • the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.
  • 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
  • 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).
  • an inducible promoter for example, an aerobic environment outside the gut.
  • “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
  • GI gastrointestinal
  • 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.
  • 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 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.
  • the genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a payload that is operably linked to a promoter that is not associated with said gene or gene cassette in nature, e.g. , a FNR-responsive promoter operably linked to a butyrogenic gene cassette.
  • the genetically engineered microorganism of the disclosure comprises a gene that is operably linked to a promoter that is not associated with said gene in nature.
  • the genetically engineered bacteria disclosed herein comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR responsive promoter (or other promoter disclosed herein) operably linked to an ant i- inflammatory or gut barrier enhancer molecule.
  • the genetically engineered virus of the disclosure comprises a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., a promoter operably linked to a gene encoding an antiinflammatory or gut barrier enhancer molecule.
  • 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. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed herein.
  • 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
  • Prokaryotic promoters are typically classified into two classes: inducible and constitutive.
  • a "constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control.
  • 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 ⁇ promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of
  • BBa_J45992 Standard Biological Parts Name BBa_J45992; BBa_J45993)
  • a constitutive Escherichia coli ⁇ 32 promoter e.g., htpG heat shock promoter (BBa_J45504)
  • a constitutive Escherichia coli ⁇ 70 promoter e.g., lacq promoter (BBa_J54200; BBa_J56015), E.
  • coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110
  • BBa_M13110 Bacillus subtilis ⁇ ⁇ promoter
  • promoter veg a constitutive Bacillus subtilis ⁇ ⁇ promoter
  • BBa_K143013 promoter 43 (BBa_K143013), P liaG (BBa_K823000), P lepA (BBa_K823002), P veg (BBa_K823003)), a constitutive Bacillus subtilis ⁇ 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_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter e.g., SP6 promoter (BBa_J64998).
  • 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.”
  • exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g. , arabinose and
  • inducible promoters include, but are not limited to, an FNR responsive promoter, a ParaC promoter, a ParaBAD promoter, and a PTetR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.
  • genetically engineered bacteria that "overproduce" a payload refer to bacteria that 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 payload than unmodified bacteria of the same subtype under the same conditions.
  • the mRNA transcript levels of one or more of the gene(s) for producing the payload in the genetically engineered bacteria are 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 higher than the mRNA transcript levels in unmodified bacteria of the same subtype under the same conditions.
  • the unmodified bacteria will not have detectable levels of the payload and/or transcription of the gene(s).
  • protein and/or transcription levels of the payload will be detectable in the corresponding genetically engineered bacterium. Transcription levels may be detected by directly measuring mRNA levels of the genes.
  • Levels of lead in the blood may be measured by atomic absorption spectrometry, anodic stripping voltammetry, and/or mass spectrometry.
  • Level of methotrexate in the blood may be measured by HPLC, immunoassay, and/or enzyme inhibition assay.
  • "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.
  • 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.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • 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.
  • the exogenous environmental conditions are specific to the small intestine of a mammal.
  • the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut.
  • exogenous exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut.
  • the environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease- state, e.g., propionate.
  • the exogenous environmental condition is a tissue-specific 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 genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter.
  • 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.
  • “environmental conditions” refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. The phrase
  • exogenous environmental conditions is meant to refer 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, and overall viability and metabolic activity of the strain during strain production.
  • exogenous and “endogenous” may be used
  • 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.
  • exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate.
  • the exogenous environmental condition is a tissue- specific or disease- specific metabolite or molecule(s).
  • the exogenous environmental condition is specific to an inflammatory disease.
  • the exogenous environmental condition is a low-pH environment.
  • the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter.
  • the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter.
  • bacteria have evolved transcription factors that are capable of sensing oxygen levels.
  • 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.
  • the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (0 2 ) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., ⁇ 21% 0 2; ⁇ 160 torr 03 ⁇ 4).
  • the term “low oxygen condition or conditions” or “low oxygen environment” refers to conditions or environments containing lo were levels of o ygen than are present in the atmosphere.
  • the term "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (0 2 ) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal.
  • the term "low oxygen” is meant to refer to a level, amount, or concentration of 0 2 that is 0-60 mmHg 0 2 (0-60 torr 0 2) (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 0 2 ), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg 0 2 , 0.75 mmHg 0 2 , 1.25 mmHg 0 2 , 2.175 mmHg 0 2 , 3.45 mmHg 0 2 , 3.75 mmHg 0 2 , 4.5 mmHg 0 2
  • low oxygen refers to about 60 mmHg 0 2 or less (e.g., 0 to about 60 mmHg 0 2 ).
  • the term “low oxygen” may also refer to a range of 0 2 levels, amounts, or concentrations between 0-60 mmHg 0 2 (inclusive), e.g., 0-5 mmHg 0 2 , ⁇ 1.5 mmHg 0 2 , 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,
  • the term "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (0 2 ) 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.
  • "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (0 2 ) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions.
  • Table A summarizes the amount of oxygen present in various organs and tissues.
  • DO amount of dissolved oxygen
  • the term "low oxygen” is meant to refer to a level, amount, or concentration of oxygen (0 2 ) 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.
  • 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 (0 2 ) 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).
  • weil-aerated solutions e.g., solutions subjected to mixing and/or stirring
  • 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 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% 0 2 saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, 0 2 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 0 2 saturation levels between 0-9%, inclusive ⁇ e.g.
  • the 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 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.
  • Microorganism refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell.
  • microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, e.g. ,
  • the microorganism is engineered ("engineered microorganism") to produce one or more therpauetic molecules, e.g. , an antinflammatory or barrier enhancer molecule.
  • 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. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides,
  • Bifidobacterium Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus
  • 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 bacteria.
  • probiotic bacteria examples include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei,
  • Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et 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.
  • stable bacterium is used to refer to a bacterial host cell carrying non- native genetic material, e.g., a gene or gene cassette for producing a payload, 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 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).
  • the stable bacterium may be a genetically engineered bacterium comprising a butyrogenic gene cassette, in which the plasmid or chromosome carrying the gene cassette is stably maintained in the bacterium, such that the gene cassette can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.
  • the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a encoding a payload, e.g., one or more anti- inflammation and/or gut barrier enhancer molecule(s), in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that the payload 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.
  • recombinant microorganism refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state.
  • a "recombinant bacterial cell” or “recombinant bacteria” 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.
  • Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids.
  • recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.
  • a "programmed or engineered microorganism” refers to a
  • a "programmed or engineered bacterial cell” or “programmed or engineered bacteria” refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function.
  • the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose.
  • the programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed.
  • 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.
  • 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 a detoxification molecule, an ant i- inflammatory molecule, or a gut barrier enhancer molecule.
  • transform 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, base substitution, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter
  • Genetic modification can include the introduction of a plasmid, e.g. , a plasmid comprising gene sequene(s) encoding a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule 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.
  • the term "transporter” is meant to refer to a mechanism, e.g. , protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di-peptide, tri-peptide, polypeptide, etc), toxic molecule, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.
  • modulate and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement.
  • modulate or modulation includes up-regulation and down-regulation.
  • a non- limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound).
  • modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen.
  • modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms.
  • modulate is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms).
  • modulate and “treat” and their cognates refer to an amelioration of a disorder or condition, e.g. , heavy metal poisoning, 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 disorder 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 disorder or condition.
  • prevent and its cognates refer to delaying the onset or reducing the risk of acquiring a given disorder or condition.
  • Those in need of treatment may include individuals already having a particular disorder or condition, as well as those at risk of having, or who may ultimately acquire the disorder or condition, e.g. , chemotherapy- induced diarrhea.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder.
  • Treatment may encompass reducing or eliminating one or more deleterious symptoms, e.g. , diarrhea, and does not necessarily encompass the elimination of the underlying disorder.
  • Treating the diseases described herein may encompass increasing levels of butyrate, increasing levels of acetate, increasing levels of butyrate and increasing GLP-2, IL- 22, and/o rIL- 10, and/or modulating levels of tryptophan and/or its metabolites (e.g. , kynurenine), and/or providing any other detoxification of a deleterious molecule and/or gut barrier enhancer molecule and does not necessarily encompass the elimination of the underlying disease.
  • tryptophan and/or its metabolites e.g. , kynurenine
  • composition refers to a preparation of genetically engineered bacteria of the invention with other components such as a
  • physiologically suitable carrier and/or excipient are physiologically suitable carriers 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.
  • 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, sodium 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, e.g., chemotherapy- induced diarrhea.
  • a therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder or condition caused by a toxic molecule, metabolite, or other deleterious molecule.
  • 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.
  • bacteriostatic or “cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure.
  • bactericidal refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.
  • secretion system or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g. , polypeptide from the microbial, e.g. , bacterial cytoplasm.
  • the secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g. ,HlyBD.
  • Non- limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g.
  • secretion systems include Sec and TAT secretion systems.
  • the polypeptide to be secreted include a "secretion tag" of either RNA or peptide origin to direct the polypeptide to specific secretion systems.
  • the secretion system is able to remove this tag before secreting the polyppetide from the engineered bacteria.
  • the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g. , OmpT cleavage thereby releasing the
  • the secretion system involves the generation of a "leaky” or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, to IB, pal, degS, degP, and nlpl.
  • Lpp functions as the primary 'staple' of the bacterial cell wall to the peptidoglycan.
  • TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype.
  • the engineered bacteria have one or more deleted or mutated membrane genes, e.g. , selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes.
  • the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g. , selected from degS, degP, and nlpl.
  • the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.
  • preventional treatment or “conventional therapy” refers to treatment or therapy that is currently accepted, considered current standard of care, and/or used by most healthcare professionals for treating a disease or disorder associated with BCAA. It is different from alternative or complementary therapies, which are not as widely used.
  • 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 genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying deleterious molecules, e.g., chemotherapeutic drugs or metabolites or byproducts thereof, nonsteroidal anti- inflammatory drugs or metabolites or byproducts thereof, or exogenous poisons.
  • the genetically engineered bacteria are further capable of expressing an anti- inflammatory molecule and/or a gut barrier enhancer molecule.
  • the genetically engineered bacteria are nonpathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments,
  • non-pathogenic bacteria are Gram-positive bacteria.
  • the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity.
  • Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides
  • the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.
  • the genetically engineered bacterium is a Gram- positive bacterium, e.g., Clostridium, that is naturally capable of producing high levels of butyrate.
  • the genetically engineered bacterium is selected from the group consisting of C. butyricum ZJUCB, C. butyricum S21, C. thermobutyricum ATCC 49875, C. beijerinckii, C. populeti ATCC 35295, C. tyrobutyricum JM1, C. tyrobutyricum CIP 1-776, C. tyrobutyricum ATCC 25755, C. tyrobutyricum CNRZ 596, and C.
  • the genetically engineered bacterium is C. butyricum CBM588, a probiotic bacterium that is highly amenable to protein secretion and has demonstrated efficacy in treating IBD (Kanai et al, 2015).
  • the genetically engineered bacterium is Bacillus, a probiotic bacterium that is highly genetically tractable and has been a popular chassis for industrial protein production; in some embodiments, the bacterium has highly active secretion and/or no toxic byproducts (Cutting, 2011).
  • 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 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.
  • 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.
  • the genetically engineered bacteria are any suitable organisms.
  • Escherichia coli strain Nissle 1917 Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the
  • E. coli Nissle lacks prominent virulence factors (e.g., E. coli a-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E.
  • E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn 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).
  • One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria. In some
  • the genetically engineered bacteria are E. coli Nissle and are naturally capable of promoting tight junctions and gut barrier function. In some embodiments, the genetically engineered bacteria are E. coli and are highly amenable to recombinant protein technologies.
  • the recombinant bacterial cell does not colonize the subject having the disorder.
  • Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum
  • Residence time in vivo may be calculated for the genetically engineered bacteria. In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention, e.g. as described herein.
  • Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009). Thus the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria.
  • the payload(s) described below are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the payload is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.
  • 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 disclosed herein.
  • the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) further comprise a kill- switch circuit, such as any of the kill-switch circuits provided herein.
  • the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)is an auxotroph.
  • the genetically engineered bacteria comprising gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.
  • the gene encoding one or more enzyme(s) capable of detoxifying a deleterious molecule is present on a plasmid in the bacterium.
  • the gene sequence(s) encoding an anti- inflammatory or gut barrier enhancer molecule is present in the bacterial chromosome.
  • a gene sequence encoding a secretion protein or protein complex, such as any of the secretion systems disclosed herein, for secreting a biomolecule e.g.
  • an enzyme capable of detoxifying a deleterious molecule, gene sequence encoding an anti- inflammatory molecule, and/or gene sequence encoding a gut barrier enhancer molecule), is present on a plasmid in the bacterium.
  • the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome.
  • the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium.
  • the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome.
  • the genetically engineered bacteria are capable of inhibiting, metabolizing, and/or detoxifying chemotherapeutic drugs or metabolites or byproducts thereof.
  • the genetically engineered bacteria detoxify the drug or metabolite or byproduct after the chemotherapeutic drug exerts its therapeutically beneficial effects, e.g., cytotoxicity in cancerous cells.
  • the genetically engineered bacteria are administered before, together with, and/or after administration of the chemotherapeutic drug.
  • the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the drug or metabolite or byproduct, thereby reducing chemotherapy-induced diarrhea, reducing chemotherapy-induced toxicity, increasing chemotherapy dosage amount, increasing chemotherapy dosage frequency, and/or increasing chemotherapy efficacy.
  • the molecule to be detoxified is a
  • chemotherapeutic drug selected from irinotecan, methotrexate, an antimetabolite, gemcitabine, cytosine arabinoside, a fluoropyrimidine, fluoro uracil, capecitabine, tegafur- uracil, a multitargeted folinic acid antagonist, pemetrexed, raltitrexed, gemcitabine, a plant alkaloid, a vinca alkaloid, vincristine, vinorelbine, a epipodophyllotoxin, etoposide, a taxane, paclitaxel, docetaxel, a topoisomerase I inhibitor, a cytotoxic antibiotic, an anthracycline, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunomycin, an alkylating agent, cyclophosphamide, a platinum, cisplatin, carboplatin, ox
  • a chemotherapeutic drug may have therapeutically beneficial effects as well as deleterious effects.
  • methotrexate is an effective antineoplastic agent, but can also cause dose-limiting diarrhea, gastrointestinal toxicity, and bone marrow toxicity (Chabner et al., 1972).
  • Methotrexate is a folate antagonist that acts by inhibiting several enzymes of the folate pathway and disrupting folate homeostasis.
  • methotrexate is rapidly degraded and virtually no n- toxic (Chabner et al, 1972).
  • no natural enzymes are capable of metabolizing methotrexate; methotrexate persists in the blood for many hours after administration and is capable of causing dose-limiting side effects.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate.
  • the payload is a small molecule that is capable of inhibiting methotrexate.
  • the payload is an enzyme that is capable of metabolizing methotrexate into non-toxic metabolites.
  • the enzyme capable of metabolizing methotrexate is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • the genetically engineered bacteria comprise a gene encoding carboxypeptidase Gi (CPD Gi) and are capable of detoxifying methotrexate.
  • the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi (Chabner et ah,
  • the genetically engineered bacteria comprise a gene encoding carboxypeptidase G 2 (CPD G 2 ) and are capable of detoxifying methotrexate.
  • CPD G 2 carboxypeptidase G 2
  • Exemplary amino acid sequences for CPD Gi and CPD G 2 are provided below.
  • the genetically engineered bacterium comprises one or more genes encoding CPD Gi ⁇ i.e., one or more cpgl genes).
  • the genetically engineered bacteria comprises a CPD Gi gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328.
  • the genetically engineered bacteria comprises a CPD Gi gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 328.
  • the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 328.
  • the genetically engineered bacterium comprises one or more genes encoding CPD G 2 ⁇ i.e., one or more cpg2 genes).
  • the genetically engineered bacteria comprises a CPD G 2 gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329.
  • the genetically engineered bacteria comprises a CPD G 2 gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 329.
  • the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329.
  • the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 329.
  • the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a constitutive promoter.
  • the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of an inducible promoter. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the exogenous
  • environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate.
  • the exogenous environmental conditions are low- oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • the gene or gene cassette for producing the methotrexate-detoxifying payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the sequence encoding CPD G 2 may be integrated into the bacterial chromosome.
  • the gene or gene cassette for producing the methotrexate-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the methotrexate-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24).
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • a gene required for survival and/or growth such as thyA (to create an auxotroph)
  • thyA to create an auxotroph
  • an active area of the genome such as near the site of genome replication
  • divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • the genetically engineered bacteria produce more of the methotrexate-detoxifying payload, e.g., CPD G 2 protein or transcript, than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria are capable of reducing local and/or systemic methotrexate levels, e.g., in the gut.
  • the genetically engineered bacteria are capable of detoxifying methotrexate in the intestinal lumen, and do not affect the therapeutically beneficial
  • the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF- ⁇ , TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S.
  • ant i- inflammatory and/or gut-barrier enhancing molecule(s) including but are not limited to, short-chain fatty acids, butyrate
  • the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating methotrexate-induced diarrhea and/or other drug-induced symptom.
  • the short-chain fatty acid is selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate.
  • engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g., a short-chain fatty acid.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying methotrexate and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene encoding Pseudomonas stutzeri CPD Gi and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically engineered bacteria comprise a gene encoding CPD G 2 and further comprise a gene cassette encoding a bio synthetic pathway for producing butyrate.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.
  • methotrexate can be used to treat certain types of cancers.
  • methotrexate can be used to treat rheumatic diseases.
  • the genetically engineered bacteria of the invention are capable of reducing methotrexate-induced diarrhea and toxicity for non-cancer indications, e.g., rheumatoid arthritis.
  • a chemotherapeutic drug may have therapeutically beneficial effects, but its metabolites or byproducts have deleterious effects.
  • irinotecan is an effective antineoplastic agent, but its metabolite SN-38 can cause dose-limiting diarrhea and gastrointestinal toxicity (Steiner et al., 2010).
  • Irinotecan is a topoisomerase I inhibitor that is used to treat colorectal cancer, non-small cell lung cancer, small cell lung cancer, and pancreatic cancer. Hepatic and peripheral carboxylesterase convert irinotecan to its active metabolite SN-38 (7-ethyl-lO-hydroxycamptothecin).
  • SN-38 is approximately 100- 1,000 times more cytotoxic than irinotecan (Steiner et al, 2010).
  • Hepatic uridine diphosphate glucuronosyltransferase-lAl UDP-GT 1A1
  • UDP-GT 1A1 Hepatic uridine diphosphate glucuronosyltransferase-lAl
  • SN-38G the much less toxic metabolite SN-38G.
  • commensal bacteria the produce ⁇ -glucuronidase deconjugate SN-38G into toxic SN-38, which can cause intestinal toxicity and diarrhea.
  • Commensal bacteria that are known to have B -glucuronidase activity include enterobacteriaceae, E.
  • irinotecan chemotherapy can cause acute diarrhea, which occurs within 24 hours of receiving the drug, and/or delayed diarrhea, which occurs after 24 hours of receiving the drug (Steiner et al, 2010).
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and/or SN-38.
  • the payload is a small molecule that inhibits ⁇ -glucuronidase to prevent the conversion of irinotecan into the toxic metabolite SN-38 (Wallace et al., 2010).
  • the small molecule that inhibits ⁇ -glucuronidase is D-saccharic acid 1.4- lactone (SAL) (Fittkau et al., 2004).
  • the payload is a small molecule that is capable of inhibiting SN-38.
  • the payload is an enzyme that is capable of metabolizing SN-38 into non-toxic metabolites.
  • the payload is an enzyme that is capable of glucuroniding SN-38, thereby converting it into non-toxic SN- 38G.
  • the enzyme is from a non-human species, e.g., a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • the payload is a molecule that is capable of inhibiting or killing the commensal bacteria that produce ⁇ -glucuronidase.
  • the payload is a molecule that promotes changes to the intestinal microflora, e.g., enhancing the survival and/or proliferation of the genetically engineered bacteria, thereby outcompeting commensal bacteria that produce ⁇ -glucuronidase.
  • the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase, which is capable of adding glucuronic acid to SN-38, and are capable of detoxifying SN-38.
  • UDP-glucuronsyltransferase which is capable of adding glucuronic acid to SN-38, and are capable of detoxifying SN-38.
  • known bacterial metabolites will be surveyed via metagenomic studies and assessed for potency to inhibit bacterial glucouronidase substrate. Bio informatics will be used to inform, select, and stratify physiologically relevant glucouronidase inhibitors.
  • An exemplary amino acid sequence of UDP-glucuronsyltransferase is provided below.
  • the genetically engineered bacterium comprises one or more genese encoding UDP-glucuronsyltransferase.
  • the genetically engineered bacteria comprises a UDP-glucuronsyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 330.
  • the genetically engineered bacteria comprises a UDP-glucuronsyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 330.
  • the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 330.
  • the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 330.
  • the gene or gene cassette for producing the SN-N-(00191] is a gene or gene cassette for producing the SN-N-(00191] in some embodiments, the gene or gene cassette for producing the SN-N-(00191] in some embodiments, the gene or gene cassette for producing the SN-N-(00191] in some embodiments, the gene or gene cassette for producing the SN-N-(00191] in some embodiments, the gene or gene cassette for producing the SN-
  • the 38-detoxifying pay load is expressed under the control of a constitutive promoter.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of an inducible promoter.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal.
  • the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g. , propionate.
  • the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • the gene or gene cassette for producing the SN-38-detoxifying payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.
  • expression from the plasmid may be useful for increasing payload expression.
  • expression from the chromosome may be useful for increasing stability of payload expression.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria.
  • the gene or gene cassette for producing the SN-38-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24.
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • a gene required for survival and/or growth such as thyA (to create an auxotroph)
  • thyA to create an auxotroph
  • an active area of the genome such as near the site of genome replication
  • divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • the genetically engineered bacteria produce more of the SN-38-detoxifying pay load than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria are capable of reducing local and/or systemic SN-38 levels, e.g., in the gut.
  • the genetically engineered bacteria are capable of detoxifying SN-38 in the intestinal lumen, and do not affect the therapeutically beneficial antineoplastic/cytotoxic effects of irinotecan on cancerous cells.
  • the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF- ⁇ , TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S.
  • ant i- inflammatory and/or gut-barrier enhancing molecule(s) including but are not limited to, short-chain fatty acids, butyrate
  • the genetically engineered bacteria encode a bio synthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating irinotecan- induced diarrhea and/or other drug-induced symptom.
  • the short-chain fatty acid is selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically
  • engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g. , a short-chain fatty acid.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying irinotecan and further comprise a gene cassette encoding a bio synthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene encoding UDP-glucuronsyltransferase and further comprise a gene cassette encoding a bio synthetic pathway for producing butyrate.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.
  • the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying NSAIDs or metabolites or byproducts thereof.
  • the genetically engineered bacteria detoxify the NSAID or metabolite or byproduct after the NSAID exerts its therapeutically beneficial effects, e.g. , reducing inflammation.
  • the genetically engineered bacteria are administered before, together with, and/or after NSAID administration (see, e.g., Cohen et al., 2013).
  • the genetically engineered bacteria are capable of detoxifying, inhibiting, and/or metabolizing the toxic NSAID or metabolite or byproduct, thereby reducing NSAID-induced diarrhea, reducing NSAID-induced toxicity, increasing NSAID dosage amount, increasing NSAID dosage frequency, and/or increasing NSAID efficacy.
  • the molecule to be detoxified is a NSAID selected from naproxen, indomethacin, ketoprofen, piroxicam, ibuprofen, diclofenac, a COX-2 inhibitor, or a metabolite or byproduct thereof.
  • An exemplary NSAID is described below.
  • cyclooxygenase- 1 COX- 1
  • COX-2 cyclooxygenase-2
  • the ant i- inflammatory and analgesic effects of NSAIDs are thought to be due to inhibition of COX-2, while the gastrointestinal toxicity of NSAIDs is thought to be due to the inhibition of COX- 1 (Scarpignato 2008).
  • NSAID-induced toxicity to the intestinal epithelium occurs after oral administration. Local sub-cellular damage occurs at the brush border cell member upon entry of the typically acidic NSAID.
  • Mitochondrial oxidative phosphorylation is disrupted, leading to ATP deficiency, increased mucosal permeability, and entrance of luminal molecules such as bacteria, pancreatic juice, bile acids, and dietary macromolecules, which causes an
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying one or more NSAIDs, e.g. , naproxen.
  • the payload is a small molecule that is capable of inhibiting one or more NSAIDs, e.g. , naproxen.
  • the payload is a proton pump inhibitor, and the genetically engineered bacteria are capable of ameliorating NSAID-induced intestinal damage (see, e.g., Scarpignato, 2008).
  • the payload is an enzyme that is capable of metabolizing the NSAID into non-toxic metabolites.
  • the enzyme capable of metabolizing the NSAID is from a non-human species, e.g. , a plant, bacterial, or other mammalian enzyme.
  • the enzyme is a synthetic or modified enzyme.
  • B -glucuronidase inhibition can alleviate NSAID- induced enteropathy (LoGuidice et al., 2012).
  • the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase (SEQ ID NO: 331) and are capable of glucuronidating and detoxifying naproxen.
  • the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase (SEQ ID NO: 331), a gene cassette for producing butyrate, and are capable of detoxifying naproxen.
  • SEQ ID NO: 331 a gene encoding glucuronosyltransferase
  • SEQ ID NO: 331 a gene encoding glucuronosyltransferase
  • a gene cassette for producing butyrate and are capable of detoxifying naproxen.
  • An exemplary amino acid sequence of glucuronosyltransferase is provided below.
  • the genetically engineered bacterium comprises one or more genes encoding glucuronosyltransferase.
  • the genetically engineered bacteria comprises a glucuronosyltransferase gene sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 331.
  • the genetically engineered bacteria comprises a glucuronosyltransferase gene sequence encoding a polypeptide
  • the genetically engineered bacteria comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 331. In some embodiments, the genetically engineered bacteria comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 331.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that promotes changes to the intestinal microflora, e.g. , enhancing the survival and/or proliferation of Gram-positive bacteria.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of inhibiting or killing Gram-negative bacteria.
  • the NSAID-detoxifying payload is expressed under the control of a constitutive promoter.
  • the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of an inducible promoter.
  • the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions.
  • the gene or gene cassette for producing the NSAID-detoxifying payload is expressed under the control of a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal.
  • the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g. , propionate.
  • the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • the gene or gene cassette for producing the NSAID-detoxifying payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the NSAID-detoxifying payload is expressed from a plasmid in the genetically engineered bacteria.
  • the gene or gene cassette for producing the NSAID-detoxifying payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 24).
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • a gene required for survival and/or growth such as thyA (to create an auxotroph)
  • thyA to create an auxotroph
  • an active area of the genome such as near the site of genome replication
  • divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • the genetically engineered bacteria produce more of the NSAID-detoxifying payload than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria are capable of reducing local and/or systemic NSAID levels, e.g., in the gut.
  • the genetically engineered bacteria are capable of detoxifying the NSAID in the intestinal lumen, and do not affect the therapeutically beneficial anti- inflammatory effects of the NSAID systemically.
  • the genetically engineered bacteria further comprise a gene cassette for producing one or more anti- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL- 10, IL-27, TGF- ⁇ , TGF- ⁇ 2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S.
  • a gene cassette for producing one or more anti- inflammatory and/or gut-barrier enhancing molecule(s) including but are not limited to, short-chain fatty acids,
  • the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating NSAID-induced diarrhea and/or other drug-induced symptom.
  • the short-chain fatty acid is selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene cassette encoding a biosynthetic pathway for producing butyrate. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying the NSAID and further comprise a gene or gene cassette for producing a payload that is capable of enhancing gut barrier function, e.g., a short-chain fatty acid. In some embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of detoxifying the NSAID and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene encoding glucuronosyltransferase and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low- copy plasmid, or a chromosome, as described above.
  • the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more heavy metals, thereby ameliorating one or more symptoms of heavy metal poisoning. In some embodiments, the genetically engineered bacteria of the invention are capable of ameliorating acute heavy metal poisoning and/or chronic heavy metal poisoning.
  • genetically engineered bacteria of the invention are capable of ameliorating one or more symptoms of aluminum poisoning, antimony poisoning, arsenic poisoning, barium poisoning, bismuth poisoning, cadmium poisoning, chromium poisoning, cobalt poisoning, copper poisoning, gold poisoning, iron poisoning, lead poisoning, lithium poisoning, manganese poisoning, mercury poisoning, nickel poisoning, phosphorous poisoning, platinum poisoning, selenium poisoning, silver poisoning, thallium poisoning, tin poisoning, and/or zinc poisoning.
  • Examples of heavy metal toxicity linked to bacteria are shown in Table 2. Table 2. Heavy metal link with bacteria/probiotics
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering a heavy metal.
  • the payload is a heavy metal chelator.
  • the payload is a plant phytochelatin.
  • the payload is from a non-human species, e.g. , a plant, bacterial, or other mammalian molecule.
  • the payload is a synthetic or modified molecule.
  • the genetically engineered bacteria are capable of expressing plant phytochelatins, particularly on the surface of the bacteria (see, e.g., Bae et al., 2000).
  • the genetically engineered bacteria of the invention are capable of binding to cadmium, thereby ameliorating one or more symptoms of cadmium poisoning.
  • the genetically engineered bacteria comprise a gene encoding a plant phytochelatin and a gene cassette for producing butyrate.
  • the genetically engineered bacteria comprise a gene encoding an enzyme involved in the production of a plant phytochelatin.
  • Exemplary plant phytochelatins include, but are not limited to, glutathione, homoglutathione, hydroxymethylglutathione, and
  • the gene or gene cassette for producing the metal- binding payload e.g., a plant phytochelatin
  • the gene or gene cassette for producing the metal-binding payload is expressed under the control of a constitutive promoter.
  • the gene or gene cassette for producing the metal-binding payload e.g., a plant phytochelatin
  • the gene or gene cassette for producing the metal-binding payload is expressed under the control of a promoter that is induced by exogenous environmental conditions.
  • the gene or gene cassette for producing the metal-binding payload is expressed under the control of a promoter that is induced by exogenous
  • the exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate.
  • the exogenous environmental conditions are low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • the gene or gene cassette for producing the metal-binding payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing payload expression. In some embodiments, expression from the chromosome may be useful for increasing stability of payload expression. In some embodiments, the gene or gene cassette for producing the metal-binding payload is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metal-binding payload is expressed from a plasmid in the genetically engineered bacteria.
  • the gene or gene cassette for producing the metal- binding payload is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T.
  • Any suitable insertion site may be used.
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • the genetically engineered bacteria produce more of the metal-binding payload than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria are capable of reducing local and/or systemic heavy metal levels.
  • the genetically engineered bacteria further comprise a gene cassette for producing one or more ant i- inflammatory and/or gut-barrier enhancing molecule(s), including but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP- 1, IL- 10, IL-27, TGF- ⁇ , TGF-p2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein and in U.S.
  • ant i- inflammatory and/or gut-barrier enhancing molecule(s) including but are not limited to, short-chain fatty acids, butyrate
  • the genetically engineered bacteria encode a biosynthetic pathway for producing a short-chain fatty acid and are capable of reducing inflammation and/or enhancing gut barrier function for treating heavy metal- induced diarrhea and/or other drug-induced symptom.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering one or more heavy metals and further comprise a gene or gene cassette for producing a payload that is capable of reducing inflammation and/or enhancing gut barrier function, e.g. , a short-chain fatty acid.
  • the genetically engineered bacteria comprise a gene or gene cassette for producing a payload that is capable of binding or sequestering one or more heavy metals and further comprise a gene cassette encoding a biosynthetic pathway for producing a short-chain fatty acid selected from butyrate, propionate, and acetate.
  • the genetically engineered bacteria comprise a gene encoding a plant phytochelatin and further comprise a gene cassette encoding a biosynthetic pathway for producing butyrate.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed under the control of a constitutive promoter, an inducible promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut, as described above.
  • the gene or gene cassette for producing the gut-barrier enhancing payload may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome, as described above.
  • the genetically engineered bacteria of the invention are capable of inhibiting, metabolizing, and/or detoxifying one or more environmental toxic molecules, such as antibiotics, anticonvulsants, mood stabilizers and sex hormones, e.g., estrogen.
  • Pseudomonas produces many enzymes that can degrade a wide array of organic compounds including drugs. Therefore, Pseudomonas can be used to identify enzymes that can degrade toxic molecules of interest and geneically engineered bacteria comprising genes encoding these enzymes can be made and used as therapeutics.
  • the genetically engineered bacteria comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule.
  • the genetically engineered bacteria comprise one or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule.
  • the genetically engineered bacteria may comprise two or more gene sequence(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule.
  • the two or more gene sequences are multiple copies of the same gene.
  • the two or more gene sequences are sequences encoding different genes.
  • the two or more gene sequences are sequences encoding multiple copies of one or more different genes.
  • the genetically engineered bacteria comprise one or more gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule.
  • the genetically engineered bacteria may comprise two or more gene cassette(s) for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule.
  • the two or more gene cassettes are multiple copies of the same gene cassette.
  • the two or more gene cassettes are different gene cassettes for producing either the same or different anti- inflammation and/or gut barrier function enhancer molecule(s).
  • the two or more gene cassettes are gene cassettes for producing multiple copies of one or more different anti- inflammation and/or gut barrier function enhancer molecule(s).
  • the anti- inflammation and/or gut barrier function enhancer molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP- 1, IL- 10 (human or viral), IL-27, TGF- ⁇ , TGF-P2, N-acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan metabolite, indole, indole metabolite, a single-chain variable fragment (scFv), antisense RNA, si
  • a molecule may be primarily anti- inflammatory, e.g. , IL- 10, or primarily gut barrier function enhancing, e.g. , GLP- 2. Alternatively, a molecule may be both anti- inflammatory and gut barrier function
  • the genetically engineered bacteria of the invention express one or more anti- inflammation and/or gut barrier function enhancer molecule(s) that is encoded by a single gene, e.g. , the molecule is elafin and encoded by the PI3 gene, or the molecule is inter leukin- 10 and encoded by the IL10 gene.
  • the genetically engineered bacteria of the invention encode one or more an anti- inflammation and/or gut barrier function enhancer molecule(s), e.g. , butyrate, that is
  • the one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.
  • expression from the plasmid may be useful for increasing expression of the anti- inflammation and/or gut barrier function enhancer molecule(s).
  • expression from the chromosome may be useful for increasing stability of expression of the anti- inflammation and/or gut barrier function enhancer molecule(s).
  • the gene sequence(s)or gene cassette(s) for producing the anti- inflammation and/or gut barrier function enhancer molecule(s) is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the butyrate biosynthesis gene cassette may be integrated into the bacterial chromosome.
  • the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is inserted into the bacterial genome at one or more of the following insertion sites in E. coli Nissle:
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • One strategy in the treatment, prevention, and/or management of disorders may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti- inflammatory effectors.
  • leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites.
  • butyrate and other SCFA e.g., derived from the microbiota
  • SCFA e.g., derived from the microbiota
  • trptophan metabolites including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehhyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostais and promote gut-barrier health.
  • These metabolites bind to aryl hydrocarbon receptor (Ahr). After agonist binding, AhR translocates to the nucleus, where it forms a heterodimer with AhR nuclear translocator (ARNT).
  • AhR-dependent gene expression includes genes involved in the production of mediators important for gut homeostasis; these mediators include IL-22, antimicrobicidal factors, increased Thl7 cell activity, and the maintenance of intraepithelial lymphocytes and RORyt-i- innate lymphoid cells.
  • Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3- dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.
  • transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3- dioxygenase (IDO), which is linked to suppression of T cell responses,
  • IP A indole 3-propionic acid
  • PXR Pregnane X receptor
  • indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.
  • the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites Butyrate
  • the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions.
  • the genetically engineered bacteria may include any suitable set of butyrogenic genes described herein.
  • Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to,
  • the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria.
  • the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from Peptoclostridium difficile, e.g., Peptoclostridium difficile strain 630: bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate.
  • Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk.
  • the genetically engineered bacteria comprise a
  • the genetically engineered bacteria comprise bcd.2, etfB3, etfA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.
  • a single gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile.
  • a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola.
  • the pbt and buk genes are replaced with tesB ⁇ e.g., from E coli).
  • a butyrogenic gene cassette may comprise ter, thiAl, hbd, crt2, and tesB.
  • the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • additional genes may be mutated or knocked out, to further increase the levels of butyrate production.
  • Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization.
  • Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
  • the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
  • the bacterial cell produces a first payload that is capable of detoxifying a deleterious molecule. In some embodiments, the bacterial cell produces a second payload that is capable of enhancing gut barrier function and anti- inflammation.
  • the first payload is carboxypeptidase Gi (CPD
  • the payload is D-saccharic acid 1, 4-lactone (SAL).
  • the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38).
  • the payload is a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), e.g. naproxen.
  • NSAID non-steroidal ant i- inflammatory drug
  • the payload is a proton pump inhibitor.
  • Proton pump inhibitors include, but are not limited to, omeprazole, aspirin, lansoprazole, dexlansoprazole, rabeprazole, pantoprazole, esomeprazole, esomeproazole magnesium/naproxen, and omeprazole/sodium bicarbonate.
  • the payload is a heavy metal chelator.
  • Heavy metal chelators include, but are not limited to, dimercaprol, dimercapto succinic acid (DMSA), dimercapto-propane sulfonate (DMPS), penicillamine, ethylenediamine tetraacetic acid (calcium disodium versenate) (CaNa2-EDTA), deferoxamine and deferasirox.
  • the payload is a plant phytochelatin.
  • the payload is a short-chained fatty acid, e.g. butyrate, propionate, or acetate.
  • the payload is the enzyme
  • the second payload is butyrate, propionate, acetate, IL-10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, IFN- ⁇ , TNF-a, 1L-1B, or tryptophan and/or its metabolites.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a deleterious molecule e.g., carboxypeptidase Gi (CPD Gi)
  • CPD Gi carboxypeptidase Gi
  • a second payload for enhancing gut barrier function e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ) in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a deleterious molecule e.g., carboxypeptidase G 2 (CPD G 2 )
  • CPD G 2 carboxypeptidase G 2
  • tryptophan e.g., tryptophan
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D- saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL) in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g.,, IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- 10-hydroxycamptothecin (SN-38), in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7- ethyl- 10-hydroxycamptothecin (SN-38) in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • SN-38 7-ethyl- 10-hydroxycamptothecin
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL-20.
  • NSAID non-steroidal ant i- inflammatory drug
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), in combination with a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • NSAID non-steroidal ant i- inflammatory drug
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , TNF- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • NSAID non-steroidal ant i- inflammatory drug
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • NSAID non-steroidal ant i- inflammatory drug
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, in
  • a second payload for enhancing gut barrier function e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a proton pump inhibitor in combination with a second payload for enhancing gut barrier function e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor) in combination with a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • t a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a proton pump inhibitor in combination with a second payload for enhancing gut barrier function e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a heavy metal chelator in combination with a second payload for enhancing gut barrier function e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a heavy metal chelator in combination with a second payload for enhancing gut barrier function e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a deleterious molecule e.g., a heavy metal chelator
  • a second payload for enhancing gut barrier function e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a plant phytochelatin in combination with a second payload for enhancing gut barrier function e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a plant phytochelatin in combination with a second payload for enhancing gut barrier function e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , SOD.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short- chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a deleterious molecule e.g., a short-chained fatty acid
  • a second payload for enhancing gut barrier function e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate in combination with a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate in combination with a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-27. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-20. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B. In some embodiments, a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IL-10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., 1L-1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas in combination with a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a deleterious molecule e.g., Pseudomonas
  • a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., Ih- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase Gi (CPD Gi), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a deleterious molecule e.g. , carboxypeptidase Gi (CPD Gi)
  • CPD Gi carboxypeptidase Gi
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., carboxypeptidase G 2 (CPD G 2 ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , carboxypeptidase G 2 (CPD G 2 ), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a deleterious molecule e.g. , carboxypeptidase G 2 (CPD G 2 )
  • CPD G 2 carboxypeptidase G 2
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., D-saccharic acid 1, 4-lactone (SAL), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co -administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4- lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , D-saccharic acid 1, 4-lactone (SAL), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a deleterious molecule e.g. , D-saccharic acid 1, 4-lactone (SAL)
  • SAL D-saccharic acid 1, 4-lactone
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding 7-ethyl- lO-hydroxycamptothecin (SN-38), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • SN-38 7-ethyl- lO-hydroxycamptothecin
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID), and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal ant i- inflammatory drug (NSAID) and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. ,, IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID) and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a molecule or enzyme capable of inhibiting, metabolizing, or glucuroniding a non-steroidal anti- inflammatory drug (NSAID), and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • NSAID non-steroidal anti- inflammatory drug
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • SOD second payload for enhancing gut barrier function
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , naproxen, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • ta first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a proton pump inhibitor, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a heavy metal chelator and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a heavy metal chelator, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a deleterious molecule e.g. , a plant phytochelatin
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a plant phytochelatin, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a plant phytochelatin is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , a short-chained fatty acid, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • TNF-a enhancing gut barrier function
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L- 1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , butyrate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL- 10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. propionate, and is coacetatered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL- 19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g. , acetate, and is coadministered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g. , TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., acetate, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., butyrate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., propionate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., acetate.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10.
  • a deleterious molecule e.g., Pseudomonas
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-22.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-27.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-20.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-24.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-19.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-19.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IL-10.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., SOD.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., GLP2.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., IFN- ⁇ .
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., TNF-a.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L-1B.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., 1L-1B.
  • a first bacterial cell produces the first payload for detoxifying a deleterious molecule, e.g., Pseudomonas, and is co- administered with a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • a deleterious molecule e.g., Pseudomonas
  • a second bacterial cell which produces a second payload for enhancing gut barrier function, e.g., tryptophan.
  • the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes.
  • bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes.
  • bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.
  • the genetically engineered bacteria comprise multiple copies of the same payload gene(s).
  • the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter.
  • the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter.
  • the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose.
  • the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut.
  • 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 molecules or metabolites that are specific to the tumor microenvironment. In some
  • the promoter is directly or indirectly induced by a molecule that is coadministered with the bacterial cell. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the tumor
  • the promoter may be tumor-specific, e.g. , hypoxia inducible. In some embodiments, the promoter may be tissue-specific.
  • the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter.
  • the bacterial cell comprises one or more gene sequence(s) for producing the payload(s), e.g., gene sequence encoding one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more antiinflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s), which is operably linked to an oxygen level-dependent promoter such that the therapeutic molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions.
  • FNR fumarate and nitrate reductase regulator
  • the oxygen level-dependent promoter is activated by a corresponding oxygen level- sensing transcriptional regulator, thereby driving production of the therapeutic molecule(s.).
  • the genetically engineered bacteria comprise one or more gene sequence(s) for producing one or more enzyme(s) (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s)expressed under the control of a fumarate and nitrate reductase regulator (FNR)-responsive promoter, an anaerobic regulation of arginine deiminiase and nitrate reduction (ANR)-responsive promoter, or a dissimilatory nitrate respiration regulator (DNR)-responsive promoter, which are capable of being regulated by the transcription factors FNR, ANR, or DNR, respectively.
  • FNR fumarate and nitrate reductase regulator
  • FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive.
  • FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
  • FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.
  • the term "payload” refers to one or more molecules produced by a genetically engineered bacterium, e.g., one or more molecules capable of detoxifying a deleterious molecule, producing one or more anti- inflammatory molecule(s), and/or producing one or more gut barrier enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, and/or tryptophan and/or its metabolites.
  • a genetically engineered bacterium e.g., one or more molecules capable of detoxifying a deleterious molecule, producing one or more anti- inflammatory molecule(s), and/or producing one or more gut barrier enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, S
  • polypeptide of interest or “polypeptides of interest”, “protein of interest”, “proteins of interest”, “payload”, “payloads” includes any or a plurality of any of the enzymes capable of detoxifying a deleterious molecule, short chain fatty acid producing enzymes, tryptophan metabolite producing enzymes, enzymes producing any gut barrier enhancer and/or anti- inflammatory metabolite, metabolite
  • the term "gene of interest” or “gene sequence of interest” includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more enzymes capable of detoxifying a deleterious molecule, short chain fatty acid producing enzymes, tryptophan metabolite producing enzymes, enzymes producing any gut barrier enhancer and/or anti- inflammatory metabolite, metabolite transporters or exporters, detox enzymes and/or any other enzyme(s) described herein.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability.
  • FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable gene or gene cassette for producing an anti- inflammation and/or gut barrier function enhancer molecule.
  • Non- limiting FNR promoter sequences are provided in Table 4. Table 4 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence.
  • the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 6, SEQ ID NO: 7, nirB l promoter (SEQ ID NO: 8), nirB2 promoter (SEQ ID NO: 9), nirB3 promoter (SEQ ID NO: 10), ydfZ promoter (SEQ ID NO: 11), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 12), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 13), fnrS, an anaerobically induced small RNA gene (fnrS l promoter SEQ ID NO: 14 or fnrS2 promoter SEQ ID NO: 15), nirB promoter fused to a crp binding site (SEQ ID NO: 16), and
  • genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or a functional fragment thereof.
  • the FNR responsive promoter comprises SEQ ID NO: 1
  • 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 payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al, 2010) or ANR (Ray et ah, 1997).
  • expression of the payload 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 tumor is a human tumor.
  • the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP.
  • CRP cyclic AMP receptor protein or catabolite activator protein or CAP
  • CRP plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Gorke and Stiilke, 2008).
  • the gene or gene cassette for producing an anti- inflammation and/or gut barrier function enhancer molecule is controlled by an oxygen level-dependent promoter fused to a CRP binding site.
  • the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site.
  • cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions.
  • an oxygen level-dependent promoter e.g., an FNR promoter fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing an payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro.
  • the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level- sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria.
  • an oxygen level- sensing transcription factor e.g., FNR, ANR or DNR
  • the heterologous oxygen- level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions.
  • the non-native oxygen- level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et ah, 2011).
  • the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity.
  • the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type 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 payload, 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.
  • a wild-type oxygen-level dependent promoter e.g., FNR, ANR, or DNR promoter
  • corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype.
  • the transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions.
  • 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).
  • both the oxygen level- sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.
  • 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 payload are present on different plasmids.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid. In some embodiments, the gene encoding the oxygen level- sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing
  • the oxygen level- sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability.
  • expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload.
  • expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload.
  • the transcriptional regulator and the payload are divergently transcribed from a promoter region.
  • the gene or gene cassette for producing the payload e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by low-oxygen conditions.
  • the gene or gene cassette for producing the payload e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present in the chromosome and operably linked to a promoter that is induced by low-oxygen conditions.
  • the gene or gene cassette for producing the payload e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline.
  • the gene or gene cassette for producing the payload e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline.
  • one or more enzyme(s) or molecules i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline.
  • expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene(s) or gene cassette(s) capable of producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s), such that the gene(s) or gene cassette(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • a bacterium may comprise multiple copies of the gene or gene cassette for producing the payload molecule(s).
  • the gene or gene cassette is expressed on a low-copy plasmid.
  • 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 or gene cassette is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing gene or gene cassette expression.
  • gene or gene cassette is expressed on a chromosome.
  • the genetically engineered bacteria may comprise multiple copies of the gene(s) or gene cassette(s) capable of producing the payload, e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s).
  • the gene(s) or gene cassette(s) capable of producing the payload e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) is present on a plasmid and operably linked to an oxygen level-dependent promoter.
  • the gene(s) or gene cassette(s) capable of producing the payload e.g.
  • gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) present in a chromosome and operably linked to an oxygen level-dependent promoter.
  • the genetically engineered bacteria produce at least one enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) in low-oxygen conditions to detoxify the toxic molecule and/or to reduce local gut inflammation by at least about 1.5-fold, at least about 2- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800- fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions.
  • Inflammation may be measured by methods known in the art, e.g. , counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Meso scale, Cayman Chemical, Qiagen).
  • the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of one more more payload(s), e.g., gene or gene cassette encoding one or more enzyme(s) or molecules (i) capable of detoxifying a deleterious molecule, (ii) for the production of one or more anti- inflammatory molecule(s), and/or (iii) for the production of one or more gut barrier enhancer molecule(s) in low-oxygen conditions than unmodified bacteria of the same subtype under the same conditions.
  • payload(s) e.g
  • Certain unmodified bacteria will not have detectable levels of the enzyme capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier enhancer molecule.
  • the enzyme capable of detoxifying a deleterious molecule, anti- inflammation and/or gut barrier enhancer molecule will be detectable in low-oxygen conditions.
  • the anti- inflammation and/or gut barrier enhancer molecule is butyrate.
  • Methods of measuring butyrate levels e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013).
  • butyrate is measured as butyrate level/bacteria optical density (OD).
  • OD optical density
  • measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production.
  • the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate
  • butyrate production is measured in the bacterial cell medium.
  • the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 ⁇ /OD, at least about 10 ⁇ /OD, at least about 100 ⁇ /OD, at least about 500 ⁇ /OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in low-oxygen conditions.
  • the anti- inflammation and/or gut barrier enhancer molecule is propionate.
  • Methods of measuring propionate levels e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Hillman, 1978; Lukovac et al., 2014).
  • measuring the activity and/or expression of one or more gene products in the propionate gene cassette serves as a proxy measurement for propionate production.
  • the bacterial cells of the invention are harvested and lysed to measure propionate production. In alternate embodiments, propionate production is measured in the bacterial cell medium.
  • the genetically engineered bacteria produce at least about 1 ⁇ , at least about 10 ⁇ , at least about 100 ⁇ , at least about 500 ⁇ , at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, or at least about 50 mM of propionate in low-oxygen conditions.
  • the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload, e.g., a detoxification molecule, an antiinflammatory molecule, and/or a gut barrier enhancer molecule, that is expressed under the control of an inducible promoter.
  • a payload e.g., a detoxification molecule, an antiinflammatory molecule, and/or a gut barrier enhancer molecule
  • the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions.
  • the gene for producing the payload is expressed under the control of an inflammatory- dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.
  • RNS reactive nitrogen species
  • RNS can cause deleterious cellular effects such as nitrosative stress.
  • RNS includes, but is not limited to, nitric oxide (NO*), peroxynitrite or peroxynitrite anion (ONOO- ), nitrogen dioxide ( ⁇ 02), dinitrogen trioxide (N203), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOC02-) (unpaired electrons denoted by ⁇ ).
  • NO* nitric oxide
  • ONOO- peroxynitrite or peroxynitrite anion
  • N203 dinitrogen trioxide
  • ONOOH peroxynitrous acid
  • ONOOC02- nitroperoxycarbonate
  • Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.
  • RNS-inducible regulatory region refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region.
  • the RNS-inducible regulatory region comprises a promoter sequence.
  • the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression.
  • the transcription factor is bound to the RNS- inducible regulatory region in the absence of RNS ; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression.
  • the RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g. , a payload gene sequence(s), e.g., any of the payloads described herein.
  • a transcription factor senses RNS and activates a corresponding RNS- inducible regulatory region, thereby driving expression of an operatively linked gene sequence.
  • RNS induces expression of the gene or gene sequences.
  • RNS-derepressible regulatory region refers to a nucleic acid sequence to which one or more RNS -sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region.
  • the RNS-derepressible regulatory region comprises a promoter sequence.
  • the RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s).
  • a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette.
  • RNS derepresses expression of the gene or genes.
  • RNS-repressible regulatory region refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region.
  • the RNS-repressible regulatory region comprises a promoter sequence.
  • the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence.
  • the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
  • the RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette.
  • a transcription factor senses RNS and binds to a corresponding RNS- repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences.
  • RNS represses expression of the gene or gene sequences.
  • a "RNS -responsive regulatory region” refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region.
  • the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 5. Table 5. Examples of RNS-sensing transcription factors and RNS-responsive genes
  • the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species.
  • the tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels.
  • the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.
  • the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes.
  • the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression.
  • the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a
  • the tunable regulatory region is a RNS-inducible regulatory region
  • the transcription factor that senses RNS is NorR.
  • NorR is an NO- responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide.
  • the genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al, 2012).
  • the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s).
  • a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload.
  • the tunable regulatory region is a RNS-inducible regulatory region
  • the transcription factor that senses RNS is DNR.
  • DNR dissimilatory nitrate respiration regulator
  • the genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008).
  • the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette.
  • a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads.
  • the DNR is Pseudomonas aeruginosa DNR.
  • the tunable regulatory region is a RNS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.
  • the tunable regulatory region is a RNS- derepressible regulatory region
  • the transcription factor that senses RNS is NsrR.
  • NsrR is "an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism" (Isabella et al., 2009).
  • the genetically engineered bacteria of the invention may comprise any suitable RNS -responsive regulatory region from a gene that is repressed by NsrR.
  • the NsrR is Neisseria gonorrhoeae NsrR.
  • the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes.
  • a gene or genes e.g., a payload gene or genes.
  • transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).
  • the genetically engineered bacteria it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria.
  • the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention.
  • the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g.
  • the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
  • the tunable regulatory region is a RNS- repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette.
  • the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
  • the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload.
  • the two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g. , encoding a payload.
  • the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette.
  • second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA.
  • a RNS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria.
  • corresponding regulatory region sequences may be present in genetically engineered bacteria.
  • the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB.
  • the genetically engineered bacteria comprise one type of RNS- sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA.
  • the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively.
  • One RNS -responsive regulatory region may be capable of binding more than one transcription factor.
  • the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al, 2009; Dunn et al, 2010; Vine et al, 2011;
  • the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter.
  • a RNS-sensing transcription factor e.g., the nsrR gene
  • an inducible promoter e.g., the GlnRS promoter or the P(Bla) promoter
  • expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule.
  • expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule.
  • the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.
  • the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS -responsive regulatory region from a different species, strain, or substrain of bacteria.
  • the heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria comprise a
  • RNS-sensing transcription factor NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae.
  • the native RNS-sensing transcription factor e.g. , NsrR
  • the native RNS-sensing transcription factor is deleted or mutated to reduce or eliminate wild- type activity.
  • the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene.
  • the gene encoding the RNS- sensing transcription factor is present on a plasmid.
  • the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids.
  • the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid.
  • the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.
  • the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g. , the NsrR gene, and a corresponding regulatory region, e.g. , a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype.
  • the mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions.
  • the genetically engineered bacteria comprise a wild-type RNS -responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g.
  • NsrR that is mutated relative to the wild- type transcription factor from bacteria of the same subtype.
  • the mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions.
  • both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.
  • the gene or gene cassette for producing a detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule is present on a plasmid and operably linked to a promoter that is induced by RNS.
  • expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.
  • 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 payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) 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.
  • the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to detoxify a toxic molecule and/or reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions.
  • Inflammation may be measured by methods known in the art, e.g. , counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Meso scale, Cayman Chemical, Qiagen).
  • the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1, 000-fold, or at least about 1, 500-fold more of pay load in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.
  • the anti- inflammation and/or gut barrier enhancer molecule is butyrate.
  • Methods of measuring butyrate levels e.g. , by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g. , Aboulnaga et al., 2013).
  • butyrate is measured as butyrate level/bacteria optical density (OD).
  • OD optical density
  • measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production.
  • the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate
  • butyrate production is measured in the bacterial cell medium.
  • the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 ⁇ /OD, at least about 10 ⁇ /OD, at least about 100 ⁇ /OD, at least about 500 ⁇ /OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of RNS.
  • the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload, e.g., detoxification molecule, an anti- inflammatory molecule, and/or a gut barrier enhancer molecule, that is expressed under the control of an inducible promoter.
  • a payload e.g., detoxification molecule, an anti- inflammatory molecule, and/or a gut barrier enhancer molecule
  • the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage.
  • the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage.
  • ROS includes, but is not limited to, hydrogen peroxide (H202), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical ( ⁇ ), superoxide or superoxide anion ( ⁇ 02-), singlet oxygen (102), ozone (03), carbonate radical, peroxide or peroxyl radical ( ⁇ 02-2), hypochlorous acid (HOC1), hypochlorite ion (OC1-), sodium hypochlorite (NaOCl), nitric oxide (NO*), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by ⁇ ).
  • Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al, 2014).
  • ROS-inducible regulatory region refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region.
  • the ROS-inducible regulatory region comprises a promoter sequence.
  • the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression.
  • the transcription factor is bound to the ROS- inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression.
  • the ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s).
  • a transcription factor e.g., OxyR
  • ROS induces expression of the gene or genes.
  • ROS-derepressible regulatory region refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region.
  • the ROS-derepressible regulatory region comprises a promoter sequence.
  • the ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s).
  • a transcription factor e.g., OhrR
  • ROS derepresses expression of the gene or gene cassette.
  • ROS-repressible regulatory region refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region.
  • the ROS-repressible regulatory region comprises a promoter sequence.
  • the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence.
  • the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
  • the ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences.
  • a transcription factor e.g., PerR
  • ROS represses expression of the gene or genes.
  • a "ROS -responsive regulatory region” refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region.
  • the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 6.
  • the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species.
  • the tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels.
  • the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.
  • the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette.
  • the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression.
  • the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.
  • the tunable regulatory region is a ROS-inducible regulatory region
  • the transcription factor that senses ROS is OxyR.
  • OxyR "functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g., "genes involved in H202 detoxification (katE, ahpCF), heme
  • the genetically engineered bacteria may comprise any suitable ROS -responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012).
  • the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene.
  • a gene e.g., a payload gene.
  • an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload.
  • OxyR is encoded by an E. coli oxyR gene.
  • the oxyS regulatory region is an E. coli oxyS regulatory region.
  • the ROS- inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.
  • the tunable regulatory region is a ROS- inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR.
  • SoxR When SoxR is "activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression" (Koo et al., 2003).
  • SoxR is known to respond primarily to superoxide and nitric oxide" (Koo et al., 2003), and is also capable of responding to H202.
  • the genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is activated by SoxR.
  • the genetically engineered bacteria of the invention comprise a ROS- inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload.
  • a gene e.g., a payload.
  • the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload.
  • the tunable regulatory region is a ROS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.
  • the tunable regulatory region is a ROS- derepressible regulatory region, and the transcription factor that senses ROS is OhrR.
  • OhrR "binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event," but oxidized OhrR is “unable to bind its DNA target" (Duarte et al., 2010).
  • OhrR is a "transcriptional repressor [that] ... senses both organic peroxides and NaOCl” (Dubbs et al., 2012) and is "weakly activated by H202 but it shows much higher reactivity for organic hydroperoxides" (Duarte et al., 2010).
  • the genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by OhrR.
  • the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g. , a payload gene.
  • a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g. , a payload gene.
  • ROS e.g. , NaOCl
  • an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload.
  • OhrR is a member of the MarR family of ROS -responsive regulators.
  • “Most members of the MarR family are transcriptional repressors and often bind to the - 10 or - 35 region in the promoter causing a steric inhibition of RNA polymerase binding" (Bussmann et al., 2010).
  • Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ.
  • the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ
  • the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g. , Dubbs et al., 2012).
  • the tunable regulatory region is a ROS- derepressible regulatory region
  • the corresponding transcription factor that senses ROS is RosR.
  • RosR is "a MarR-type transcriptional regulator” that binds to an "18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is "reversibly inhibited by the oxidant H202" (Bussmann et al., 2010).
  • RosR is capable of repressing numerous genes and putative genes, including but not limited to "a putative polyisoprenoid-binding protein (cgl322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cgl426), two putative FMN reductases (cgl l50 and cgl850), and four putative monooxygenases (cg0823, cgl848, cg2329, and cg3084)" (Bussmann et al., 2010).
  • a putative polyisoprenoid-binding protein cgl322
  • gene upstream of and divergent from rosR a sensory histidine kinase
  • cgtS9 a putative transcriptional regulator of the Crp/F
  • the genetically engineered bacteria of the invention may comprise any suitable ROS- responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010).
  • the genetically engineered bacteria of the invention comprise a ROS- derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload.
  • ROS e.g., H202
  • a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.
  • the genetically engineered bacteria it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria.
  • the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention.
  • the genetically engineered bacterium of the invention is Escherichia coli
  • the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR.
  • the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
  • the tunable regulatory region is a ROS- repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette.
  • the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
  • the tunable regulatory region is a ROS- repressible regulatory region
  • the transcription factor that senses ROS is PerR.
  • PerR In Bacillus subtilis, PerR "when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho et al., 2014).
  • PerR is a "global regulator that responds primarily to H202" (Dubbs et al., 2012) and "interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes" (Marinho et al, 2014).
  • PerR is capable of binding a regulatory region that "overlaps part of the promoter or is immediately downstream from it" (Dubbs et al., 2012).
  • the genetically engineered bacteria of the invention may comprise any suitable ROS -responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et ah, 2012).
  • the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload, e.g., detoxification molecule, an ant i- inflammatory molecule, and/or a gut barrier enhancer molecule.
  • the two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload.
  • the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette.
  • second repressors useful in these embodiments include, but are not limited to, TetR, CI, and LexA.
  • the ROS-sensing repressor is PerR.
  • the second repressor is TetR.
  • a PerR- repressible regulatory region drives expression of TetR
  • a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload.
  • tetR In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.
  • a ROS -responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria.
  • OxyR is primarily thought of as a transcriptional activator under oxidizing conditions.
  • OxyR can function as either a repressor or activator under both oxidizing and reducing conditions
  • OxyR has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)" (Zheng et al., 2001).
  • the genetically engineered bacteria of the invention may comprise any suitable ROS- responsive regulatory region from a gene that is repressed by OxyR.
  • OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon.
  • the genetically engineered bacteria comprise any suitable ROS -responsive regulatory region from a gene that is activated by RosR.
  • the genetically engineered bacteria comprise any suitable ROS- responsive regulatory region from a gene that is activated by PerR.
  • corresponding regulatory region sequences may be present in genetically engineered bacteria.
  • “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012).
  • the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS.
  • the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG.
  • the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively.
  • One ROS -responsive regulatory region may be capable of binding more than one transcription factor.
  • the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.
  • nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 7. OxyR binding sites are underlined and bolded.
  • genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NOs: 18, 19, 20, or 21, or a functional fragment thereof.
  • Table 7. Nucleotide sequences of exemplary OxyR-regulated regulatory regions
  • the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter.
  • a ROS-sensing transcription factor e.g., the oxyR gene
  • a promoter that is stronger than the native promoter e.g., the GlnRS promoter or the P(Bla) promoter
  • expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the payload molecule.
  • expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the payload molecule.
  • the ROS- sensing transcription factor and payload molecule e.g., therapeutic molecule, are divergently transcribed from a promoter region.
  • the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS -responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS -responsive regulatory region from a different species, strain, or substrain of bacteria.
  • the heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria comprise a
  • ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS from Escherichia coli.
  • the native ROS-sensing transcription factor e.g., OxyR
  • the native ROS- sensing transcription factor e.g. , OxyR
  • the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene.
  • the gene encoding the ROS- sensing transcription factor is present on a plasmid.
  • the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids.
  • the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same.
  • the gene encoding the ROS-sensing transcription factor is present on a chromosome.
  • the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.
  • the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g. , the soxR gene, and a corresponding regulatory region, e.g. , a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype.
  • the mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions.
  • the genetically engineered bacteria comprise a wild-type ROS -responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g.
  • both the ROS- sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.
  • the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some
  • the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some
  • expression is further optimized by methods known in the art, e.g. , by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.
  • the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s).
  • the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS -responsive regulatory region.
  • the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS -responsive regulatory region.
  • the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.
  • an oxygen level-dependent promoter a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload 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 payload.
  • the gene encoding the payload is expressed on a low-copy plasmid.
  • 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 payload is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing expression of the payload.
  • the gene encoding the payload is expressed on a chromosome.
  • the genetically engineered bacteria of the invention produce at least one detoxification molecule, anti- inflammation and/or gut barrier enhancer molecule in the presence of ROS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20- fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600- fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions.
  • Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g. , by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g. , by qPCR; PCR arrays; transcription factor phosphorylation assays;
  • the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of an of a detoxification molecule, anti- inflammation and/or gut barrier enhancer molecule in the presence of ROS than unmodified bacteria of the same subtype under the same conditions.
  • Certain unmodified bacteria will not have detectable levels of the anti- inflammation and/or gut barrier enhancer molecule.
  • the anti-inflammation and/or gut barrier enhancer molecule will be detectable in the presence of ROS.
  • the anti- inflammation and/or gut barrier enhancer molecule is butyrate.
  • butyrate is measured as butyrate level/bacteria optical density (OD).
  • measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production.
  • the bacterial cells of the invention are harvested and lysed to measure butyrate production.
  • butyrate production is measured in the bacterial cell medium.
  • the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 ⁇ /OD, at least about 10 ⁇ /OD, at least about 100 ⁇ /OD, at least about 500 ⁇ /OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of ROS.
  • the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.
  • the constitutive promoter is active under in vivo conditions, e.g. , the gut, or in the presence of metabolites associated with certain diseases, such as toxicity-induced diarrhea or other toxicity- induced condition, as described herein.
  • the promoter is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein.
  • the constitutive promoter is active under in vivo conditions, e.g., the gut and/or in the presence of metabolites associated with certain diseases, such as toxicity-induced diarrhea or other toxicity-induced condition, as described herein, and under in vitro conditions, e.g. , various cell culture and/or cell production and/or manufacturing conditions, as described herein.
  • the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions ⁇ e.g. , in vivo and/or in vitro and/or production/manufacturing conditions).
  • the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal.
  • the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal.
  • the constitutive promoter is active in low- oxygen or anaerobic conditions such as the environment of the mammalian gut.
  • the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is co- administered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.
  • Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables. The strength of the constitutive promoter can be further fine-tuned through the selection of ribosome binding sites of the desired strengths.
  • the gene sequence(s) encoding a propionate catabolism enzyme is operably linked to a Escherichia coli ⁇ 70 promoter.
  • Exemplary E. coli ⁇ 70 promoters are listed in Table 8.
  • the gene sequence(s) encoding a detoxification molecule and/or anti-inflammation molecule and/or gut-barrier enhancing molecule is operably linked to a Escherichia coli ⁇ 70 promoter.
  • Exemplary E. coli ⁇ 70 promoters are listed in Table 6A.
  • BBa_J23104 35 NO: 162 family member agctagctcagtcctaggtattgtgctagc

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Abstract

L'invention concerne des bactéries obtenues par génie génétique, des compositions pharmaceutiques les comprenant, et des procédés de détoxification de molécules délétères.
PCT/US2017/012982 2016-01-11 2017-01-11 Bactéries modifiées pour détoxifier les molécules délétères Ceased WO2017123610A2 (fr)

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PCT/US2016/020530 WO2016141108A1 (fr) 2015-03-02 2016-03-02 Bactéries modifiées pour traiter des maladies pour lesquelles une diminution de l'inflammation intestinale et/ou une plus grande imperméabilité de la muqueuse intestinale s'avèrent bénéfiques
PCT/US2016/032565 WO2016183532A1 (fr) 2015-05-13 2016-05-13 Bactéries modifiées pour traiter une maladie ou un trouble
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CN116585360A (zh) * 2023-05-24 2023-08-15 微康益生菌(苏州)股份有限公司 一种改善慢性肾病的益生菌剂及其应用
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WO2019165345A1 (fr) * 2018-02-23 2019-08-29 Innovate Biopharmaceuticals, Inc. Compositions et méthodes de traitement ou de prévention de la perméabilité paracellulaire intestinale
US12016889B2 (en) 2018-02-23 2024-06-25 9 Meters Biopharma, Inc. Compositions and methods for treating or preventing intestinal paracellular permeability
WO2020181009A1 (fr) * 2019-03-04 2020-09-10 Northwestern University Conversion enzymatique bactérienne d'agents chimiothérapeutiques à base d'anthracycline
US20220184147A1 (en) * 2019-03-04 2022-06-16 Northwestem University Bacterial enzymatic conversion of anthracycline chemotherapeutics to reduce toxicity and promote diversity among the intestinal microbiota
WO2020247594A1 (fr) 2019-06-04 2020-12-10 Cocoon Biotech Inc. Produits à base de soie, formulations et procédés d'utilisation
CN116064627A (zh) * 2020-08-14 2023-05-05 中国环境科学研究院 一种特异去除砷的工程菌
CN116585360A (zh) * 2023-05-24 2023-08-15 微康益生菌(苏州)股份有限公司 一种改善慢性肾病的益生菌剂及其应用
CN116585360B (zh) * 2023-05-24 2023-11-14 微康益生菌(苏州)股份有限公司 一种改善慢性肾病的益生菌剂及其应用
WO2025085957A1 (fr) * 2023-10-23 2025-05-01 Centenary Institute Of Cancer Medicine And Cell Biology Récepteurs de virus adéno-associés (aav) et leurs utilisations

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