EP3411051A2 - 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 - Google Patents

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

Info

Publication number
EP3411051A2
EP3411051A2 EP17705544.9A EP17705544A EP3411051A2 EP 3411051 A2 EP3411051 A2 EP 3411051A2 EP 17705544 A EP17705544 A EP 17705544A EP 3411051 A2 EP3411051 A2 EP 3411051A2
Authority
EP
European Patent Office
Prior art keywords
gene
bacterium
promoter
disease
butyrate
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.)
Pending
Application number
EP17705544.9A
Other languages
German (de)
English (en)
Inventor
Paul F. Miller
Vincent M. ISABELLA
Jonathan W. KOTULA
Dean Falb
Adam B. FISHER
Yves Millet
Ning Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synlogic Operating Co Inc
Original Assignee
Synlogic Operating Co Inc
Priority date (The priority date 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 date listed.)
Filing date
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 US15/260,319 external-priority patent/US11384359B2/en
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 Operating Co Inc filed Critical Synlogic Operating Co Inc
Publication of EP3411051A2 publication Critical patent/EP3411051A2/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • compositions and therapeutic methods for inhibiting inflammatory mechanisms in the gut, restoring and tightening gut mucosal barrier function, and/or treating and preventing autoimmune disorders.
  • the disclosure relates to genetically engineered bacteria that are capable of reducing inflammation in the gut and/or enhancing gut barrier function.
  • the genetically engineered bacteria are capable of reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing an autoimmune disorder.
  • the compositions and methods disclosed herein may be used for treating or preventing autoimmune disorders as well as diseases and conditions associated with gut inflammation and/or compromised gut barrier function, e.g., diarrheal diseases, inflammatory bowel diseases, and related diseases.
  • IBDs Inflammatory bowel diseases
  • TNF tumor necrosis factor
  • Compromised gut barrier function also plays a central role in autoimmune diseases pathogenesis (Lerner et al., 2015a; Lerner et al., 2015b; Fasano et al., 2005;
  • a single layer of epithelial cells separates the gut lumen from the immune cells in the body.
  • the epithelium is regulated by intercellular tight junctions and controls the equilibrium between tolerance and immunity to nonself- antigens (Fasano et al., 2005). Disrupting the epithelial layer can lead to pathological exposure of the highly reactive epithelial cells.
  • autoimmune disease (Fasano, 2012).
  • Rheumatoid arthritis and celiac disease are autoimmune disorders that are thought to involve increased intestinal permeability (Lerner et al., 2015b).
  • dysregulation of intercellular tight junctions can lead to disease onset (Fasano, 2012).
  • the loss of protective function of mucosal barriers that interact with the environment is necessary for autoimmunity to develop (Lerner et al., 2015a).
  • the genetically engineered bacteria disclosed herein are capable of producing therapeutic anti-inflammation and/or gut barrier enhancer molecules.
  • the genetically engineered bacteria are functionally silent until they reach an inducing environment, e.g., a mammalian gut, wherein expression of the therapeutic molecule is induced.
  • the genetically engineered bacteria are naturally non-pathogenic and may be introduced into the gut in order to reduce gut inflammation and/or enhance gut barrier function and may thereby further ameliorate or prevent an autoimmune disorder.
  • the anti-inflammation and/or gut barrier enhancer molecule is stably produced by the genetically engineered bacteria, and/or the genetically engineered bacteria are stably maintained in vivo and/or in vitro.
  • the invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of treating diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier function, e.g., an inflammatory bowel disease or an autoimmune disorder.
  • the genetically engineered bacteria of the invention produce one or more therapeutic molecule(s) under the control of one or more promoters induced by an environmental condition, e.g., an environmental condition found in the mammalian gut, such as an inflammatory condition or a low oxygen condition.
  • an environmental condition e.g., an environmental condition found in the mammalian gut, such as an inflammatory condition or a low oxygen condition.
  • the genetically engineered bacteria produce one or more therapeutic molecule(s) 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.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the therapeutic molecule is butyrate; in an inducing environment, the butyrate biosynthetic gene cassette is activated, and butyrate is produced.
  • Local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells.
  • the genetically engineered bacteria produce their therapeutic effect only in inducing environments such as the gut, thereby lowering the safety issues associated with systemic exposure.
  • a butyrate-producing bacterium comprising at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene.
  • Such bacterium is capable of producing butyrate, but does not produce acetate.
  • the bacterium further has at least one mutation in or deletion of an endogenous adhE gene.
  • the bacterium further has at least one mutation in or deletion of an endogenous IdhA gene.
  • the bacterium further has at least one mutation in or deletion of an endogenous frd gene.
  • the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous IdhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous IdhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous IdhA gene.
  • the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non- native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or IdhA gene and/or frd gene.
  • the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
  • the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut.
  • the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous adhE gene.
  • the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous pta gene.
  • the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous gene selected from frd and/or ldhA and/or adhE and/or pta.
  • the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene, frd gene, adhE gene, and pta gene.
  • the bacterium described above comprises an endogenous pta gene and produces acetate.
  • the bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has an endogenous pta gene.
  • Such bacterium is capable of producing butyrate and acetate.
  • the bacterium further has at least one mutation in or deletion of an endogenous adhE gene.
  • the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene.
  • the bacterium further has at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous ldhA gene.
  • the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non-native bio synthetic pathways for producing butyrate, wherein the bacteria produces acetyl Co A and wherein the bacterium has an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or frd gene.
  • the at least one gene or gene cassette for producing butyrate may comprise ter, thiAl, hbd, crt2, pbt, and buk genes.
  • the at least one gene or gene cassette for producing butyrate may comprise ter, thiAl, hbd, crt2, and tesB genes.
  • the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
  • the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut.
  • an acetate-producing bacterium that produces acetate but not butyrate.
  • the acetate-producing bacterium produces acetyl CoA and comprises a wild-type pta gene.
  • the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an ldhA gene and at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene and at least one mutation in or deletion of an frd gene.
  • the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene and at least one mutation in or deletion of an frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene, at least one mutation in or deletion of an frd gene, and at least one mutation in or deletion of an ldhA gene.
  • the bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and has an endogenous frd gene and/or endogenous ldhA gene and/or endogenous adhA gene.
  • the bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and does not comprise at least one mutation in or deletion of an ldhA gene, an adhE gene, and/or a frd gene.
  • the promoter may be induced under low-oxygen or anaerobic conditions.
  • the promoter is selected from an FNR- responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter.
  • the promoter is an FNR-responsive promoter.
  • the promoter may be induced by the presence of reactive nitrogen species.
  • the promoter is selected from an NsrR-responsive promoter, NorR- responsive promoter, and a DNR-responsive promoter.
  • the promoter may be induced by the presence of reactive oxygen species.
  • the promoter is selected from an OxyR-responsive promoter, PerR- responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a RosR- responsive promoter.
  • the gene and/or gene cassette is located on a chromosome in the bacterium. In some embodiments, the at least one gene and/or gene cassette is located on a plasmid in the bacterium.
  • the bacterium is a probiotic bacterium.
  • the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
  • thebacterium is Escherichia coli strain Nissle.
  • the bacterium is an an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
  • the bacterium may be an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway.
  • composition comprising one or more of any of the bacterium disclosed herein; and a pharmaceutically acceptable carrier.
  • the composition is formulated for oral or rectal administration.
  • Disclosed herein is a method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, a composition disclosed herein.
  • Disclosed herein is a method of treating a disease or condition associated with gut inflammation and/or compromised gut barrier function comprising the step of administering to a patient in need thereof, a composition.
  • the autoimmune disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, 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, Beh
  • encephalomyelitis Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome,
  • Granulomatosis with Polyangiitis Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere's disease,
  • Postpericardiotomy syndrome Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombo
  • the autoimmune disorder may be selected from the group consisting of type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis.
  • the disease or condition may be selected from an inflammatory bowel disease, including Crohn's disease and ulcerative colitis, and a diarrheal disease.
  • FIG. 1A, FIG. IB, FIG 1C, FIG. ID, FIG. IE, FIG. IF, FIG. 1G, FIG. 1H, FIG. II, FIG. 1J, and FIG. IK depict schematics of E. coli that are genetically engineered to express a propionate biosynthesis cassette (FIG. 1A), a butyrate
  • FIG. IB an acetate biosynthesis cassette (FIG. 1C), a cassette for the expression of GLP-2 (FIG. ID), a cassette for the expression of human IL-10 (FIG. IE) or v-IL-22 or hIL-22 (FIG. IF) under the control of a FNR-responsive promoter.
  • the genetically engineered E. coli depicted in FIG. ID, FIG. IE, and FIG. IF may further comprise a secretion system for secretion of the expressed polypeptide out of the cell.
  • FIG. 1G depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in ldhA
  • FIG. II depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in adhE and frdA (FIG. II).
  • FIG. 1J depicts bacteria overexpressing acetate by deletion in ldhA.
  • FIG. IK depicts bacteria overexpressing GLP-2 in combination with a deletion in adhE and pta.
  • 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 tet promoter on a plasmid.
  • a "bdc2 cassette” or “bdc2 butyrate cassette” refres 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 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, buk.
  • 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 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 control of an inducible promoter other than tetracycline.
  • 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.
  • 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 (circle, "NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk) is expressed.
  • 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 recombinant bacterium of the invention and its induction in the presence of H202.
  • 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) is expressed.
  • the OxyR transcription factor interacts with H202 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 disclosure.
  • 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. 3A, FIG 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F.
  • FIG. 3A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, and FIG. 3F depict schematics of the gene organization of exemplary bacteria of the disclosure.
  • 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. 3A, FIG 3B, FIG. 3C, FIG. 3D, FIG. 3E,
  • FIG. 4A 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"). Therefore, none of the butyrate biosynthesis enzymes ⁇ ter, thiAl, hbd, crt2, pbt, and buk; white boxes) is expressed.
  • 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. 4D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence 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) is 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) is 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 disclosure.
  • 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) is 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 NO.
  • the NsrR transcription factor ( "NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB) is expressed.
  • NsrR NsrR transcription factor
  • 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. 6A and FIG. 6B depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • FIG. 6A depicts relatively low propionate 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 propionate biosynthesis enzymes (pet, IcdA, IcdB, IcdC, etfA, acrB, acrC) is expressed.
  • FIG. 6B depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • propionate production is induced by NO or H 2 O 2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C-4F, FIG. 5C- 5F.
  • FIG. 7 depicts an exemplary propionate biosynthesis gene cassette.
  • FIG. 8A, FIG. 8B, and FIG. 8C depict schematics of the gene
  • FIG. 8A depicts relatively low propionate 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 propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, Ipd) is expressed.
  • FIG. 8A depicts relatively low propionate 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 propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, Ipd) is expressed.
  • FIG. 8B depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • FIG. 8C depicts an exemplary propionate biosynthesis gene cassette. In other embodiments, propionate production is induced by NO or H 2 O 2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C- 4F, FIG. 5C-5F.
  • FIG. 9A and FIG. 9B depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • FIG. 9A and FIG. 9B depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • FIG. 9A depicts relatively low propionate 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"). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, Ipd, tesB) is expressed.
  • FIG. 9B depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • propionate production is induced by NO or H 2 0 2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C-3F, FIG. 4C- 4F, FIG. 5C-5F.
  • FIG. 10A, FIG. 10B, and FIG. IOC depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.
  • FIG. 10A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production.
  • 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.
  • FIG. 10B and FIG. IOC depict schematics of the gene organization of another exemplary engineered bacterium of the invention and its induction of propionate production under low-oxygen conditions.
  • FIG. 10A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production.
  • 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.
  • FIG. 10B depicts relatively low propionate 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"). Therefore, none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfli) is expressed.
  • FIG. IOC depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • propionate production is induced by NO or H 2 0 2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 3C- 3F, FIG. 4C-4F, FIG. 5C-5F.
  • FIG. 11 depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure.
  • FIG. 11 shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, 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. 12 depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure.
  • FIG. 12 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. 13 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. 14A, FIG. 14B, FIG.14C, and FIG. 14D depict schematics and graphs showing butyrate or biomarker production of a butyrate producing circuit under the control of an FNR promoter.
  • FIG. 14A depicts a schematic showing a butyrate producing circuit under the control of an FNR promoter.
  • FIG. 14B 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. 14C 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.
  • 14D depict levels of mouse lipocalin 2 (left) and calprotectin (right) quantified by ELISA using the fecal samples in an in vivo model.
  • SYN-501 reduces inflammation and/or protects gut barrier function as compared to wild type Nissle control.
  • FIG. 15 depicts a graph measuring gut-barrier function in dextran sodium sulfate (DSS)-induced mouse models of IBD.
  • DSS dextran sodium sulfate
  • FIG. 16 depicts serum levels of FITC-dextran analyzed by
  • FITC-dextran is a readout for gut barrier function in the DSS -induced mouse model of IBD.
  • FIG. 17 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H20, 100 mM butyrate in H20, 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 H20 (+) 200 mM butyrate.
  • FIG. 18 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. 19A 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, SYNIOOI and SYN1002 both integrated at the agal/rsml locus) gave comparable butyrate production to the plasmid strain SYN501.
  • FIG. 19B and FIG. 19C depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYN1001, on alkaline phosphatase activity in HT-29 cells represented in bar (FIG. 19B) and nonlinear fit (FIG. 19C) graphical formats.
  • FIG. 20A and FIG. 20B depicts the construction and gene organization of an exemplary plasmids.
  • FIG. 20A 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. 20B 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. 21 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).
  • FIG. 22 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. 23 depicts the construction and gene organization of an exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic031-oxyS- butyrogenic gene cassette).
  • FIG. 24 depicts the construction and gene organization of another exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette
  • FIG. 25 depicts a schematic illustrating a strategy for increasing butyrate and acetate production in engineered bacteria. Aerobic metabolism through the citric acid cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of the colon. E. coli makes high levels of acetate as an end production of fermentation. To improve acetate production, while still maintaining highlevels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic
  • Non- limiting examples of competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
  • Deletions of interest therefore include deletion of adhE, ldh, and frd.
  • the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
  • FIG. 26A and FIG. 26B depict line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG. 26A) and in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26B).
  • Acetate production of an engineered E. coli Nissle strain comprising a deletion in the endenous ldh gene (SYN2001) was compared with streptomycin resistant Nissle (SYN94).
  • FIG. 26C and FIG. 26D depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 26C) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 26D).
  • Deletions in endogenous adhE (Aldehyde- alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes.
  • SYN2006 comprises a FNRS ter-tesB cassette integrated at the HAl/2 locus and a deletion in the endogenous adhE gene.
  • SYN2007 comprises a FNRS ter-tesB cassette integrated at the HAl/2 locus and a deletion in the endogenous ldhA gene.
  • SYN2008 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous adhE gene.
  • SYN2003 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous ldhA gene.
  • FIG. 26E depicts a bar graph showing acetate and butyrate production at the indicated time points post induction in 0.5% glucose MOPS (pH6.8).
  • a strain comprising a FNRS-ter-tesB butyrate cassette integrated at the HAl/2 locus of the chromosome (SYN1004) was compared with a strain comprising the same integrated cassette and additionally a deletion in the endogenous frd gene (SYN2005).
  • FIG. 26F depicts a bar graph showing acetate and butyrate production at 18 hours in 0.5% glucose MOPS (pH6.8), comparing three strains engineered to produce short chain fatty acids.
  • SYN2001 comprises a deletion in the endenous ldh gene;
  • SYN2002 comprises a FNRS-ter-tesB butyrate cassette integrated at the HAl/2 locus and deletions in the endogenous adhE and pta genes.
  • SYN2003 comprises FNRS-ter-pbt-buk butyrate cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene.
  • FIG. 26G and FIG. 26H depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFNy secretion in primary human PBMC cells from donor 1 (Dl) (Fig. 26G ) and donor 2 (D2) (FIG. 26H).
  • FIG. 27 depicts a schematic of an exemplary propionate biosynthesis gene cassette.
  • FIG. 28 depicts a schematic of a construct comprising the sleeping beauty mutase operon from E. coli under the control of a heterologous FnrS promoter.
  • FIG. 29 depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 28.
  • FIG. 30A, FIG. 30B, and FIG. 30C 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, GLP-2, IL- 10, and IL-22, is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (FIG. 30A and FIG. 30B) or a tet-inducible promoter (FIG. 30C).
  • an inducible promoter such as oxygen level-dependent promoters (e.g.
  • FNR-inducible promoter FNR-inducible promoter
  • promoters induced by IBD specific molecules or 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 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. 30B and FIG. 30D.
  • FIG. 31A and FIG. 31B 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 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. 31A) or an inducible promoter, such as oxygen level-dependent promoters (e.g. , FNR- inducible promoter, FIG.
  • promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response e.g., can be
  • FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, and FIG. 32E depict schematics of non- limiting examples of constructs for the expression of GLP2 for bacterial secretion.
  • FIG. 32A depicts a schematic of a human GLP2 construct inserted into the FliC locus, under the control of the native FliC promoter.
  • FIG. 32B depicts a schematic of a human GLP2 construct, including the N terminal 20 amino acids of FliC, inserted into the FliC locus under the control of the native FliC promoter.
  • FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, and FIG. 32E depict schematics of non- limiting examples of constructs for the expression of GLP2 for bacterial secretion.
  • FIG. 32A depicts a schematic of a human GLP2 construct inserted into the FliC locus, under the control of the native FliC promoter.
  • FIG. 32B depicts a schematic of a human
  • FIG. 32C depicts a schematic of a human GLP2 construct, including the N-terminal 20 amino acids of FliC, inserted into the FliC locus under the control of a tet inducible promoter.
  • FIG. 32D depicts a schematic of a human GLP2 construct with a N terminal OmpF secretion tag (sec-dependent secretion system) under the control of a tet inducible promoter.
  • FIG. 32E depicts a schematic of a human GLP2 construct with a N terminal TorA secretion tag (tat secretion system) under the control of a tet inducible promoter.
  • FIG. 33A and FIG. 33B depict line graphs of ELISA results.
  • FIG. 33A depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum- starved Colo 205 cells treated with supernatants from engineered bacteria comprising a PAL deletion and an integrated construct encoding hIL-22 with a phoA secretion tag. The data demonstrate that hIL-22 secreted from the engineered bacteria is functionally active.
  • FIG. 33B depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA showing a antibody completion assay.
  • Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody.
  • the data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.
  • FIG. 33C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation.
  • FIG. 33E depicts a Western blot analysis of bacterial supernatants from strain SYN2980 and SYN2982, using IL- 10 antibody (IL- 10 (D13A11) XP® Rabbit rnAb #12163, Cell Signaling Technology).
  • the secreted polypepetide has the same molecular weight as the standards, indicating that the signal sequence is cleaved from the native peptide.
  • FIG. 34 depicts a schematic of tryptophan metabolism along the kynurenine and the serotonin arms in humans.
  • the abbreviations for the enzymes are as follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase; AAAD: aromatic -amino acid decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase; HIOMT, hydroxyl-O-methyltransferase; IDO, indoleamine 2,3- dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3- monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan 2,3- dioxygenase; TPH, tryptophan hydroxylase;
  • FIG. 35 depicts a schematic of bacterial tryptophan catabolism machinery, which is genetically and functionally homologous to IDOl enzymatic activity, as described in Vujkovic-Cvijin et al., Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism; Sci Transl Med. 2013 July 10; 5(193): 193ra91, the contents of which is herein incorporated by reference in its entirety.
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 35.
  • the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 35, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole- 3 acetaldehyde.
  • FIG. 36A and FIG. 36B depict schematics of indole metabolite mode of action (FIG.36A) and indole biosynthesis (FIG. 36B).
  • FIG.36A depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function.
  • PXR pregnane X receptors
  • I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production.
  • AhR aryl hydrocarbon receptor
  • Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections.
  • Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon- like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).
  • FIG. 36B 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).
  • 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 FIGs. 36A and 36B.
  • the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 36A and 36B, including but not limited to, kynurenine, indole-3-aldehyde, indole- 3 -acetic acid, and/or indole-3 acetaldehyde.
  • FIG. 37A and FIG. 37B depict diagrams of bacterial tryptophan metabolism pathways.
  • FIG. 37A 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 (ECl.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2- monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0).
  • FIG. 37B Depicts a schematic of tryptophan derived pathways.
  • Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole- 3 -acetaldehyde; IAA:
  • Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2), EC 1.13.11.11 (Idol); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (ladl), EC 1.2.3.7 (Aaol); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclbl, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125 (CYP79B2, CYP79B3); 9.
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIGs. 37A and 37B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIGs. 37A and 37B. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome.
  • 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. 38 depicts a schematic of the E. coli tryptophan synthesis pathway.
  • tryptophan is bio synthesized from chorismate, the principal common precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and enterobactin (enterochelin), as shown in the superpathway of chorismate metabolism.
  • Five genes encode five enzymes that catalyze tryptophan biosynthesis from chorismate.
  • the five genes trpE trpD trpC trpB trpA form a single transcription unit, the trp operon.
  • a weak internal promoter also exists within the trpD structural gene that provides low, constitutive levels of mRNA.
  • FIG. 39 depicts one embodiment of the disclosure in which the E. coli TRP synthesis enzymes are expressed from a construct under the control of a tetracycline inducible system.
  • FIG. 40A, FIG. 40B, FIG. 40C, and FIG. 40D depicts schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan.
  • the genetically engineered bacteria comprise circuits for the production of tryptophan.
  • Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter.
  • the one or more cassettes are under the control of constitutive promoters.
  • 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), 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.
  • the bacteria may also include an auxotrophy, e.g. , deletion of thyA ( ⁇ thyA; thymidine dependence).
  • Tryptophan is produced from its precursor, chorismate, 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.
  • Optional knockout of the tryptophan repressor trpR is also depicted.
  • Optional production of chorismate through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown.
  • the bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40B, and/or FIG. 40C, and/or FIG. 40D.
  • FIG. 40B 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. 40A and/or described in the description of FIG. 40A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40C, and/or FIG. 40D.
  • trpR and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • 40C 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.
  • serA tryptophan production is improved.
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 40A and/or described in the description of FIG. 40A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40B, and/or FIG. 40D.
  • Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • the bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter.
  • FIG. 40D depicts a non- limiting example of 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 optionally comprises either a wild type or a feedback resistant SerA gene.
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 40A and/or described in the description of FIG.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 40B, and/or FIG. 40C.
  • Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • the bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter.
  • the bacteria may also comprise a deletion in PheA, which prevents conversion of chorismate into phenylalanine and thereby promotes the production of anthranilate and tryptophan. [077] FIG. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG.
  • FIG. 41G, and FIG. 41H depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included.
  • FIG. 41A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan.
  • the one or more cassettes are under the control of inducible promoters. In certain embodiments the one or more cassettes are under the control of constitutive promoters.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for Tryptophan
  • FIG. 41B depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L- tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L- tryptophan oxidase, e.g., from streptomyces sp.
  • aro9 L-tryptophan aminotransferase, e.g., from S. cerevisae
  • aspC aspartate aminotransferase, e.g., from E. coli
  • taal L- tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana
  • staO L- tryptophan oxidase,
  • FIG. 41C depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole- 3 -acetaldehyde and FICZ from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E.
  • FIG. 41D depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for cyp79B2, (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), which together convert tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • FIG. 41E depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising
  • ID01 indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3- dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine— oxoglutarate transaminase, e.g., from S.
  • FIG. 41F depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising
  • IDOl indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TD02 (tryptophan 2,3- dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine— oxoglutarate transaminase, e.g., from S.
  • GOT2 Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens or AADAT (Kynurenine/alpha- aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB 1 (Kynurenine— oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2
  • FIG. 41G depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan.
  • the bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 40A and/or and/or FIG. 40B, and/or FIG. 40C, and/or FIG. 40D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from E. coli), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • FIG. 41H 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.
  • DIM diindolylmethane
  • ICZ indolo(3,2-b) carbazole
  • 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. 41A, FIG. 41B, FIG. 41D, FIG. 41D, FIG. 41E, FIG. 41F, FIG. 41G and FIG. 41H may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid.
  • the one or more cassettes are under the control of inducible promoters.
  • the one or more cassettes are under the control of constitutive promoters.
  • the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L-tryptophan- pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp.
  • trpDH Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-21078
  • ipdC Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae
  • iadl Indole-3-acetaldehyde dehydrogenase, e.g., from
  • Ustilago maydis) or AAOl (Indole- 3 -acetaldehyde oxidase, e.g., from Arabidopsis thaliana) which together produce indole- 3 -acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • an inducible promoter e.g., an FNR promoter.
  • FIG. 42B the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes) ot tynA (Monoamine oxidase, e.g., from E.
  • tdc Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes
  • ot tynA Monoamine oxidase, e.g., from E.
  • FIG. 42C the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate aminotransferase, e.g., from E. coli, or taal (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp.
  • TP-A0274 or trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 ( indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana) e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • an inducible promoter e.g., an FNR promoter.
  • FIG. 42D the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • IaaM Tryptophan 2-monooxygenase e.g., from Pseudomonas savastanoi
  • iaaH Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi
  • FIG. 42E the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described
  • the genetically engineered bacteria comprise a circuit comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana) or cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana and cyp71al3 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nitl (Nitrilase, e.g., from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an F
  • FIG. 42A, FIG. 42B, FIG. 42C, FIG. 42D, and FIG. 42E may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • auxotrophy e.g., in one example, the thyA gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • FIG. 42F the optional circuits for tryptophan production are as depicted and described in FIG. 40A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 40B and/or FIG. 40C and/or FIG. 40D.
  • tryptophan can be imported through a transporter.
  • the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole- 3 -acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iadl (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole- 3 -acetaldehyde into indole- 3 -acetate.
  • trpDH Trptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108
  • ipdC Indole-3-pyruvate decarboxylase, e.g.
  • FIG. 43A, FIG. 43B, and FIG. 43C 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.
  • 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.
  • the one or more cassettes are under the control of constitutive promoters.
  • 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), 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.
  • the bacteria may also include an auxotrophy, e.g., deletion of thyA ( ⁇ thyA; thymidine dependence).
  • FIG. 43A a depicts non- limiting example of a tryptamine producing strain.
  • Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.
  • FIG. 43B depicts a non-limiting example of an indole-3- acetate producing strain.
  • Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole- 3 -acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iadl (Indole- 3 -acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole- 3 -acetaldehyde into indole-3-acetate.
  • FIG. 43C depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 40A and/or FIG. 40B and/or FIG. 40C and/or FIG. 40D. Additionally, the strain comprises a circuit as described in FIG.
  • trpDH Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108, which produces (indol- 3yl)pyruvate from tryptophan
  • fldA indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes, which converts converts indole- 3 -lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA
  • fldB and fldC indole-3-lactate dehydratase e.g., from Clostridium sporogenes, which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or Acul:
  • the circuits further comprise fldHl and/or fldH2 (indole- 3 -lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes), which converts (indol-3-yl)pyruvate into indole- 3 -lactate).
  • fldHl and/or fldH2 indole- 3 -lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes
  • FIG. 44A and FIG. 44B depict schematics showing exemplary
  • Phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino-heptulosonate 7- phosphate (DAHP).
  • DAHP 3-deoxy-D-arabino-heptulosonate 7- phosphate
  • DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon.
  • Trp tryptophan
  • chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe).
  • Tyr tyrosine
  • Phe phenylalanine
  • DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-deoxy- D-arabino-heptulosonate 7-phosphate (DAHP).
  • DAHP 3-deoxy- D-arabino-heptulosonate 7-phosphate
  • AroB Dehydroquinate synthase (DHQ synthase) is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonic acid 7- phosphate (DAHP) to dehydroquinate (DHQ).
  • DAHP 3-deoxy-D-arabino-heptulosonic acid 7- phosphate
  • DHQ dehydroquinate dehydratase (DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the conversion of DHQ to 3 -dehydro shikimate and introduces the first double bond of the aromatic ring.
  • AroE, YdiB E. coli expresses two shikimate dehydrogenase paralogs, AroE and YdiB. Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme converts 3- dehydro shikimate to shikimate by catalyzing the NADPH linked reduction of 3-dehydro- shikimate.
  • AroL/AroK Shikimate kinase is involved in the fifth step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP.
  • EPSP synthase 3 -Phospho shikimate- 1- carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids.
  • EPSP synthase catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of phosphate to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP).
  • AroC Chorismate synthase
  • AroC Chorismate synthase
  • This enzyme catalyzes the conversion of 5- enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system.
  • TrpEDCAB E coli trp operon
  • TrpE anthranilate synthase converts chorismate and L- glutamine into anthranilate, pyruvate and L-glutamate.
  • TrpD Anthranilate phosphoribosyl transferase catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5'-phosphoribosyl)- anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional
  • TrpC phosphoribosylanthranilate isomerase / indole-3-glycerol phosphate synthase
  • TrpC carboxyphenylaminodeoxyribulose phosphate.
  • the indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure of this product to yield indole-3-glycerol phosphate.
  • the TrpA polypeptide (TSase a) functions as the a subunit of the tetrameric ( ⁇ 2- ⁇ 2) tryptophan synthase complex.
  • TrpB polypeptide functions as the ⁇ subunit of the complex, which catalyzes the synthesis of L- tryptophan from indole and L- serine, also termed the ⁇ reaction.
  • TnaA Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L-tryptophan to indole, pyruvate and NH4+.
  • PheA Bifunctional chorismate mutase / prephenate dehydratase (PheA) carries out the shared first step in the parallel bio synthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis.
  • TyrA Bifunctional chorismate mutase / prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis.
  • TyrB, ilvE, AspC Tyrosine aminotransferase (TyrB), also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis.
  • TyrB catalyzes the transamination of 2-ketoisocaproate, p-hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and phenylalanine, respectively.
  • TyrB overlaps with the catalytic activities of branched-chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine.
  • SerA D-3-phosphoglycerate dehydrogenase catalyzes the first committed step in the biosynthesis of L-serine.
  • SerC The serC-encoded enzyme, phosphoserine/phosphohydroxythreonine aminotransferase, functions in the biosythesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5'-phosphate is a cofactor for both enzyme activities.
  • SerB Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated.
  • FIG. 44B depicts a schematic showing exemplary engineering strategies which can improve tryptophan production.
  • bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEfbr, and/or TrpD, and/or TrpC, and/or Trp A, and/or TrpB.
  • endogenous TnaA is knocked out through mutation(s) and/or deletion(s).
  • bacteria are engineered to comprise one or more additional copies of SerA.
  • bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter.
  • endogenous PheA is knocked out through mutation(s) and/or deletion(s).
  • two or more of the strategies depicted in the schematic of FIG. 44B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.
  • FIG.45A and FIG. 45B and FIG. 45C depict bar graphs showing tryptophan production by various engineered bacterial strains.
  • FIG.45A depicts a bar graph showing tryptophan production by various tryptophan producing strains.
  • the data show expressing a feedback resistant form of AroG (AroG r ) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE fbr ) has a positive effect on tryptophan production.
  • AroG r AroG r
  • trpE fbr feedback resistant trpE
  • FIG. 45B shows tryptophan production from a strain comprising a tet-trpE fbr DCBA, tet-aroG fb construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically).
  • FIG. 45C depicts a bar graph showing improved tryptophan production by engineered strain comprising AtrpRAtnaA, tet-trpE ⁇ DCBA, tet-aro ' r through the addition of serine.
  • FIG. 46 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.
  • AtrpRAtnaA AtrpRAtnaA, tet-aroGfbr.
  • SYN2339 comprises AtrpRAtnaA, tet- aroGfbr, tet-trpEfbrDCBA.
  • SYN2473 comprises AtrpRAtnaA, tet-aroGfbr-serA, tet- trpEfbrDCBA.
  • SYN2476 comprises AtrpRAtnaA, tet-trpEfbrDCBA. Results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production.
  • FIG. 47 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 converts indole- 3 -acrylic acid into IPA.
  • the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein.
  • AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria.
  • trpR and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • FIG. 48 depicts a schematic of indole-3-propionic acid (IP A), indole acetic acid (IAA), and tryptamine synthesis(TrA) circuits.
  • 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 Clostridium sporogenes
  • FldD indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes
  • Acul acrylyl- CoA reductase, e.g., from Rhodobacter sphaeroides.
  • lpdC Indole-3-pyruvate
  • decarboxylase e.g., from Enterobacter cloacae; ladl: Indole-3-acetaldehyde
  • dehydrogenase e.g., from Ustilago maydis
  • Tdc Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
  • 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 (IPyA), NH 3 , NAD(P)H and H + .
  • IPyA indol-3- yl)pyruvate
  • NH 3 NH 3
  • NAD(P)H H +
  • H + Indole-3-lactate dehydrogenase
  • 1.1.1.110 e.g., Clostridium sporogenes or Lactobacillus casei converts (indol- 3yl)pyruvate (IpyA) and NADH and H+ to indole- 3 -lactate (ILA) and NAD+.
  • 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.
  • Indole-3-pyruvate decarboxylase (lpdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld) ladl: Indole-3- acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA).
  • the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 40 (A-D) and FIG. 44 and as described elsewhere herein.
  • AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria.
  • trpR and/or the tnaA gene are replaced with feedback resistant versions to improve tryptophan production in the
  • FIG. 49 depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342.
  • SYN2126 comprises AtrpR and AtnaA (AtrpRAtnaA).
  • SYN2339 comprises circuitry for the production of tryptophan (AtrpRAtnaA, tetR-Ptet-trpEfbrDCBA (pSOOl), tetR-Ptet-aroGfbr (pl5A)).
  • SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iadl incorporated at the end of the second construct
  • FIG. 50 depicts a bar graph showing tryptophan and tryptamine production for strains SYN2339, SYN2340, and SYN2794.
  • SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine and comprises AtrpRAtnaA, tetR- P tet -trpE ⁇ DCBA (pSClOl), tetR-P tet -aroG* 1 (pl5A).
  • SYN2340 comprises AtrpRAtnaA,
  • FIG. 51A, FIG. 51B, FIG. 51C, FIG. 51D, FIG. 51E depict schematics of non-limiting examples of genetically engineered bacteria of the disclosure which comprises one or more gene sequence(s) and/or gene cassette(s) as described herein.
  • FIG. 52 depicts a map of integration sites within the E. coli 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. 53 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).
  • FIG. 54A and FIG. 54B depict schematics of bacterial chromosomes, for example the E. coli Nissle 1917 Chromosome.
  • FIG. 54A depicts a schematic of an engineered bacterium comprising, a circuit for butyrate production, a circuit for propionate production, and a circuit for production of one or more interleukins relevant to IBD.
  • Fig. 54B depicts a schematic of an engineered bacterium comprising three circuits, a circuit for butyrate production, a circuit for GLP-2 expression and and a circuit for production of one or more interleukins relevant to IBD.
  • FIG. 55 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. 56 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
  • 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. 57 depicts a schematic of a type I secretion system, which
  • HlyB an ATP-binding cassette transporter
  • HlyD a membrane fusion protein
  • TolC an outer membrane protein
  • FIG. 58 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. 59 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).
  • FIGs. 60A- 60C depict other non-limiting embodiments 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 ParaBAD promoter (ParaBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin.
  • ParaBAD ParaBAD promoter
  • TetR Tet repressor
  • FIG. 60A 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.
  • FIG. 60B depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous
  • 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.
  • 60C 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.
  • TetR 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. 61 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. 62 depicts another no n- limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin.
  • expression of the anti-toxin is turned off.
  • the toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
  • FIG. 63 depicts another no n- 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. 64 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. 65 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., "GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS Synthetic Biology (2015) 4: 307-316.
  • FIG. 66 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 in the tables (Pfnrl-5).
  • FNR- responsive promoters were used to create a library of anaerobic-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.
  • FIGs. 67A-67C depict a schematic representation of the lacZ gene under the control of an exemplary FNR promoter and corresponding graphical data.
  • FIGs. 67A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (Pfnrs)- LacZ encodes the ⁇ -galactosidase enzyme and is a common reporter gene in bacteria.
  • FIG. 67B depicts FNR promoter activity as a function of ⁇ -galactosidase activity in SYN340.
  • SYN340 an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen.
  • FIG. 67C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
  • FIGs. 68A-68D depict bar graphs, schematic, and dot blot, respectively, showing the structure or activity of reporter constructs.
  • FIG. 68A and FIG. 68B depict bar graphs of reporter constructs activity.
  • FIG. 68A depicts a graph of an ATC-inducible reporter construct expression
  • FIG. 68B depicts a graph of a nitric oxide-inducible reporter construct expression.
  • These constructs when induced by their cognate inducer, lead to expression of GFP.
  • FIG. 68C depicts a schematic of the constructs.
  • FIG. 68D depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter.
  • DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice.
  • FIG. 69 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. 70 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. 71 A and FIG. 71B depict a schematic diagrams of a wild-type clbA construct (FIG. 71 A) and a schematic diagram of a clbA knockout construct (FIG. 71B).
  • FIG. 72 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animla disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen.
  • FIG. 73 depicts a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.
  • Step 1 depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm.
  • Step 2 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
  • Step 3 depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours.
  • Step 4 depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS.
  • Step 5 depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
  • Fig. 74 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. 75A depicts a graph showing bacterial cell growth of a Nissle thyA auxotroph strain (thyA knock-out) in various concentrations of thymidine.
  • chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in LB + lOmM thymidine at 37C. The next day, cells were diluted 1: 100 in 1 mL LB + lOmM thymidine, and incubated at 37C for 4 hours. The cells were then diluted 1: 100 in 1 mL LB + varying concentrations of thymidine in triplicate in a 96-well plate. The plate is incubated at 37C with shaking, and the OD600 is measured every 5 minutes for 720 minutes. This data shows that Nissle thyA auxotroph does not grow in environments lacking thymidine.
  • Fig. 75B depicts a bar graph of Nissle residence in vivo of wildtype Nissle versus Nissle thyA auxotroph (thyA knock-out). Streptomycin- resistant Nissle (wildtype or thyA auxotroph) 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
  • Each bar represents the number of Nissle recovered from the fecal samples each day for 7 consecutive days. There were no bacteria recovered in fecal samples from mice gavaged with Nissle thyA auxotroph bacteria after day 3. This data shows that the Nissle thyA auxotroph does not persist in vivo in mice.
  • Fig. 76 depicts a one non- limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived antitoxin and a long-lived toxin.
  • 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.
  • Figs. 77A-77D depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 77A and Fig. 77B), which also contains a chromosomal component (shown in Fig. 77C and Fig. 77D).
  • the bosafety plasmid system vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 77A) and thyA (Fig. 77B) and promoter elements driving expression of these components.
  • bla is knocked out and replaced with one or more constructs described herein, in which a first protein of interest (POI1) and/or a second protein of interest, e.g., a transporter (POI2), and/or a third protein of interest (POI3) are expressed from an inducible or constitutive promoter.
  • Fig. 77C and Fig. 77D depict schematics of the gene organization of the chromosomal component of a biosafety system.
  • Fig. 77C 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. 77D 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. 77A)
  • the chromosomal constructs shown in Fig. 77C and Fig. 77D are knocked into the DapA locus.
  • the plasmid containing the functional ThyA is used (as shown in Fig. 77B)
  • 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. 78 depicts a schematic of a polypeptide of interest displayed on the surface of the bacterium.
  • a non-limiting example of such a therapeutic protein is a scFv.
  • the polypeptide is expressed as a fusion protein, which comprises a outer membrane anchor from another protein, which was developed as part of a display system.
  • Non- limiting examples of such anchors are described herein and include LppOmpA,
  • NGIgAsig-NGIgAP NGIgAsig-NGIgAP, InaQ, Intimin, Invasin, pelB-PAL, and blcA/BAN.
  • a bacterial strain which has one or more diffusible outer membrane phenotype ("leaky membrane”) mutation, e.g., as described herein.
  • Fig. 79 depicts the gene organization of exemplary construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.
  • Fig. 80A depicts a "Oxygen bypass switch" useful for aerobic pre- induction of a strain comprising one or proteins of interest (POI), e.g., one or more anticancer molecules or immune modulatory effectors (POIl) and a second set of one or more proteins of interest (POI2), e.g., one or more transporter(s)/importer(s) and/or exporter(s), 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.
  • a strain with active effector molecules prior to administration. This can be done by pre-inducing the expression of these effectors as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration.
  • strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more effectors or 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).
  • 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 pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs.
  • This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.
  • 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. 80B 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. 80C 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. 81 depicts the gene organization of an exemplary construct, e.g., comprised in SYN-PKU401, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene.
  • Fig. 82 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of one or more POIs.
  • this construct is useful for pre-induction and preloading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG.
  • 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 construct expressing a second POI, e.g., a transporter, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG.
  • a second POI e.g., a transporter
  • 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. 77 A, Fig. 77B, Fig. 77C, and Fig. 77D.
  • the construct is integrated into the genome at one or more locations described herein.
  • Fig. 83A, Fig. 83B, and Fig. 83C depict schematics of non-limiting examples of constructs for the expression of proteins of interest POI(s).
  • Fig 83A 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 a POI1 and/or a POI2 prior to in vivo
  • 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 a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive.
  • the construct is used in combination with a POI3 expression construct.
  • 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. 84A 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. 84B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1 : 100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction.
  • the Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions.
  • 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.
  • 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. 85A depicts a schematic diagram of a wild-type clbA construct.
  • Fig. 85B depicts a schematic diagram of a clbA knockout construct.
  • the present disclosure includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of reducing gut inflammation, enhancing gut barrier function, and/or treating or preventing autoimmune disorders.
  • the genetically engineered bacteria comprise at least one non-native gene and/or gene cassette for producing a non-native anti- inflammation and/or gut barrier function enhancer molecule(s).
  • the at least one gene and/or gene cassette is further operably linked to a regulatory region that is controlled by a
  • the genetically engineered bacteria are capable of producing the anti- inflammation and/or gut barrier function enhancer molecule(s) in inducing environments, e.g., in the gut.
  • the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used to treat or prevent autoimmune disorders and/or diseases or conditions associated with gut inflammation and/or compromised gut barrier function, including IBD.
  • diseases and conditions associated with 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
  • 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
  • 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, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyper lipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocyto
  • encephalomyelitis Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen s
  • 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.
  • NAPEs N-acylphosphatidylethanolamines
  • elafin also called peptidase inhibitor 3 and SKALP
  • trefoil factor melatonin
  • tryptophan PGD 2
  • kynurenic acid
  • Such molecules may also include compounds that inhibit proinflammatory 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.
  • proinflammatory 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 PXR agonists (e.
  • Such molecules also include HDAC inhibitors (e.g., butyrate), activators of GPR41 and/or GPR43 (e.g., butyrate and/or propionate and/or acetate), activtators of GPR109A (e.g., butyrate), inhibitors of NF- kappaB signaling (e.g., butyrate), and modulators of PPARgamma (e.g., butyrate), activators of AMPK signaling (e.g., acetate), and modulators of GLP-1 secretion.
  • HDAC inhibitors e.g., butyrate
  • activators of GPR41 and/or GPR43 e.g., butyrate and/or propionate and/or acetate
  • activtators of GPR109A e.g., butyrate
  • inhibitors of NF- kappaB signaling e.g., butyrate
  • modulators of PPARgamma e.g., buty
  • a molecule may be primarily anti- 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”.
  • the term "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.
  • 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. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell.
  • Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids.
  • recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.
  • a "programmed or engineered microorganism” refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state to perform a specific function.
  • 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 “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 menat 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 any of the ant i- 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-p2, N- acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as others.
  • 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.
  • transgene refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.
  • a "non-native" nucleic acid sequence refers to a nucleic acid sequence not normally present in a microorganism, 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 virus, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria or virus 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 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 anti- inflammatory 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.
  • regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures.
  • the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed herein.
  • a "gene cassette" or “operon" encoding a biosynthetic pathway refers to the two or more genes that are required to produce an ant i- inflammatory or gut barrier enhancer molecule.
  • the gene cassette or operon 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
  • the genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a
  • 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-acety transferase, 3-hydroxybutyryl- CoA dehydrogenase, crotonase, phosphate butyry transferase, 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 Treponema denticola.
  • the butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.
  • 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,
  • 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 Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation).
  • acrylate pathway propionate biosynthesis genes e.g., pet, IcdA, IcdB, IcdC, et
  • 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 homolog 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, dihydrolipo amide 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).
  • 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).
  • 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).
  • carboxytransferase (mmdA, PFREUD_18870, beep) which converts methylmalonyl-CoA to propionyl-CoA.
  • 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).
  • 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). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art.
  • the acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or
  • 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 may be operably linked to a promoter that is induced under low-oxygen conditions.
  • "Operably linked” refers a nucleic acid sequence, e.g., a gene or gene cassette for producing an anti- inflammatory or gut barrier enhancer molecule, 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 "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 ant i- 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 ant i- inflammatory or gut barrier enhancer molecule. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be "directly linked" to a regulatory sequence in a manner which allows expression of the gene.
  • a gene may be "indirectly linked" to a regulatory sequence in a manner which allows expression of the gene.
  • two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes.
  • a regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3 ' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • a "promoter” as used herein refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5' of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. 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, Ptac promoter, BBa_J23100, a constitutive Escherichia coli GS promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli ⁇ 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)), a constitutive Bacillus subtilis ⁇ promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis ⁇ promoter (e.g., promoter etc).
  • a constitutive Bacillus subtilis ⁇ promoter e.
  • BBa_K143010 promoter gsiB (BBa_K143011)
  • promoter gsiB BBa_K143011
  • Salmonella promoter e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)
  • a Salmonella promoter e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)
  • a Salmonella promoter e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)
  • T7 promoter e.g., T7 promoter (BBa_I712074; BBa_I719005;
  • BBa_Z0253 BBa_Z0253
  • 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 tetracycline.
  • 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.
  • stable bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a gene encoding one or more anti-inflammation and/or gut barrier enhancer molecule(s), 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.
  • non-native genetic material e.g., a gene encoding one or more anti-inflammation and/or gut barrier enhancer molecule(s)
  • 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 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
  • copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.
  • the term "expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.
  • 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 an antiinflammatory or 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 modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not.
  • Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising an anti- inflammatory or 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.
  • 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), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.
  • a mechanism e.g., protein, proteins, or protein complex
  • a molecule e.g., amino acid, peptide (di-peptide, tri-peptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.
  • exogenous environmental condition or “exogenous environment signal” refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced.
  • exogenous environmental conditions is meant to refer to the environmental conditions external to the engineered micororganism, 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
  • 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
  • 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.
  • oxygen level-dependent transcription factors include, but are not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR.
  • FNR fluarate and nitrate reductase
  • ANR anaerobic nitrate respiration
  • DNR dissimilatory nitrate respiration regulator
  • 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 transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression.
  • PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA.
  • PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS.
  • Factor promoters and/or regulatory regions:
  • 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). In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous
  • the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides.
  • 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 combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter.
  • the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure.
  • the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response).
  • the loss of exposure to an exogenous environmental condition inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut).
  • "Gut" refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste.
  • the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine.
  • the gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas.
  • the upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine.
  • the lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal.
  • Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.
  • the 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 lower levels of oxygen than are present in the atmosphere.
  • the term "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (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 rnmHg 0 2 (0-60 torr 03 ⁇ 4 (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
  • 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 IB summarizes the amount of oxygen present in various organs and tissues.
  • DO dissolved oxygen
  • the term "low oxygen” is meant to refer to a level, amount, or concentration of oxygen (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., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way.
  • the level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (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).
  • Well-aerated solutions e.g., solutions subjected to mixing and/or stirring
  • oxygen producers or consumers are 100% air saturated.
  • the term "low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.
  • any and all incremental fraction(s) thereof e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%,
  • any range of air saturation levels between 0-40%, inclusive e.g., 0-5%, 0.05 - 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 - 2.0%, 0-10%, 5-10%, 10- 15%, 15-20%, 20-25%, 25-30%, etc.
  • the exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
  • the term "low oxygen” is meant to refer to 9% 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%.
  • Ileum (mid- small intestine) -10 torr; -6% oxygen in ambient air (e.g., 11 +/- 3
  • Distal sigmoid colon ⁇ 3 torr (e.g., 3 +/- 1 torr)
  • tumor ⁇ 32 torr (most tumors are ⁇ 15 torr)
  • 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., Saccharomyces, and protozoa.
  • 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 do not contain lipopolysaccharides (LPS).
  • LPS lipopolysaccharides
  • non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to certain strains belonging to the genus Bacillus, Bacteroides, Bifidobacterium,
  • Brevibacteria Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis,
  • Non-pathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut.
  • the disclosure further includes non-pathogenic Saccharomyces, such as Saccharomyces boulardii.
  • Naturally pathogenic bacteria may be genetically engineered to 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.
  • the probiotic bacteria are Gram-negative bacteria.
  • the probiotic bacteria are Gram-positive bacteria. 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, certain strains belonging to the genus Bifidobacteria, Escherichia Coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, and 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.
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et ah, 2012; Cuevas-Ramos et al, 2010; Olier et al, 2012; Nougayrede et al, 2006).
  • Nonpathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • 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).
  • auxotroph refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient) to support its growth.
  • An "auxotrophic modification” is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient.
  • essential gene refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).
  • module and “treat” a disease and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof.
  • modulate and “treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient.
  • modulate and “treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.
  • module and “treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition.
  • prevent and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.
  • Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder.
  • 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.
  • Treating autoimmune disorders and/or diseases and conditions associated with gut inflammation and/or compromised gut barrier function may encompass reducing or eliminating excess inflammation and/or associated symptoms, and does not necessarily encompass the elimination of the underlying disease.
  • 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 anti-inflammation 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
  • a "pharmaceutical composition” refers to a preparation of genetically engineered microorganism of the disclosure, e.g., genetically engineered bacteria or virus, with other components such as a physiologically suitable carrier and/or excipient.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial or viral 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.
  • terapéuticaally 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., infla m mation, dia rrhea . an autoimmune disorder.
  • 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 an autoimmune a disorder and/or a disease or condition associated with gut inflammation and/or compromised gut barrier function.
  • 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.
  • toxin refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure.
  • the term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins.
  • the term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases.
  • anti-toxin or
  • anti-toxin refers to a protein or enzyme which is capable of inhibiting the activity of a toxin.
  • anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.
  • payload refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus.
  • the payload is a therapeutic payload, e.g. and 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.
  • the payload comprises a repressor element, such as a kill switch.
  • the payload comprises an antibiotic resistance gene or genes.
  • the payload is encoded by a gene, multiple genes, gene cassette, or an operon.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism.
  • the genetically engineered microorganism comprises two or more payloads.
  • 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.
  • 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.
  • polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • Recombinantly produced polypeptides and proteins expressed in host cells including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e.
  • fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments.
  • Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.
  • Polypeptides also include fusion proteins. As used herein, the term
  • variant includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide.
  • fusion protein refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques.
  • Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins.
  • “Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
  • Similarity between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein
  • 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
  • variable light chain VL
  • variable heavy chain VH
  • antibody 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.
  • the term “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),
  • 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.
  • single-chain antibody or “single-chain antibodies” typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an
  • 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. 4,946,778.
  • the 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
  • 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.). Long 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.
  • 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 mimetics.
  • 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 "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.
  • linker refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains.
  • synthetic refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.
  • codon-optimized 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
  • secretion system or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a
  • secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g.,HlyBD.
  • secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation- division (RND) multi-drug efflux pumps, various single membrane secretion systems.
  • Non- liming examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems.
  • 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. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm into the periplasmic compartment by the native Sec system.
  • 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.
  • 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.
  • microorganisms such as genetically engineered bacteria of the disclosure are capable of producing one or more non-native anti- inflammation and/or gut barrier function enhancer molecules.
  • the genetically engineered bacteria are obligate anaerobic bacteria.
  • the genetically engineered bacteria are facultative anaerobic bacteria.
  • the genetically engineered bacteria are aerobic bacteria.
  • the genetically engineered bacteria are Gram- positive bacteria.
  • the genetically engineered bacteria are Gram- positive bacteria and lack LPS.
  • the genetically engineered bacteria are Gram-negative bacteria.
  • the genetically engineered bacteria are Gram-positive and obligate anaerobic bacteria.
  • the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria,
  • Mycobacterium Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum,
  • Bifidobacterium breve UCC2003 Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum,
  • Clostridium butyricum M-55 Clostridium cochlearum, Clostridium felsineum,
  • Clostridium histolyticum Clostridium multifermentans, Clostridium novyi-NT,
  • Clostridium paraputrificum Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes,
  • the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei,
  • Lactobacillus plantarum Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii, Clostridium clusters IV and XlVa of Firmicutes (including species of Eubacterium), Roseburia, Faecalibacterium, Enterobacter,
  • Faecalibacterium prausnitzii Clostridium difficile, Subdoligranulum, Clostridium sporogenes, Campylobacter jejuni, Clostridium saccharolyticum, Klebsiella, Citrobacter, Pseudobutyrivibrio, and Ruminococcus.
  • the genetically engineered bacteria are selected from Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli, 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 thetaiotao micron 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
  • the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell.
  • the genetically engineered bacteria are 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. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic
  • E. coli Nissle 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).
  • 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 (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration.
  • the genetically engineered bacteria may require continued administration.
  • 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.
  • 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 an ant i- inflammatory or gut barrier enhancer molecule further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein.
  • 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. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more
  • 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 ParaBAD.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacteria is an auxotroph comprising gene sequence encoding an anti- inflammatory or gut barrier enhancer molecule and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
  • the gene encoding an anti- inflammatory or gut barrier enhancer 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 anti- inflammatory or 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. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome.
  • 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 enhancing.
  • 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. In alternate
  • 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 synthesized by a bio synthetic pathway requiring multiple genes.
  • an anti-inflammation and/or gut barrier function enhancer molecule(s) e.g., butyrate
  • 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. In some embodiments, expression from the plasmid may be useful for increasing expression of the anti- inflammation and/or gut barrier function enhancer molecule(s). In some
  • 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.
  • 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.
  • 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: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g., Fig. 52 for exemplary insertion sites).
  • 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.
  • One strategy in the treatment, prevention, and/or management of inflammatory bowel 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 ant i- 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
  • intestinal integrity e.g., as reviewed in Thorburn et al., Diet, Metabolites, and "Western- Lifestyle” Inflammatory Diseases; Immunity Volume 40, Issue 6, 19 June 2014, Pages 833-842).
  • A SCFA-induced promotion of mucus by gut epithelial cells, possibly through signaling through metabolite sensing GPCRs;
  • B SCFA-induced secretion of IgA by B cells;
  • C SCFA-induced promotion of tissue repair and wound healing;
  • D SCFA-induced promotion of Treg cell development in the gut in a process that presumably facilitates immunological tolerance;
  • E SCFA- mediated enhancement of epithelial integrity in a process dependent on inflammasome activation (e.g., via NALP3) and IL-18 production; and
  • F ant i- inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and inhibition of NF-KB.
  • GPR43 and GPR109A are expressed by the colonic epithelium, by inflammatory leukocytes (e.g. neutrophils and marcophages) and by Treg cells. These receptors signal through G proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin- pathway, leading to NFkappa B inhibition.
  • Other effects can be ascribed to SCFA-mediated HDAC inhibition, e.g. butyrate, which may regulate macrophage function and promote TReg cells.
  • tryptophan metabolites including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehyde, 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+ innate lymphoid cells intraepithelial lymphocytes and RORyt+ 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.
  • the genetically engineered bacteria of the invention comprise an acetate gene cassette and are capable of producing acetate.
  • the genetically engineered bacteria may include any suitable set of acetate biosynthesis genes.
  • the bacteria eomprise an endogenous acetate biosynthetic gene or gene cassette and naturally produce acetate.
  • Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale, 2008), and these endogenous acetate biosynthesis pathways may be a source of genes for the genetically engineered bacteria of the invention.
  • the genetically engineered bacteria of the invention comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria.
  • the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced.
  • the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli.
  • the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and/or Thermoacetogenium.
  • the genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis.
  • the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes.
  • the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate.
  • one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production.
  • the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions.
  • the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.
  • E. coli Nissle acetate is generated as an end product of fermentation.
  • glucose fermentation occurs in two steps, (1) the glycolysis reactions and (2) the NADH recycling reactions, i.e. these reactions re-oxidize the NAD+ generated during the fermentation process.
  • E. coli employs the "mixed acid” fermentation pathway (see, e.g., FIG 25).
  • the "mixed acid” pathway E coli generates several alternative end products and in variable amounts (e.g., lactate, acetate, formate, succinate, ethanol, carbon dioxide, and hydrogen) though various arms of the fermentation pathway, e.g., as shown in FIG. 25.
  • prevention or reduction of flux through one or more metabolic arm(s) generating metabolites other than acetate e.g.
  • deletions in gene(s) encoding such enzymes increase acetate production.
  • Such enzymes include fumarate reductase (encoded by the frd genes), lactate dehydrogenase (encoded by the ldh gene), and aldehyde- alcohol dehydrogenase (encoded by the adhE gene).
  • LdhA is a soluble NAD-linked lactate dehydrogenase (LDH) that is specific for the production of D-lactate and is a homotetramer and shows positive homotropic cooperativity under higher pH conditions.
  • LDH lactate dehydrogenase
  • the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous ldhA gene.
  • AdhE is a homopolymeric protein with three catalytic functions: alcohol dehydrogenase, coenzyme A-dependent acetaldehyde dehydrogenase, and pyruvate formate-lyase deactivase. During fermentation, AdhE has catalyzes two steps towards the generation of ethanol: (1) the reduction of acetyl-CoA to acetaldehyde and (2) the reduction of acetaldehyde to to ethanol.
  • the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous adhE gene.
  • the fumarate reductase enzyme complex encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation.
  • Frd A is one of two catalytic subunits in the four subunit fumarate reductase complex.
  • FrdB is the second catalytic subinut of the complex.
  • FrdC and FrdD are two integral membrane protein components of the fumarate reductase complex.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in one or more endogenous genes selected from in the IdhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous IdhA and rdA genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous IdhA genes and adhE genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous IdhA, the frdA, and adhE genes.
  • the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty- fold, thirty- fold, forty- fold, or fifty- fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle.
  • an engineered or naturally occurring strain e.g., E. coli Nissle.
  • one or more mutations and/or deletions in one or more gene(s) encoding one or more enzyme(s) which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate.
  • Phosphate acetyltransferase catalyzes the reversible conversion between acetyl-CoA and acetylphosphate, a step in the metabolism of acetate (Campos- Bermudez et al., Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation; FEBS J. 2010 Apr;277(8): 1957-66). Both pyruvate and phosphoenolpyruvate activate the enzyme in the direction of acetylphosphate synthesis and inhibit the enzyme in the direction of acetyl- CoA synthesis.
  • acetyl-CoA I pathway has been the target of metabolic engineering to reduce the flux to acetate and increase the production of commercially desired end products (see, e.g., Singh, et al., Manipulating redox and ATP balancing for improved production of succinate in E. coli; Metab Eng. 2011 Jan;13(l):76- 81).
  • a pta mutant does not grow on acetate as the sole source of carbon (Brown et al., The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli; J Gen Microbiol. 1977 Oct;102(2):327-36).
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene.
  • the gentically engineered bacteria produce butyrate.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene and also in one or more endogenous genes selected from the IdhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and adhE genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and IdhA genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, IdhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, IdhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutationand/or deletion in the endogenous pta, IdhA, frdA, and adhE genes. In some embodiments, the gentically engineered bacteris produce butyrate.
  • the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • 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 ⁇ see, e.g., Table 2 and Table 3).
  • Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio,
  • 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: bcd2, 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 combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate.
  • 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.
  • Table 2 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.
  • etfB3 ACAGGCGATAGATGGAGATACTGCACAAGTTGGACCTCAAATAGCTGAAC SEQ ID NO: 2 ATTTAAATCTTCCATCAATAACATATGCTGAAGAAATAAAAACTGAAGGT
  • SEQ ID NO: 8 CACACATGGGTGGAGGAGTTTCTGTTGGAGCTCATAAAAATGGTAAAATA
  • the gene products of the bcd2, etfA3, and etfB3 genes in Clostridium difficile form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant.
  • the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen- limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut.
  • the genetically engineered bacteria comprise a ter gene, e.g., from
  • Treponema denticola which can functionally replace all three of the bcd.2, etfB3, and elf A3 genes, e.g., from Peptoclostridium difficile.
  • the genetically engineered bacteria comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce 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..
  • the genetically engineered bacteria of the invention comprise thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile; ter, e.g., from Treponema denticola; one or more of bcd.2, etfB3, and elf A3, e.g., from
  • Peptoclostridium difficile and produce 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 is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production 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.
  • the gene products of pbt and buk convert butyrylCoA to Butyrate.
  • the pbt and buk genes can be replaced by a tesB gene.
  • tesB can be used to cleave off the CoA from butyryl-coA.
  • the genetically engineered bacteria comprise bcd2, etfB3, etfA3, thiAl, hbd, and crt2, e.g., from Peptoclostridium difficile, and tesB from E.
  • the genetically engineered bacteria comprise ter gene (encoding trans-2- enoynl-CoA reductase) e.g., from Treponema denticola, thiAl, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E.
  • Coli and produce butyrate in low- oxygen conditions, in the presence of specific 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 is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as 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.
  • the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells.
  • the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis.
  • local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders.
  • the bcd.2 gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the bcd.2 gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the bcd.2 gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.
  • the bcd.2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.
  • the bcd.2 gene comprises the sequence of SEQ ID NO: 1.
  • the bcd.2 gene consists of the sequence of SEQ ID NO: 1.
  • the etfB3 gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2.
  • the etfB3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2.
  • the etfB3 gene comprises the sequence of SEQ ID NO: 2.
  • the etfB3 gene consists of the sequence of SEQ ID NO: 2.
  • the elf A3 gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the elf A3 gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 3. In another embodiment, the elf A3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3.
  • the etfA3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3.
  • the elf A3 gene comprises the sequence of SEQ ID NO: 3.
  • the etfA3 gene consists of the sequence of SEQ ID NO: 3.
  • the thiAl gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the thiAl gene has at least about 95% identity with SEQ ID NO: 4. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4.
  • the thiAl gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4.
  • the thiAl gene comprises the sequence of SEQ ID NO: 4.
  • the thiAl gene consists of the sequence of SEQ ID NO: 4.
  • the hbd gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 5. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5.
  • the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5.
  • the hbd gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 5.
  • the crt2 gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6.
  • the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6.
  • the crt2 gene comprises the sequence of SEQ ID NO: 6.
  • the crt2 gene consists of the sequence of SEQ ID NO: 6.
  • the pbt gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 7. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7.
  • the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7.
  • the pbt gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 7.
  • the buk gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8.
  • the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8.
  • the buk gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 8.
  • the ter gene has at least about 80% identity with SEQ ID NO: 9. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 9. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9.
  • the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9.
  • the ter gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 9.
  • the tesB gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10.
  • the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10.
  • the tesB gene comprises the sequence of SEQ ID NO: 10.
  • the tesB gene consists of the sequence of SEQ ID NO: 10.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 11 through SEQ ID NO: 20.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 11 through SEQ ID NO: 20.
  • one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene.
  • the butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.
  • one or more targeted deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production).
  • Non- limiting examples of such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), IdhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
  • Deletions which may be introduced therefore include deletion of adhE, ldh, and frd.
  • the genetically engineered bacteria comprise one or more butyrate-producing cassette(s) and further comprise mutations and/or deletions in one or more of frdA, IdhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more butyrate producing cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the IdhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the IdhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous IdhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous IdhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous IdhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous IdhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the IdhA gene, the frdA gene and the adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous IdhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous IdhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous IdhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiAl-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty- fold, thirty- fold, fourty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the need may arise to prevent and/or reduce acetate production of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of butyrate production.
  • an engineered or naturally occurring strain e.g., E. coli Nissle
  • one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate.
  • a non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate.
  • Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for butyrate production.
  • one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentaion, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for butyrate synthesis.
  • Such mutations and/or deletions include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the IdhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the IdhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and IdhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and IdhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt- buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta, IdhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and IdhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and IdhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta, IdhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, IdhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, IdhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, IdhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd- crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiAl, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiAl-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or twofold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen- fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate.
  • one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production.
  • the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation.
  • 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.
  • the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the butyrate gene cassette in nature.
  • the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.
  • the butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell.
  • the butyrate gene cassette is located on a plasmid in the bacterial cell.
  • the butyrate gene cassette is located in the chromosome of the bacterial cell.
  • a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell.
  • the butyrate gene cassette is expressed on a low- copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high- copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate.
  • the genetically engineered bacteria of the invention are capable of producing an ant i- inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.
  • an ant i- inflammatory or gut barrier enhancer molecule e.g., propionate
  • the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions.
  • the genetically engineered bacteria may express any suitable set of propionate biosynthesis genes ⁇ see, e.g., Table 4, Table 5, Table 6, Table 7).
  • 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 comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria.
  • the genetically engineered bacteria comprise the genes pet, led, and acr from Clostridium propionicum.
  • the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC.
  • the rate limiting step catalyzed by the Acr enzyme is replaced by the Acul from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA.
  • the propionate cassette comprises pet, IcdA, IcdB, IcdC, and acul.
  • the homo log of Acul in E coli, yhdH is used.
  • This the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH.
  • the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA ⁇ , thrB, thrC, ilvA ⁇ , aceE, aceF, and Ipd, and optionally further comprise tesB.
  • the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from E.
  • 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 converts succinyl CoA to L-methylmalonylCoA
  • ygfG converts L-methylmalonylCoA into PropionylCoA
  • ygfH converts propionylCoA into propionate and succinate into succinylCoA.
  • the genes may be codon-optimized, and translational and transcriptional elements may be added.
  • Table 4-6 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette.
  • Table 7 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.
  • acrC AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGA SEQ ID NO: 27 CCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGAT

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Des bactéries génétiquement modifiées, des compositions pharmaceutiques les comprenant ainsi que des méthodes de traitement ou de prévention de troubles auto-immuns, de l'inhibition de mécanismes inflammatoires dans l'intestin, et/ou de renforcement de la fonction de barrière de la muqueuse intestinale sont divulguées.
EP17705544.9A 2016-02-04 2017-02-03 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 Pending EP3411051A2 (fr)

Applications Claiming Priority (18)

Application Number Priority Date Filing Date Title
US201662291461P 2016-02-04 2016-02-04
US201662291470P 2016-02-04 2016-02-04
US201662291468P 2016-02-04 2016-02-04
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
US201662347576P 2016-06-08 2016-06-08
US201662347508P 2016-06-08 2016-06-08
US201662348620P 2016-06-10 2016-06-10
US201662354682P 2016-06-24 2016-06-24
PCT/US2016/039444 WO2016210384A2 (fr) 2015-06-25 2016-06-24 Bactéries manipulées pour traiter des maladies métaboliques
US201662362954P 2016-07-15 2016-07-15
US201662385235P 2016-09-08 2016-09-08
US15/260,319 US11384359B2 (en) 2014-12-22 2016-09-08 Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
PCT/US2016/050836 WO2017074566A1 (fr) 2015-10-30 2016-09-08 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
US201662423170P 2016-11-16 2016-11-16
US201662439871P 2016-12-28 2016-12-28
PCT/US2016/069052 WO2017123418A1 (fr) 2016-01-11 2016-12-28 Bactéries modifiées pour traiter des maladies métaboliques
PCT/US2017/016603 WO2017136792A2 (fr) 2016-02-04 2017-02-03 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

Publications (1)

Publication Number Publication Date
EP3411051A2 true EP3411051A2 (fr) 2018-12-12

Family

ID=59500293

Family Applications (1)

Application Number Title Priority Date Filing Date
EP17705544.9A Pending EP3411051A2 (fr) 2016-02-04 2017-02-03 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

Country Status (4)

Country Link
EP (1) EP3411051A2 (fr)
AU (1) AU2017213646A1 (fr)
CA (1) CA3013770A1 (fr)
WO (1) WO2017136792A2 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11291693B2 (en) 2015-06-25 2022-04-05 Synlogic Operating Company, Inc. Bacteria engineered to treat metabolic diseases
US10392674B2 (en) 2015-07-22 2019-08-27 President And Fellows Of Harvard College Evolution of site-specific recombinases
WO2017019895A1 (fr) 2015-07-30 2017-02-02 President And Fellows Of Harvard College Évolution des talen
US11913044B2 (en) 2018-06-14 2024-02-27 President And Fellows Of Harvard College Evolution of cytidine deaminases
US20220257732A1 (en) 2019-04-29 2022-08-18 Synlogic Operating Company, Inc. Enumeration of genetically engineered microorganisms by live cell counting techniques
WO2021242897A1 (fr) * 2020-05-26 2021-12-02 Synlogic Operating Company, Inc. Bactéries recombinées pour la production d'acide indole-3-acétique (iaa) et leurs utilisations
WO2022067219A1 (fr) * 2020-09-28 2022-03-31 The Regents Of The University Of Michigan Méthodes et compositions pour inflammation intestinale
CN112458033B (zh) * 2020-11-16 2023-01-24 四川农业大学 减毒鼠伤寒沙门菌及其构建方法与应用
US20240110191A1 (en) 2020-12-02 2024-04-04 Synlogic Operating Company, Inc. Engineered microorganisms
EP4377455A1 (fr) * 2021-07-28 2024-06-05 The Broad Institute Inc. Procédés d'évolution continue assistée par phage périplasmique
EP4162946A1 (fr) * 2021-10-05 2023-04-12 Institut national de recherche pour l'agriculture, l'alimentation et l'environnement Kynurénine-aminotransférase et produits associés pour le traitement de maladies intestines inflammatoires
CN114369146B (zh) * 2022-01-14 2023-05-23 上海交通大学医学院附属仁济医院 一种阿克曼氏菌Amuc_2172蛋白及其制备方法和用途
EP4295859A1 (fr) * 2022-06-20 2023-12-27 Institut national de recherche pour l'agriculture, l'alimentation et l'environnement Kynurénine-aminotransférase et produits associés pour le traitement des maladies arthritiques

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
GB9107305D0 (en) 1991-04-08 1991-05-22 Unilever Plc Probiotic
US6203797B1 (en) 1998-01-06 2001-03-20 Stephen C. Perry Dietary supplement and method for use as a probiotic, for alleviating the symptons associated with irritable bowel syndrome
EP1034787A1 (fr) 1999-03-11 2000-09-13 Société des Produits Nestlé S.A. Souches de lactobacillus capables de prévenir la diarrhée causée des bactéries pathogènes
US7731976B2 (en) 2003-08-29 2010-06-08 Cobb And Company, Llp Treatment of irritable bowel syndrome using probiotic composition
BRPI0719748A2 (pt) * 2006-12-01 2013-12-10 Gevo Inc Microrganismo modificados por engenharia para produzir n-butanol e métodos relacionados
EP2655624B1 (fr) 2010-12-23 2017-11-29 Biogen MA Inc. Peptides coupleurs et polypeptides les comportant
WO2016141108A1 (fr) * 2015-03-02 2016-09-09 Synlogic, Inc. 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
US20160206666A1 (en) * 2014-12-22 2016-07-21 Synlogic, Inc. Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tighten gut mucosal barrier

Also Published As

Publication number Publication date
WO2017136792A2 (fr) 2017-08-10
AU2017213646A1 (en) 2018-08-23
WO2017136792A8 (fr) 2017-09-28
CA3013770A1 (fr) 2017-08-10
WO2017136792A3 (fr) 2017-09-08

Similar Documents

Publication Publication Date Title
US20230043588A1 (en) Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
US11384359B2 (en) Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
US11685925B2 (en) Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
US20240110192A1 (en) Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
EP3411051A2 (fr) 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
US11896627B2 (en) Bacteria engineered to treat metabolic diseases
AU2018290278B2 (en) Bacteria for the treatment of disorders
US20190010506A1 (en) Bacteria engineered to treat metabolic diseases
JP2018532412A5 (fr)
WO2017136795A1 (fr) Bactéries modifiées pour traiter des maladies associées au metabolisme du tryptophane
WO2017123418A1 (fr) Bactéries modifiées pour traiter des maladies métaboliques
WO2016210373A2 (fr) Bactéries recombinantes modifiées pour la biosécurité, compositions pharmaceutiques, et leurs procédés d'utilisation
US20180325963A1 (en) Bacteria engineered to treat disorders in which oxalate is detrimental
WO2017139697A1 (fr) Bactéries modifiées en vue du traitement de maladies associées à l'hyperammoniémie
WO2017139708A1 (fr) Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna)
WO2017123610A2 (fr) Bactéries modifiées pour détoxifier les molécules délétères
US20210161976A1 (en) Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier
EP3313371A2 (fr) Bactéries manipulées pour traiter des maladies métaboliques

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20180817

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20191219

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230514