WO2017139708A1 - Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna) - Google Patents

Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna) Download PDF

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Publication number
WO2017139708A1
WO2017139708A1 PCT/US2017/017563 US2017017563W WO2017139708A1 WO 2017139708 A1 WO2017139708 A1 WO 2017139708A1 US 2017017563 W US2017017563 W US 2017017563W WO 2017139708 A1 WO2017139708 A1 WO 2017139708A1
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WIPO (PCT)
Prior art keywords
gene
bacterium
producing
gene cassette
cassette
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PCT/US2017/017563
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English (en)
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WO2017139708A8 (fr
Inventor
Paul F. Miller
Vincent M. ISABELLA
Dean Falb
Jonathan W. KOTULA
Suman MACHINANI
Yves Millet
Adam B. FISHER
Ning Li
Alex TUCKER
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Synlogic Inc
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Synlogic Inc
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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
Priority claimed from PCT/US2017/013074 external-priority patent/WO2017123676A1/fr
Priority claimed from PCT/US2017/012946 external-priority patent/WO2017123592A1/fr
Priority claimed from PCT/US2017/016603 external-priority patent/WO2017136792A2/fr
Priority claimed from PCT/US2017/016609 external-priority patent/WO2017136795A1/fr
Application filed by Synlogic Inc filed Critical Synlogic Inc
Publication of WO2017139708A1 publication Critical patent/WO2017139708A1/fr
Publication of WO2017139708A8 publication Critical patent/WO2017139708A8/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • Non-alcoholic fatty liver disease describes a range of conditions caused by a build-up of fat within liver cells.
  • the first stage of NAFLD is simple fatty liver - also called hepatic steatosis, which often does not cause severe symptoms in the liver;
  • Non-alcoholic steatohepatitis is a severe form of NAFLD, where excess fat accumulation in the liver results in chronic inflammation and damage. NASH affects approximately 3-5% of the population in America, especially in those identified as obese. NASH is characterized by such abnormalities as advanced lipotoxic metabolites, proinflammatory substrate, fibrosis, and increased hepatic lipid deposition. If left untreated, NASH can lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • NASH tumor necrosis factor-alpha
  • adiponectin ratio oxidative stress resulting from mitochondrial abnormalities
  • methionine and choline deficiency have been described in in vivo animal models to cause increased hepatocellular injury and weight loss in addition to inflammation, oxidative stress, and fibrosis. See, for example, Caballero, et ah, J. Bio. Chem.,
  • Colonic propionate delivery has also been shown to reduce intrahepatocellular lipid content in NASH patients, including improvements in weight gain and intra-abdominal fat deposition (see, for example, Chambers et ah, Gut, gutjnl-2014), and GLP-1 administration has been shown to reduce the degree of lipotoxic metabolites and pro-inflammatory substrates, both of which have been shown to speed NASH development, as well as reduce hepatic lipid deposition (see, for example, Bernsmeier et ah, PLoS One, 9(l):e87488, 2014 and Armstrong et ah, J. Hepatol., 2015). Studies have also suggested that rapid weight loss through bariatric surgery ⁇ e.g.
  • gastric bypass is effective in decreasing steatosis, hepatic inflammation, and fibrosis.
  • Other treatments have involved using anti-diabetic medications such as metformin, rosiglitazone, and pioglitazone. Though inconclusive, the studies suggest that the
  • the present disclosure provides engineered bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with nonalcoholic steatohepatitis (NASH).
  • the engineered bacteria comprise gene sequence(s) encoding one or more enzymes present in a biosynthetic pathway for producing a short chain fatty acid, e.g. , butyrate, propionate, and/or acetate.
  • the engineered bacteria comprise one or more gene cassette(s) encoding a biosynthetic pathway for producing a short chain fatty acid, e.g., butyrate, propionate, and/or acetate.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate. In some embodiments, the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate. In some embodiments, the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more GLP- 1 peptides. In some embodiments, the engineered bacteria comprise genetic circuitry for reducing bile salt.
  • the engineered bacteria comprise gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s) and/or gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise one or more gene cassettes which modulate typtophan levels, e.g. , in the serum and/or in the gut.
  • tryptophan catabolized into one or more of its metabolites collectively called kynurenine and indole metabolites herein.
  • the engineered bacteria comprise one or more gene cassettes which modulate kynurenine levels, e.g. , in the serum and/or in the gut.
  • the engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Table 12 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in Fig. 29.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein,including, but not limited to those listed in Table 12 and elsewhere herein, in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in Fig. 29.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 12, and elsewhere herein, in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in Fig. 16.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels.
  • the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels.
  • the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels.
  • the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios.
  • the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios
  • the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) selected from one or more of the following: gene cassette(s) encoding a biosynthetic pathway for producing a short chain fatty acid, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s) and at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. acetate biosynthetic cassette(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthetic cassette(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g.
  • propionate biosynthesis gene cassette(s) at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), and at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. acetate biosynthetic cassette(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more GLP- 1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more GLP- 1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more GLP- 1
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more GLP-1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more GLP- 1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate e.g., acetate biosynthesis gene cassette(s), and gene sequence(s) encoding one or more GLP- 1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, and gene sequence(s) encoding one or more GLP- 1 polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g., acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g., acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s), and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), for producing GLP- 1, and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the bacteria comprise gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s), and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • butyrate biosynthesis gene cassette(s) at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, and gene sequence(s) encoding one or more bile salt transporter(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), and gene sequence(s) encoding GLP- 1 and a transporter.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s), and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), gene sequence(s) encoding GLP- 1, gene sequences encoding a transporter and gene sequence(s) encoding one or more bile salt hydrolase polypeptide(s). .
  • the engineered bacteria comprise at least one gene sequence(s) encoding GLP- 1 and one or more bile salt hydrolase polypeptide(s).
  • the engineered bacteria comprise at least one gene sequence(s) encoding GLP- 1 and a transporter.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s) and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g., acetate biosynthesis gene cassette(s) and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g., acetate biosynthesis gene cassette(s), and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g.
  • butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, and at least one gene cassette for the production or catabolism of tryptophan and/or one of its metabolites.
  • the genetically engineered bacteria may further comprise one or more cassettes for the consumption of ammonia.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s) and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3- propionic acid.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s) and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g., propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s) and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g. , butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, e.g. , acetate biosynthesis gene cassette(s), and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • a tryptophan metabolite selected from tryptamine and/or indole- 3 -acetic acid and indole-3-propionic acid.
  • the engineered bacteria comprise at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing butyrate, e.g., butyrate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing propionate, e.g. , propionate biosynthesis gene cassette(s), at least one gene cassette(s) encoding one or more biosynthetic pathway(s) for producing acetate, and at least one gene cassette for the production of a tryptophan metabolite selected from tryptamine and/or indole-3-acetic acid and indole-3-propionic acid.
  • a tryptophan metabolite selected from tryptamine and/or indole-3-acetic acid and indole-3-propionic acid.
  • the engineered bacteria further compriseoptional genetic circuitry designed to ensure the safety and non-colonization of a subject that is administered the engineered bacteria, such as, for example, one or more auxotrophies, kill switches, and combinations thereof. These engineered bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect.
  • a bacterial cell disclosed herein has been genetically engineered to comprise one or more synthetic circuits selected from a propionate biosynthesis gene cassette, a butyrate biosynthesis gene cassette, GLP- 1 sequence, and combinations thereof, and is capable of processing propionate, butyrate, and/or GLP- 1 in low-oxygen environments, e.g., the gut.
  • compositions comprising the bacterial cells disclosed herein may be used to treat and/or prevent liver disease, such as nonalcoholic steatohepatitis (NASH).
  • liver disease such as nonalcoholic steatohepatitis (NASH).
  • NASH nonalcoholic steatohepatitis
  • butyrate expression functions to improve hepatic inflammation and intestinal barrier function.
  • Propionate expression functions to reduce intrahepatocellular lipid content
  • GLP- 1 expression functions to decrease lipotoxicity in NASH patients.
  • a pharmaceutical composition comprising an engineered bacterial cell, wherein the engineered bacterial cell comprises a heterologous propionate gene cassette; a heterologous butyrate gene cassette; or a heterologous GLP- 1 gene operably linked to a first inducible promoter, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to a second inducible promoter, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the heterologous propionate gene cassette; the heterologous butyrate gene cassette; or the heterologous GLP-1 gene.
  • a pharmaceutical composition comprising an engineered bacterial cell, wherein the engineered bacterial cell comprises a heterologous propionate gene cassette; a heterologous butyrate gene cassette; or a heterologous GLP-1 gene operably linked to a first inducible promoter, a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to a second inducible promoter, and a pharmaceutically acceptable carrier, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the heterologous propionate gene cassette; the heterologous butyrate gene cassette; or the heterologous GLP-1 gene.
  • the pharmaceutical composition comprises a heterologous propionate gene cassette and a heterologous butyrate gene cassette. In one embodiment, the pharmaceutical composition comprises a heterologous propionate gene cassette and a heterologous GLP-1 gene. In one embodiment, the pharmaceutical composition comprises a heterologous butyrate gene cassette and a heterologous GLP-1 gene. In one embodiment, the pharmaceutical composition comprises a heterologous propionate gene cassette, a
  • heterologous butyrate gene cassette and a heterologous GLP-1 gene.
  • the heterologous propionate gene cassette is from
  • the heterologous propionate gene cassette comprises pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC genes.
  • the heterologous propionate gene cassette comprises thrA ⁇ , thrB, thrC, ilvA ⁇ , aceE, aceF, and Ipd genes.
  • the heterologous butyrate gene cassette is from
  • the heterologous butyrate gene cassette comprises bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk genes.
  • the heterologous GLP- 1 gene is from Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus gasseri, Bifidobacterium longum, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Streptomyces lividans, or Homo sapiens.
  • the heterologous GLP-1 gene has a sequence with at least 90% identity to a nucleic acid sequence encoding SEQ ID NO:40. In one embodiment, the heterologous GLP-1 gene comprises a nucleic acid sequence encoding SEQ ID NO:40. In one embodiment, the heterologous GLP-1 gene consists of a nucleic acid sequence encoding SEQ ID NO:40.
  • the engineered bacterial cell is not capable of colonizing the gut of a mammal.
  • the heterologous propionate gene cassette the heterologous propionate gene cassette
  • heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a plasmid in the bacterial cell.
  • the heterologous propionate gene cassette, the heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a chromosome in the bacterial cell.
  • the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are different promoters.
  • the first inducible promoter is not operably linked with the heterologous propionate gene cassette, the heterologous butyrate gene cassette, or the heterologous GLP-1 gene in nature.
  • the second inducible promoter is not operably linked with the heterologous gene encoding the substance that is toxic to the bacterial cell in nature.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each directly induced by environmental conditions.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each indirectly induced by environmental conditions.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each directly or indirectly induced by environmental conditions specific to the small intestine of a mammal.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each directly or indirectly induced by low-oxygen or anaerobic conditions.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each an FNR responsive promoter.
  • the FNR responsive promoter is a promoter selected from the group consisting of a promoter comprising SEQ ID NO: l, a promoter comprising SEQ ID NO:2, a promoter comprising SEQ ID NO:3, a promoter comprising SEQ ID NO:4, a promoter comprising SEQ ID NO:5, a promoter comprising SEQ ID NO:6, a promoter comprising SEQ ID NO:7, a promoter comprising SEQ ID NO:8, a promoter comprising SEQ ID NO:9, a promoter comprising SEQ ID NO: 10, a promoter comprising SEQ ID NO: 11, and a promoter comprising SEQ ID NO: 12.
  • the engineered bacterial cell is an engineered probiotic bacterial cell.
  • the engineered bacterial cell is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium,
  • the engineered bacterial cell is of the genus Escherichia. In one embodiment, the engineered bacterial cell is of the species Escherichia coli strain Nissle.
  • the engineered bacterial cell is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
  • the mammalian gut is a human gut.
  • the engineered bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine
  • the second inducible promoter is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.
  • a method for treating nonalcoholic steatohepatitis (NASH) in a subject comprising administering a pharmaceutical composition comprising a programmed engineered bacterial cell to the subject, wherein the programmed engineered bacterial cell expresses: a heterologous propionate gene cassette; a heterologous butyrate gene cassette; a heterologous GLP-1 gene; a heterologous propionate gene cassette and a heterologous butyrate gene cassette; a heterologous propionate gene cassette and a heterologous GLP-1 gene; a heterologous butyrate gene cassette and a heterologous GLP-1 gene; or a heterologous propionate gene cassette, a heterologous butyrate gene cassette, and a heterologous GLP-1 gene; in response to an exogenous environmental condition in the subject, thereby treating nonalcoholic steatohepatitis (NASH) in the subject.
  • a pharmaceutical composition comprising a programmed engineered bacterial cell to the subject, wherein the programmed engineered bacterial
  • the programmed engineered bacterial cell further comprises a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to a second inducible promoter, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the heterologous propionate gene cassette; the heterologous butyrate gene cassette; or the heterologous GLP-1 gene.
  • the heterologous propionate gene cassette is from
  • the heterologous propionate gene cassette comprises pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC genes.
  • the heterologous propionate gene cassette comprises hrA ⁇ , thrB, thrC, ilvA ⁇ , aceE, aceF, and Ipd genes.
  • the heterologous butyrate gene cassette is from
  • the heterologous butyrate gene cassette comprises bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk genes.
  • the heterologous GLP-1 gene is at least 90% identical to a nucleic acid sequence encoding SEQ ID NO:40. In one embodiment, the heterologous GLP- 1 gene comprises a nucleic acid sequence encoding SEQ ID NO:40.
  • the engineered bacterial cell is not capable of colonizing the gut of the subject. In one embodiment, the engineered bacterial cell does not colonize the gut of the subject.
  • the heterologous propionate gene cassette the heterologous propionate gene cassette
  • heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a plasmid in the bacterial cell.
  • the heterologous propionate gene cassette, the heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a chromosome in the bacterial cell.
  • the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are different promoters.
  • the first inducible promoter is not operably linked with the heterologous propionate gene cassette, the heterologous butyrate gene cassette, or the heterologous GLP-1 gene in nature.
  • the second inducible promoter is not operably linked with the heterologous gene encoding the substance that is toxic to the bacterial cell in nature.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each directly induced by environmental conditions. In one embodiment, the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter, are each indirectly induced by environmental conditions. In one embodiment, the
  • environmental conditions are environmental conditions specific to the small intestine of a mammal.
  • the environmental conditions specific to the small intestine of a mammal are low-oxygen or anaerobic conditions.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each an FNR responsive promoter.
  • the FNR responsive promoter is a promoter selected from the group consisting of a promoter comprising SEQ ID NO: l, a promoter comprising SEQ ID NO:2, a promoter comprising SEQ ID NO:3, a promoter comprising SEQ ID NO:4, a promoter comprising SEQ ID NO:5, a promoter comprising SEQ ID NO:6, a promoter comprising SEQ ID NO:7, a promoter comprising SEQ ID NO:8, a promoter comprising SEQ ID NO:9, a promoter comprising SEQ ID NO: 10, a promoter comprising SEQ ID NO: 11, and a promoter comprising SEQ ID NO: 12.
  • the engineered bacterial cell is an engineered probiotic bacterial cell.
  • the engineered bacterial cell is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium,
  • the engineered bacterial cell is of the genus Escherichia. In one embodiment, the engineered bacterial cell is of the species Escherichia coli strain Nissle.
  • the engineered bacterial cell is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
  • the mammalian gut is a human gut.
  • the engineered bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine
  • the second inducible promoter is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.
  • a method for treating nonalcoholic steatohepatitis (NASH) in a subject comprising administering a pharmaceutical composition described herein to the subject, thereby treating nonalcoholic steatohepatitis (NASH) in the subject.
  • the pharmaceutical composition is administered orally.
  • the subject is fed a meal within one hour of administering the pharmaceutical composition.
  • the subject is fed a meal concurrently with administering the pharmaceutical composition.
  • an engineered bacterial cell comprising a heterologous propionate gene cassette, a heterologous butyrate gene cassette, and/or a heterologous GLP-1 gene operably linked to a first inducible promoter, wherein the first inducible promoter is a fumarate and nitrate reductase (FNR) responsive promoter or a propionate promoter.
  • FNR fumarate and nitrate reductase
  • the engineered bacterial cell further comprises a heterologous gene encoding a substance that is toxic to the bacterial cell that is operably linked to a second inducible promoter, wherein expression of the substance that is toxic to the bacterial cell is delayed in time as compared to the expression of the heterologous propionate gene cassette; the heterologous butyrate gene cassette; or the heterologous GLP-1 gene.
  • the heterologous propionate gene cassette is from
  • the heterologous propionate gene cassette comprises pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC genes.
  • the heterologous propionate gene cassette comprises thrA ⁇ , thrB, thrC, ilvA ⁇ , aceE, aceF, and Ipd genes.
  • the heterologous butyrate gene cassette comprises bcd.2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk genes.
  • the heterologous GLP-1 gene has at least 90% identity to a nucleic acid sequence encoding SEQ ID NO:40. In one embodiment, the heterologous GLP-1 gene comprises a nucleic acid sequence encoding SEQ ID NO:40.
  • the engineered bacterial cell is not capable of colonizing the gut of a mammal.
  • the mammal is a human.
  • the heterologous propionate gene cassette the heterologous propionate gene cassette
  • heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a plasmid in the bacterial cell.
  • the heterologous propionate gene cassette, the heterologous butyrate gene cassette, and/or the heterologous GLP-1 gene is located on a chromosome in the bacterial cell.
  • the first inducible promoter and the second inducible promoter are separate copies of the same inducible promoter. In one embodiment, the first inducible promoter and the second inducible promoter are different promoters.
  • the first inducible promoter is not operably linked with the heterologous propionate gene cassette, the heterologous butyrate gene cassette, or the heterologous GLP-1 gene in nature.
  • the second inducible promoter is not operably linked with the heterologous gene encoding the substance that is toxic to the bacterial cell in nature.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each directly induced by environmental conditions. In one embodiment, the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter, are each indirectly induced by environmental conditions. In one embodiment, the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter, are each directly or indirectly induced by environmental conditions specific to the small intestine of a mammal. In one embodiment, the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter, are each directly or indirectly induced by low-oxygen or anaerobic conditions.
  • the first inducible promoter, the second inducible promoter, or the first inducible promoter and the second inducible promoter are each an FNR responsive promoter.
  • the FNR responsive promoter is a promoter selected from the group consisting of a promoter comprising SEQ ID NO: l, a promoter comprising SEQ ID NO:2, a promoter comprising SEQ ID NO:3, a promoter comprising SEQ ID NO:4, a promoter comprising SEQ ID NO:5, a promoter comprising SEQ ID NO:6, a promoter comprising SEQ ID NO:7, a promoter comprising SEQ ID NO:8, a promoter comprising SEQ ID NO:9, a promoter comprising SEQ ID NO: 10, a promoter comprising SEQ ID NO: 11, and a promoter comprising SEQ ID NO: 12.
  • the engineered bacterial cell is an engineered probiotic bacterial cell.
  • the engineered bacterial cell is a member of a genus selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium,
  • the engineered bacterial cell is of the genus Escherichia. In one embodiment, the engineered bacterial cell is of the species Escherichia coli strain Nissle.
  • the engineered bacterial cell is an auxotroph in a gene that is complemented when the engineered bacterial cell is present in a mammalian gut.
  • the mammalian gut is a human gut.
  • the engineered bacterial cell is an auxotroph in diaminopimelic acid or an enzyme in the thymine
  • the second inducible promoter is directly or indirectly induced by an environmental condition not naturally present in the mammalian gut.
  • a pharmaceutical composition comprising an engineered bacterial cell and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for oral administration.
  • a method for treating nonalcoholic steatohepatitis (NASH) in a subject comprising administering a pharmaceutical composition described herein to the subject, thereby treating nonalcoholic steatohepatitis (NASH) in the subject.
  • the pharmaceutical composition is administered orally.
  • the subject is fed a meal within one hour of administering the pharmaceutical composition.
  • the subject is fed a meal concurrently with administering the pharmaceutical composition.
  • the subject is fed a meal before administering the pharmaceutical composition.
  • the subject is fed a meal after administering the pharmaceutical composition.
  • Figs. 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, and 1J depict schematics of exemplary circuits described herein, which are inducible under anaerobic or low oxygen conditions, e.g. , a butyrate circuit (Fig. 1A), a propionate circuit (Fig. IB), and a GLP- 1 gene that is inducible under anaerobic or low oxygen conditions (Fig. 1C).
  • Fig. ID depicts a schematic of a bile salt hydrolase enzyme and/or bile salt transporter construct(s) inducible under anaerobic or low oxygen conditions.
  • the recombinant bacterial cell may further comprise an auxotrophic mutation, a secretion system, e.g. , leaky membrane system or type III secretion system, and/or a kill switch, as further described herein.
  • the recombinant bacterial cell may further optionally comprise an auxotrophy.
  • Figs. IE, IF, and 1G depict exemplary dual circuits described herein, including, for example, a propionate circuit and a butyrate circuit that are inducible under anaerobic or low oxygen conditions (Fig. IE); a propionate circuit and a GLP- 1 gene that are inducible under anaerobic or low oxygen conditions (Fig.
  • Circuits include, for example, a propionate circuit, a butyrate circuit, and a GLP- 1 gene that are inducible under anaerobic or low oxygen conditions (Fig. 1H); a bile salt hydrolase and/or bile salt importer circuit, a butyrate circuit, and a GLP-1 gene that are inducible under anaerobic or low oxygen conditions (Fig. 1H); a bile salt hydrolase and/or bile salt importer circuit, a butyrate circuit, and a GLP-1 gene that are inducible under anaerobic or low oxygen conditions (Fig.
  • a bile salt hydrolase a circuit, a butyrate circuit, and a GLP-1 gene that are inducible under anaerobic or low oxygen conditions (Fig. 1J).
  • the engineered bacterium shown in any of Figs. 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, and 1J 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.
  • Figs. 2A, 2B, 2C, 2D, 2E, 2F, and 2G depict schematics of exemplary circuits described herein, which are inducible under anaerobic or low oxygen conditions.
  • Fig. 2A depicts a schematic showing an exemplary Kynurenine Degradation Circuit. Kynurenine is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynureninase is expressed to metabolize Kynurenine to Anthranilic acid in the cell. Both the transporter and kynureninase genes are optionally expressed from an inducible promoter, e.g., a FNR- inducible promoter.
  • FIG. 2B depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynurenine biosynthetic cassette is expressed to produce Kynurenine. Both the transporter and Kynurenine biosynthetic cassette genes are optionally expressed from an inducible promoter, e.g., a FNR- inducible promoter.
  • Fig. 2C depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or
  • Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Tryptophan is synthesized and then Kynurenine is synthesized from the synthesized tryptophan or from tryptophan imported into the cell. Both the transporter and kynureninase biosynthetic genes are optionally expressed from an inducible promoter, e.g., a FNR- inducible promoter.
  • Fig. 2D depicts a schematic of an E.
  • Fig. 2E depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein.
  • Export mechanism for kynurenine and/or tryptophan is also expressed or provided.
  • Fig. 2E depicts a schematic of an E. coli that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein.
  • FIG. 2F depicts a schematic of an E. coli that is genetically engineered to produce butyrate, tryptophan metabolites, and tryptophan (which can be converted to bioactive tryptophan metabolites or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for tryptophan and/or tryptophan metabolites is also expressed or provided.
  • Fig. 2G depicts a schematic of an E.
  • coli that is genetically engineered to produce butyrate, and propionate, kynurenine and/or other tryptophan metabolites, and GLP-1, under the control of a FNR-responsive promoter and further comprising a secretion system, e.g., for GLP-1 secretion as known in the art or described herein.
  • a secretion system e.g., for GLP-1 secretion as known in the art or described herein.
  • kynurenine/or tryptophan metabolites is also expressed or provided.
  • the engineered bacterium shown in any of Figs. 2A, 2B, 2C, 2D, 2E, 2F, and 2G may also have an auxo trophy, e.g., in one example, the thy A gene can be been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • Figs. 3A, 3B, 3C, and 3D depict the pathway and a schematic of different butyrate producing circuits.
  • Fig3A depicts a metabolic pathway for butyrate production
  • Figs. 3B and 3C depict two schematics of two different butyrate producing circuits (found in SYN- UCD503 and SYN-UCD504), both under the control of a tetracycline inducible promoter.
  • Fig. 3D depicts a schematic of a third butyrate gene cassette (found in SYN-UCD505) under the control of a tetracycline inducible promoter.
  • SYN-UCD503 comprises 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.
  • SYN-UCD504 comprises 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.
  • SYN-UCD505 comprises 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.
  • Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by molecules or metabolites indicative of liver damage (e.g., bilirubin), 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.
  • Figs. 4A, 4B, 4C, 4D, 4E, and 4F depict schematics of the gene organization of exemplary bacteria of the disclosure. Figs.
  • FIG. 4A and 4B depict the gene organization of an exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions.
  • Fig. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk; white boxes) is expressed.
  • Fig. 4A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB
  • FIG. 4B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • Figs. 4C and 4D 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
  • butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, buk; white boxes) 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 gray arrows and black squiggles) and ultimately to the production of butyrate.
  • Figs. 4E and 4F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H202.
  • the OxyR transcription factor (gray 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; white boxes) 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 gray arrows and black squiggles) and ultimately to the production of butyrate.
  • FIGs. 5A, 5B, 5C, 5D, 5E, and 5F depict schematics of the gene organization of exemplary bacteria of the disclosure.
  • Figs. 5A and 5B 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. 4.
  • Fig. 5A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter").
  • Fig. 5B depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two grey boxed "FNR"s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • Figs. 5C and 5D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In Fig.
  • FIG. 5E and 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 (gray circle, "OxyR") binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, pbt, buk; white boxes) is expressed.
  • the OxyR transcription factor interacts with H 2 0 2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 6A, 6B, 6C, 6D, 6E, and 6F depict schematics of the gene organization of exemplary bacteria of the disclosure.
  • Figs. 6A and 6B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.
  • Fig. 6A depicts relatively low butyrate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey 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; white boxes) is expressed.
  • FIG. 6B depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed "FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • Figs. 6C and 6D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO.
  • the NsrR transcription factor (gray circle, "NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiAl, hbd, crt2, tesB; white boxes) is expressed.
  • the NsrR in the presence of NO, the NsrR
  • Figs. 6E and 6F 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 (gray circle, "OxyR") binds to, but does not induce, the oxyS promoter.
  • Figs. 7A, 7B, and 7C depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • Fig. 7A depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (grey 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; white boxes) is expressed.
  • Fig. 7A depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (grey boxed "FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the propionate biosynthesis enzymes (pet, IcdA, IcdB, I
  • FIG. 7B depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two grey 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. 7C depicts an exemplary propionate biosynthesis gene cassette.
  • propionate production is induced by NO or H 2 O 2 as depicted and described for the butyrate cassette(s) in the preceding Fig. 4C-4F, 5C-5F, 6C-6F.
  • Figs. 8A, 8B, and 8C depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • Fig. 8A depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by "X") FNR (grey 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; white boxes) is expressed.
  • Fig. 8B depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two grey 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.
  • propionate production is induced by NO or H 2 O 2 as depicted and described for the butyrate cassette(s) in the preceding Fig. 4C-4F, 5C-5F, 6C-6F.
  • Figs. 8D, 8E, and 8F depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.
  • Fig. 8D depicts relatively low propionate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey 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; white boxes) is expressed.
  • Fig. 8E depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two grey 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. 8F depicts an exemplary propionate biosynthesis gene cassette.
  • propionate production is induced by NO or H 2 0 2 as depicted and described for the butyrate cassette(s) in the preceding Fig. 4C-4F, 5C-5F, 6C-6F.
  • Fig. 9A, 9B, and 9C depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.
  • Fig. 9A 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.
  • Figs. 9A and 9B 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. 9A, 9B, and 9C depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.
  • Fig. 9A 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
  • FIG. 9A depicts relatively low propionate production under aerobic conditions in which oxygen (0 2 ) prevents (indicated by "X") FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter ("FNR promoter"). Therefore, none of the propionate biosynthesis enzymes ⁇ sbm, ygfD, ygfG, ygftt; white boxes) is expressed.
  • Fig. 9B depicts increased propionate production under low- oxygen or anaerobic conditions due to FNR dimerizing (two grey 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. 4C-4F, 5C-5F, 6C-6F.
  • Fig. 10 depicts bile salt metabolism.
  • Bile salts are synthesized from cholesterol in the liver and stored in the gallbladder. After release into the duodenum, microbial bile salt hydrolase activity in the small intestine deconjugates the glycine or taurine molecules to produce primary bile acids (also known as unconjugated bile acids). Most bile acids are reabsorbed into the enterohepatic portal system, but some enter the large intestine where they are further metabolized by microbial 7a-dehydroxylase to produce secondary bile acids. Excess bile acids are also lost in the stool (200 mg - 600 mg per day).
  • Fig. 11 depicts the structure of bile salts and the location at which bile salt hydrolase enzymes deconjugate the bile salts. BSH activity has been detected in
  • Lactobacillus spp Lactobacillus spp, Bifidobacterium spp, Enterococcus spp, Clostridium spp, and Bacteroides spp.
  • BSH positive bacteria are gram positive with the exception of two Bacteroides strains. BSH in has been detected in pathogenic bacteria, e.g., Listeria monocytogenes and
  • E. coli does not demonstrate BSH actvity nor contain bsh homolog in genome
  • Fig. 12 depicts the state of one non-limiting embodiment of the bile salt hydrolase enzyme construct under inducing conditions. Expression of the bile salt hydrolase enzyme and a bile salt transporter are both induced by the FNR promoter in the absence of oxygen.
  • the thyA gene has been mutated in the E. coli Nissle genome, so thymidine must be supplied in the culture medium to support growth.
  • the recombinant bacterial cell may further comprise an auxotrophic mutation, a type III secretion system, and/or a kill switch, as further described herein.
  • Fig. 13 depicts a schematic of tryptophan metabolism 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; QPRT, quinolinic acid phosphoric
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the tryptophan metabolism enzymes depicted in Fig. 13, or bacterial functional homo logs thereof. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes which produce one or more of the tryptophan metabolites depicted in Fig. 13. 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 liver damage, e.g., as seen in NASH, , 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. 14 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 (IP A) 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. I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production.
  • IP A Indole-3-propionate
  • I3A Indole- 3 -aldehyde
  • I3A acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function.
  • I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune
  • AhR 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. 15 depicts a schematic of a 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. 39.
  • the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in Fig. 39, including but not limited to, kynurenine, indole-3-aldehyde, indole- 3 -acetic acid, and/or indole-3 acetaldehyde.
  • 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 liver damage, e.g., as seen in NASH, , 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 tryptophan metabolism enzymes which are encoded by the genetically engineered bacteria are derived from genera within phyla, which include, but are not limited to, proteobacteria, actinobacteria, firmicutes, bacteroidetes, chloroflexi, cyanobacteria, an euryarchaeota (e.g. , as described in Vujkovic-Cvijin et al.).
  • Fig. 16 depicts a schematic of the trypophan metabolic pathway. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled,
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in Fig. 16. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in Fig.
  • 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 liver damage, e.g., as seen in NASH, , 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. 17A and Fig. 17B depict diagrams of bacterial tryptophan metabolism pathways.
  • Fig. 17A depicts a schematic of the bacterial tryptophan metabolism, as described, e.g. , in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase
  • Trp decarboxylase EC 4.1.1.28
  • tryptamine oxidase EC 1.4.3.4
  • Trp side chain oxidase EC 4.1.1.43
  • indole acetaldehyde dehydrogenase EC 1.2.1.3
  • 11 indole acetic acid oxidase
  • Trp 2-monooxygenase EC 1.13.12.3
  • indole acetamide hydrolase EC 3.5.1.0.
  • the dotted lines ( ) indicate a spontaneous reaction.
  • Fig. 17B 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: Indole- 3 -acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA:
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in Fig. 17. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in Fig. 17. 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 liver damage, e.g., as seen in NASH, , 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. 18 depicts schematic of the E. coli tryptophan synthesis pathway, including genes, enzymes, and reactions involved. The seven genes, or genetic segments, seven enzymes, or enzyme domains, and seven reactions, involved in tryptophan formation are shown. Only one of the reactions is reversible. The products of four other pathways contribute carbon and/or nitrogen during tryptophan formation. Two of the tryptophan pathway enzymes often function as polypeptide complexes: anthranilate synthase, consisting of the TrpG and TrpE polypeptides, and tryptophan synthase, consisting of the TrpB and TrpA polypeptides.
  • Fig. 19. shows a schematic depicting an exemplary Tryptophan circuit.
  • Tryptophan is produced from the Chorismate precursor through expression of the trpE, trpG- D, trpC-F, trpB and trpA genes.
  • Optional knockout of the tryptophan Repressor trpR is also depicted.
  • Optional production of the Chorismate precursor through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. All of these genes are optionally expressed from an inducible promoter, e.g., a FNR- inducible promoter.
  • the bacteria may also include an auxotrophy, e.g., deletion of thyA ( ⁇ thyA; thymidine dependence).
  • the bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan.
  • Non limiting example of a bacterial strain is listed.
  • Figs. 20A-20D depicts schematics of exemplary embodiments of the disclosure, in which 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. In certain embodiments 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. 20A shows a schematic depicting an exemplary Tryptophan circuit.
  • 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.
  • 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. 20B, and/or FIG. 20C, and/or FIG. 20D.
  • FIG. 20B 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. 20A and/or described in the description of FIG. 20A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 20C, and/or FIG. 20D.
  • trpR and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • FIG. 20C 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.
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 20A and/or described in the description of FIG. 20A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 20B, and/or FIG. 20D.
  • 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. 20D 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. 20A and/or described in the description of FIG. 20A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 20B, and/or FIG. 20C.
  • 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.
  • FIG. 21A 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.
  • 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for Tryptophan decarboxylase, e.g., from Catharanthus roseus, which converts tryptophan to tryptamine, e.g., under the control of an inducible promoter e.g., an FNR promoter.
  • FIG. 21B 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. 20A and/or FIG. 20B, and/or FIG. 20C, and/or FIG.
  • 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.
  • FIG. 21C 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D 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. 21D 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D 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. 21E 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D 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
  • FIG. 21F 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D 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. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g. , from homo sapiens or AADAT
  • FIG. 21G 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. 20A and/or and/or FIG. 20B, and/or FIG. 20C, and/or FIG. 20D 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.
  • 21H depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-carbinol, indole-3-aldehyde, 3,3' diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet.
  • the genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g. , from Arabidopsis thaliana) under the control of an inducible promoter, e.g. an FNR promoter.
  • the engineered bacterium shown in any of FIG. 21A, FIG. 21B, FIG. 21D, FIG. 21D, FIG.
  • FIG. 21E, FIG. 21F, FIG. 21G and FIG. 21H 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.
  • Figs. 22A-22F 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. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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.
  • 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 oxida
  • trpDH Trptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-21048
  • ipdC Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae
  • iadl Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis
  • AAOl Indole- 3 -acetaldehyde oxidase, e.g., from Arabidopsis thaliana
  • an inducible promoter e.g., an FNR promoter.
  • the optional circuits for tryptophan production are as depicted and described in FIG. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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.
  • FIG. 22C the optional circuits for tryptophan production are as depicted and described in FIG. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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. 22D the optional circuits for tryptophan production are as depicted and described in FIG. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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. 22E the optional circuits for tryptophan production are as depicted and described in FIG. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG
  • 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 FNR promoter, the engineered
  • FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E 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. 22F the optional circuits for tryptophan production are as depicted and described in FIG. 20A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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. 23A, Fig. 23B, and Fig. 23C depict schematics of exemplary
  • 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. 23A 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. 20A and/or FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.
  • FIG. 23B 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. 20A and/or FIG. 20B and/or FIG. 20C and/or FIG. 20D.
  • 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. 23C 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. 20A and/or FIG. 20B and/or FIG. 20C and/or FIG. 20D. 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: (indole-3-
  • 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. 24A and Fig. 24B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production.
  • FIG. 24A depicts a schematic showing intermediates in tryptophan biosynthesis and the gene products catalyzing the production of these intermediates.
  • 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.
  • chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe).
  • Teyr tyrosine
  • Phe phenylalanine
  • D-3-phosphoglycerate is converted to serine, which can also be a source for tryptophan biosynthesis.
  • AroG, AroF, AroH DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D- erythrose 4-phosphate to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate
  • DAHP DAHP
  • RhoF tyrosine
  • AroG phenylalanine
  • RhoH tryptophan(AroH)
  • 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).
  • AroD 3 -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-dehydroshikimate and introduces the first double bond of the aromatic ring.
  • AroE, YdiB: E. coli expresses two shikimate
  • 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-dehydroshikimate 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.
  • shikimate kinase enzymes I (AroK) and II (AroL).
  • AroA 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) is involved in the 7th and last step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. 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.
  • TrpEDCAB E coli trp operon: TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L-glutamate.
  • Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a
  • TrpC Bifunctional phosphoribosylanthranilate isomerase / indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway.
  • TrpC phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into
  • 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 (a2- ⁇ 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 bio synthetic 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
  • 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),
  • FIG. 24B depicts a schematic showing exemplary engineering strategies which can improve tryptophan production. Each of these exemplary strategies can be used alone or two or more strategies can be combined to increase tryptophan production. Intervention points are in bold, italics and underlined.
  • 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).
  • 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.
  • TrpE e.g. TrpEfbr
  • TrpD e.g. TrpEfbr
  • TrpD e.g. TrpEfbr
  • TrpD e.g. TrpEfbr
  • TrpD e.g. TrpEfbr
  • TrpD e.g. TrpD
  • TrpC Trp A
  • TrpB e.g. TrpA
  • TrpA e.g. TrpAfbr
  • 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. 24B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.
  • FIG. 25A, Fig. 25B, and Fig. 25C depict bar graphs showing tryptophan production by various engineered bacterial strains.
  • FIG.25A depicts a bar graph showing tryptophan production by various tryptophan producing strains.
  • the data show expressing a feedback resistant form of AroG (AroG fbr ) is necessary to get tryptophan production.
  • FIG. 25B 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).
  • PTS Phosphotransferase system
  • FIG. 25C 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. 26 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.
  • SYN2126 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. 27 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-acrylate reductase (e.g., from Clostridum botulinum).
  • WAL Tryptophan ammonia lyase
  • indole- 3-acrylate reductase e.g., from Clostridum botulinum
  • Tryptophan ammonia lyase converts tryptophan to indole- 3 -acrylic acid
  • 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 FIGS. 20 (A-D) and FIG. 24 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. 28 depicts a schematic of indole-3-propionic acid (IPA), 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.
  • FldD indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes
  • Acul acrylyl-CoA reductase, e.g., from Clostridium sporogenes
  • Rhodobacter sphaeroides Rhodobacter sphaeroides.
  • lpdC Indole-3-pyruvate decarboxylase, e.g. , from Enterobacter cloacae;
  • ladl Indole- 3 -acetaldehyde dehydrogenase, e.g., from Ustilago maydis;
  • Tdc 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), NH3, NAD(P)H and H+.
  • Indole- 3 -lactate dehydrogenase (EC 1.1.1.110, e.g. , Clostridium sporogenes or Lactobacillus casei) converts (indol-3yl)pyruvate (IpyA) and NADH and H+ to indole- 3 -lactate (ILA) and NAD+.
  • Indole-3-propionyl-CoA:indole-3- lactate CoA transferase converts indole- 3 -lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IP A) and indole-3-lactate-CoA.
  • Indole-3-acrylyl-CoA reductase (FldD ) and acrylyl-CoA reductase (Acul) convert indole-3-acrylyl-CoA to indole-3- propionyl-CoA.
  • Indole- 3 -lactate dehydratase 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 FIGS. 20 (A-D) and FIG. 24 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. 29 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
  • 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. 30 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-Ptet- trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr (pl5A).
  • SYN2340 comprises AtrpRAtnaA, tetR- Ptet-trpEfbrDCBA (pSClOl), tetR-Ptet-aroGfbr-tdcCr (pl5A).
  • SYN2794 comprises
  • Fig. 31A and Fig. 31B depict bar graphs showing butyrate production of butyrate producing strains of the disclosure.
  • Fig. 31A 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. 31B 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. 32 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
  • preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.
  • Fig. 33A depicts a schematic of a butyrate producing circuit under the control of an FNR promoter.
  • Fig. 33B 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. Cells were washed and resuspended in minimal media w/ 0.5% glucose and incubated microaerobically to monitor butyrate production over time.
  • SYN-UCD501 led to significant butyrate production under anaerobic conditions.
  • Fig. 33C depicts SYN-UCD501 in the presence and absence of glucose and oxygen in vitro.
  • SYN-UCD501 comprises pSClOl PydfZ-ter butyrate plasmid;
  • SYN- UCD500 comprises pSClOl PydfZ-bcd butyrate plasmid;
  • SYN-UCD506 comprises pSClOl nirB-bcd butyrate plasmid.
  • Fig. 33D depicts levels of mouse lipocalin 2 and calprotectin quantified by ELISA using the fecal samples in an in vivo model of HE.
  • SYN-UCD501 reduces inflammation and/or protects gut barrier function as compared to wild type Nissle control.
  • Fig. 34A and Fig. 34B depicts bar graphs showing in vitro arginine (Fig. 34A) and butyrate (Fig. 34B) production for (1) butyrate producing strain; (2) arginine producing strain (ammonia consuming strain), and (3) strain that produces butyrate and also consumes ammonia.
  • SYN-UCD501 butyrate producing strain comprising Logic 156 (pSClOl PydfZ-ter butyrate plasmid; amp resistance)
  • SYN-UCD305 arginine producing/ammonia consuming strain comprising AArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, and AThyA, with no antibiotic resistance
  • SYN- UCD601 butyrate producing and arginine producing/ammonia consuming strain comprising AArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, AThyA, and Logicl56 (pSClOl PydfZ-ter butyrate plasmid; amp resistance)).
  • the data show that SYN- UCD601 is able to produce similar levels of arginine as SYN-UCD305 and similar levels of butyrate as SYN-UCD501 in vitro.
  • Fig. 35 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. 36 depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB -butyrate construct (SYN-UCD507) or the pLogic046- nsrR-norB -butyrate construct (SYN-UCD508), which produce more butyrate as compared to wild-type Nissle.
  • Fig. 37 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. 38A 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.38B and Fig. 38C depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYNIOOI, on alkaline phosphatase activity in HT-29 cells represented in bar (Fig. 38B) and nonlinear fit (Fig. 38C) graphical formats.
  • Fig. 39 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. 40A and 40B depicts a propionate production strategy of the disclosure.
  • Fig. 40A depicts a schematic of a construct comprising the sleeping beauty mutase operon from E. coli under the control of a heterologous FnrS promoter.
  • Fig. 40B depicts a bar graph of propionate 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. 40A.
  • Fig. 41A- 41F depict graphs comparing mice fed a choline deficient, L-amino acid defined, high- fat diet (CDAHFD) for nine days to mice on normal chow with respect to weight and various markers of hepatic inflammation and fibrosis.
  • Fig. 41A depicts a graph showing changes in body weight over the 9 day time course in CDAHFD fed mice and mice fed a normal chow.
  • Fig. 41B depicts a bar graph showing serum MCP-1 levels in day nine in CDAHFD fed mice and mice fed a normal chow, as determined by ELISA.
  • Fig. 41C-41F depict fold changes in expression of CollAl (Collagen Type I Alpha 1 ; Fig.
  • Col3Al Collagen Type III Alpha 1 ; Fig. 41D
  • CoWAl Collagen Type IV Alpha 1 ; Fig. 41E
  • ACTA2 Actin, Alpha 2, Smooth Muscle, Aorta; Fig. 41F
  • Fig. 42 depicts a map of integration sites within the E. coli Nissle
  • chromosome chromosomes. 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. 43 depicts three bacterial strains which constitutively express red fluorescent protein (RFP).
  • RFP red fluorescent protein
  • strains 1-3 the rfp gene was inserted into different sites in the bacterial chromosome, and resulted in varying degrees of brightness under fluorescent light.
  • Unmodified E. coli Nissle strain 4 is non-fluorescent.
  • Fig. 44 depicts an exemplary schematic of the E. coli 1917 Nissle
  • chromosome comprising multiple mechanisms of action (MoAs).
  • Figs. 45A, 45B, 45C, 45D, 45E, 45F, 45G, 45H, and 451 depict schematics of bacterial chromosomes, for example the E. coli Nissle 1917 Chromosome.
  • Fig. 45A, 45B, and 45C each depict schematics of an engineered bacterium comprising one circuit, a circuit for butyrate production (Fig. 45A), a circuit for propionate production (Fig. 45B), and a circuit for GLP-1 expression (Fig. 45C).
  • Fig. 45A, 45A a circuit for butyrate production
  • Fig. 45B a circuit for propionate production
  • Fig. 45C a circuit for GLP-1 expression
  • 45D, 45E, 4545F and 45G each depict a schematic of an engineered bacterium comprising two circuits, a circuit for BSH expression and a circuit for expression of a bile salt importer (Fig.45D), a circuit for butryate production and a circuit for GLP-1 expression (Fig.45E), a circuit for butryate production and a circuit for propionate production (Fig.45F), a circuit for propionate production and a circuit for GLP-1 expression (Fig.45G).
  • Fig. 45H depicts a schematic of an engineered bacterium comprising three circuits, including a circuit for butyrate production, propionate production, and GLP-1 expression.
  • Fig. 451 depicts a schematic of an engineered bacterium comprising four circuits, including a circuit for butyrate production, GLP-1 expression, BSH expression, and expression of a bile salt importer.
  • Fig. 46 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 Tables 52-56 (Pfnrl-5).
  • FNR-responsive promoters selected from the exemplary FNR promoters shown in Tables 52-56 (Pfnrl-5).
  • Different 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 (-O2). Samples were removed at 4 hrs and the promoter activity based on ⁇ -galactosidase levels was analyzed by performing standard ⁇ -galactosidase colorimetric assays.
  • Fig. 47 A 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. 47B depicts a bar graph of 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. Values for standard ⁇ -galactosidase colorimetric assays are expressed in Miller units (Miller, 1972).
  • Fig. 47C depicts a line graph of the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
  • 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.
  • it is desirable to pre-load a strain with active payload(s) prior to administration This can be done by pre-inducing the expression of these enzymes as the strains are propagated, ⁇ e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration.
  • strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more proteins of interest.
  • FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ, The 02 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar
  • 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
  • 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 48B 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 FnrS24Y 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. 48C 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. 49 depicts the gene organization of an exemplary construct, comprising a cloned protein of interest (POI) gene under the control of a Tet promoter sequence and a Tet repressor gene.
  • POI protein of interest
  • FIG. 50 depicts the gene organization of an exemplary construct comprising Lacl in reverse orientation, and a IPTG inducible promoter driving the expression of a protein of interest (POI, e.g., one or more metabolic effector(s) described herein).
  • POI protein of interest
  • this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG.
  • inducer e.g., IPTG.
  • this construct is used alone.
  • the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs.
  • the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.
  • FIG. 51 depicts a construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.
  • FIG. 52A, FIG. 52B, and FIG. 52C depict schematics of non- limiting examples of constructs expressing a protein of interest (POI).
  • FIG. 52A 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.
  • the construct comprises SEQ ID NO: 101. In some embodiments, this construct is used alone.
  • the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs.
  • the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration.
  • the construct provides in vivo activity.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid.
  • the construct is located on a plasmid component of a biosafety system.
  • the construct is integrated into the bacterial chromosome at one or more locations.
  • the construct is used in combination with other POI constructs, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • a temperature sensitive system can be used to set up a conditional auxotrophy.
  • a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).
  • FIG. 52B depicts a schematic of a non- limiting example of the organization of a construct for POI expression under the control of a rhamnose inducible promoter.
  • a rhamnose inducible promoter For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some
  • the construct allows pre-induction and pre-loading of one or more POIs prior to in vivo administration.
  • the construct is useful for pre-induction and is combined with low-oxygen inducible constructs.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid.
  • the construct is located on a plasmid component of a biosafety system.
  • the construct is integrated into the bacterial chromosome at one or more locations.
  • FIG. 52C depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control of an arabinose inducible promoter.
  • the arabinose inducible POI construct comprises AraC (in reverse orientation), a region comprising an Arabinose inducible promoter, and the POI gene. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of one or more POI(s) prior to in vivo administration.
  • the construct is useful for pre-induction and is combined with low- oxygen inducible constructs.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid.
  • the construct is located on a plasmid component of a biosafety system.
  • the construct is integrated into the bacterial chromosome at one or more locations.
  • FIG. 53A 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. 53B depicts a line 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 dap A 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 (bio safety switch).
  • Fig. 54A, 54B, and 54C depict ATC (Fig. 54A) or nitric oxide-inducible (Fig. 54B) reporter constructs. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible P nsr R-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units.
  • Fig. 54C depicts a schematic of the constructs.
  • Fig. 55 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. 56 depicts a schematic of another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal, e.g. , low-oxygen conditions.
  • an exogenous environmental signal e.g. , low-oxygen conditions.
  • 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 araB AD promoter, which induces expression of TetR (tet repressor) and an anti-toxin.
  • Fig. 56 also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araB AD promoter and the bacterial cell cannot survive.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araB AD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell.
  • Fig. 57A, 57B, and 57C depict schematics of non-limiting examples of the disclosure.
  • Fig. 57A depicts a schematic of 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 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 araB AD promoter, which induces expression of TetR, thus preventing expression of a toxin.
  • Fig. 57B depicts a schematic of a repression- based kill switch in which the AraC transcription factor is activated in the presence of arabinose and induces expression of TetR and an anti-toxin. TetR prevents the expression of the toxin.
  • Fig. 57C 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 In the presence of arabinose, the AraC
  • transcription factor undergoes a conformational change that allows it to bind to and activate the araB AD promoter, which induces expression of TetR (tet repressor) 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).
  • toxin which is under the control of a promoter having a TetR binding site.
  • arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell.
  • the araC gene is under the control of a constitutive promoter in this circuit.
  • Fig. 58 depicts a schematic of one non-limiting embodiment of the disclosure, where an exogenous environmental condition, e.g., low-oxygen conditions, 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. 59 depicts a schematic of another non-limiting embodiment of the disclosure, where an exogenous environmental condition, e.g., low-oxygen conditions, 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.
  • the toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
  • Fig. 60 depicts aschematic of one non-limiting embodiment of the disclosure, in which the genetically engineered bacteria produces equal amount of a Hok toxin and a short-lived Sok anti-toxin.
  • the cell loses the plasmid, the anti-toxin decays, and the cell dies.
  • the cell produces equal amounts of toxin and anti-toxin and is stable.
  • the center panel the cell loses the plasmid and anti-toxin begins to decay.
  • the anti-toxin decays completely, and the cell dies.
  • Fig. 61 depicts a schematic of another non-limiting embodiment of the disclosure, where an exogenous environmental condition, e.g., low-oxygen conditions, 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. 62 depicts a schematic of another non- limiting embodiment of the disclosure, where an exogenous environmental condition, e.g., low-oxygen conditions, 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.
  • an exogenous environmental condition e.g., low-oxygen conditions, 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 toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
  • Fig. 63 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. 64 depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N- terminal secretion signal, a linker and the beta-domain of an autotransporter.
  • the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence.
  • the beta-domain is recruited to the Bam complex where the beta- domain is folded and inserted into the outer membrane as a beta-barrel structure.
  • the therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence.
  • the therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker.
  • Fig. 65 depicts a schematic of a type I secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP- binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes.
  • HlyB an ATP- binding cassette transporter
  • HlyD a membrane fusion protein
  • TolC an outer membrane protein
  • Fig. 66 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. 67 depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen.
  • An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell.
  • An inducible promoter small arrow, bottom
  • a FNR-inducible promoter drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons).
  • Fig. 68 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.
  • Figs. 69A-69D depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (Fig. 69A and Fig. 69B), which also contains a chromosomal component (shown in Fig. 69C and Fig. 69D).
  • the bosafety plasmid system vector comprises Kid Toxin and R6K minimal ori, dapA (Fig. 69A) and thyA (Fig. 69B) 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 (POD) are expressed from an inducible or constitutive promoter.
  • Fig. 69C and Fig. 69D depict schematics of the gene organization of the chromosomal component of a biosafety system.
  • Fig. 69C 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. 69D 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. 69A)
  • the chromosomal constructs shown in Fig. 69C and Fig. 69D are knocked into the DapA locus.
  • the plasmid containing the functional ThyA is used (as shown in Fig. 69B)
  • 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. 70 depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days.
  • Fig. 71 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. 72A 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. 72B 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 gastrointestinal tract. 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. 73 depicts the prpR propionate-responsive inducible promoter.
  • the sequence for one propionate-responsive promoter is also disclosed herein as SEQ ID NO: 584.
  • Fig. 74 depicts a schematic of a wild-type clbA construct and a clbA knock-out construct.
  • Figs. 75A-75E depict a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.
  • Fig. 75A depicts the parameters for starter culture 1 (SCI): loop full - glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm.
  • Fig. 75B 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
  • 75C depicts the parameters for the production bioreactor: inoculum - SC2, temperature 37° C, pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours.
  • Fig. 75D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS.
  • Fig. 75E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
  • Fig. 76 depicts a simple, robust, and rapid platform for generating and characterizing synthetic biotics, comprising steps 1 through 10.
  • Step 1 comprises designing one orm more disease pathway(s);
  • Step 2 comprises identifying one or more target metabolite(s);
  • Step 3 comprises designing on e or more gene circuit(s);
  • Step 4 comprises building the synthetic biotic;
  • Step 5 comprises activating the one or more circuit(s) in vitro;
  • Step 6 comprises characterizing circuit activation kinetics;
  • Step 7 comprises optimizing in vitro productivity to the disease threshold;
  • Step 8 comprises testing the optimized circuit(s) in animal disease model(s);
  • Step 9 comprises assimilating into the microbiome;
  • Step 10 comprises developing understanding of the in vivo PK and dosing regimen.
  • FIG. 77A, FIG. 77B, and FIG. 77C depict schematics of the gene
  • a therapeutic polypeptide of interest is assembled behind a fliC-5'UTR, and is driven by the native fliC and/or fliD promoter (FIG. 77 A and FIG. 77B) or a tet-inducible promoter (FIG. 77C).
  • an inducible promoter such as oxygen level-dependent promoters (e.g., FNR- inducible promoter), and promoters induced by a metabolite that may or may not be naturally present (e.g. , can be exogenously added) in the gut, e.g. , arabinose can be used.
  • the one or more cassettes are under the control of constitutive promoters.
  • 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. 77B and FIG. 77C.
  • FIG. 78A and FIG. 78B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., metabolic and/or satiety effector and/or immune modulator polypeptides described herein, 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. 78A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR- inducible promoter, FIG. 78B), 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.
  • the one or more cassettes are under the control of constitutive promoters.
  • Fig. 79 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, 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. 80 depicts a schematic of a construct comprising GLP-1 (1-37) under the control of the FliC promoter and 5'UTR containing the N-terminal flagellar secretion signal for secretion.
  • FIG. 81A, FIG. 81B, FIG. 81C, and FIG. 81D depict schematics of the organization of exemplary GLP-1 secretion constructs with phoA (FIG. 81A and FIG. 81B) or OmpA (FIG. 81C and FIG. 81D) secretion tags.
  • Three different RBS binding sites, 20K (FIG. 81A and FIG. 81C), 100K (FIG. 81B), and 67K (FIG. 81D) with varying strength (20 ⁇ 67 ⁇ 100) are used.
  • the Tet inducible promoter and the TetR sequence is replaced by a different inducible promoter system or a constitutive promoter in these constructs.
  • the background of the strain which contains these constructs and from which GLP-1 is secreted comprises a deletion or mutation in 1pp.
  • FIG. 81A depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -PhoA-Glpl.
  • FIG. 81B depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-lOOK RBS -PhoA-Glpl.
  • FIG. 81C depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and comprises TetR-pTet-20K RBS -OmpF-Glpl.
  • FIG. 81D depicts a schematic of a GLP-1 secretion construct which is expressed by the genetically engineered bacteria and
  • FIG. 82A and FIG. 82B depict schematics of the genetically engineered strains SYN2627 (comprising TetR-pTet-20K RBS -PhoA-Glpl) and SYN2643 (comprising TetR-pTet-20K RBS -PhoA-Glpl). Both strains comprise a deletion or mutation in 1pp.
  • FIG. 82C depicts a bar graph showing the intracellular and secreted levels of GLP-1 as detected by ELISA assay for strains SYN2627 and SYN2643.
  • FIG. 83A and FIG. 83B depict line graphs of ELISA results.
  • FIG. 83A depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum-starved Colo205 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. 83B 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
  • FIG. 83C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation.
  • FIG. 84A and FIG. 84B depict line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG. 84A) and in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 84B).
  • 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. 84C and FIG. 84D depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 84C) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 84D). Deletions in endogenous adhE
  • 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. 84E 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
  • FIG. 84F 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 HAl/2 locus and a deletion in the endogenous ldhA gene.
  • FIG. 84G and FIG. 84H depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFND secretion in primary human PBMC cells from donor 1 (Dl) (FIG. 84G ) and donor 2 (D2) (FIG. 84H).
  • FIG. 85 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.
  • TCA cycle citric acid cycle
  • 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. 86A and FIG. 86B depict bar graphs showing Acetate/Butyrate production in 0.5% glucose MOPS (pH6.8) (FIG. 86A) and Acetate/Butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 86B).
  • 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.
  • FIG. 87 depicts a schematic of an exemplary propionate biosynthesis gene cassette.
  • FIG. 88 depicts exemplary circuit designs for the recombinant bacteria of the disclosure.
  • two bile salt hydrolase (BSH) genes from Lactobacillus salivarius (BSH1 and BSH2) are under the control of an aTc-inducible promoter in a single operon.
  • two bile salt hydrolase (BSH) genes (BSH1 and BSH2) are each under the control of an aTc-inducible promote for individual expression.
  • the BSH1 and BSH2 genes encode the same bile salt hydrolase enzyme.
  • the BSH1 and BSH2 genes encode different bile salt hydrolase enzymes.
  • non-alcoholic fatty liver disease includes a wide spectrum of liver abnormalities which range from simple steatosis to non-alcoholic steatohepatitis (NASH).
  • NASH is a severe form of NAFLD, where excess fat accumulation in the liver results in chronic inflammation and damage.
  • Nonalcoholic fatty liver disease is a component of metabolic syndrome and a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH).
  • Simple liver steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH, which tends to progress to cirrhosis in up to 20% of patients and can subsequently lead to liver failure or hepatocellular carcinoma.
  • NASH affects approximately 3-5% of the population in America, especially in those identified as obese. NASH is characterized by such abnormalities as advanced lipotoxic metabolites, pro-inflammatory substrate, fibrosis (e.g. , in which collagen deposition is manifested in a particular perivenular and/or pericellular pattern) , and increased hepatic lipid deposition. If left untreated, NASH can lead to cirrhosis, liver failure, and hepatocellular carcinoma.
  • NASH neurodegenerative disease
  • cytokine imbalance specifically, an increase in the tumor necrosis factor-alpha (TNF-a)/adiponectin ratio
  • oxidative stress resulting from mitochondrial abnormalities include insulin resistance, cytokine imbalance (specifically, an increase in the tumor necrosis factor-alpha (TNF-a)/adiponectin ratio), and oxidative stress resulting from mitochondrial abnormalities.
  • liver biopsy is needed for the accurate assessment of the graduation of steatosis, necroinflammatory changes, and fibrosis and allows NASH and steatosis to be distinguished (Sanches et al., Nonalcoholic Steatohepatitis: A Search for Factual Animal Models BioMed Research International Volume 2015).
  • At least two grading systems for NASH have been developed, which take into consideration the severity of hepatic steatosis, portal and lobular inflammation, and collagen deposition.
  • hepatic steatosis is scored as follows: Grade 0 (minimal or no steatosis ( ⁇ 5% of hepatocytes affected); grade 1 (mild steatosis (5 to 32% of hepatocytes affected)); grade 2 (moderate to severe steatosis (33 to 66% of hepatocytes affected)); grade 3 (severe steatosis (>66% of hepatocytes affected)).
  • the portal and lobular inflammation is also scored as follows: grade 0 (minimal or no inflammation) ;grade 1 (mild); grade 2 (moderate to severe); grade 3 (severe).
  • the collagen deposition is scored as follows: Grade 0 (minimal or no evidence of fibrosis); grade 1 (mild fibrosis); grade 2 (moderate to severe fibrosis); grade 3 (severe fibrosis).
  • the SAF system is a second way of grading NASH, which also consistes of a semiquantitative score of steatosis (S), inflammatory activity (A), and fibrosis (F) Bedossa et al.,
  • genetically engineered bacteria may be useful in some embodiments to improve the grade of hepatic steatosis, portal and lobular inflammation, and collagen deposition.
  • one strategy in the treatment, prevention, and/or management of NASH may include approaches to help promote the feeling of satiety in the patient, e.g. through the administration of a satiety effector, examples of which can be found infra.
  • Another strategy in the treatment, prevention, and/or management of NASH may include approaches to reduce liver triglyceride content, e.g., by increasing fatty acid oxidation, decreasing lipogenesis, and improving hepatic glucose metabolism.
  • glucagon-like peptide 1 an incretin secreted by L-cells in the small intestine in response to food intake
  • GLP- 1 analogs have been used to stimulate insulin secretion in the treatment of type-two diabetes and non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • LPS LPS in the liver, mitochondrial dysfunction, oxidative stress, and/or or proinflammatory cytokines (TNF-alpha, interleukins, e.g. , IL- 1JL- 6, and IL-8), more recently, a concept of multiple hits has been developed. Moreover, inflammation can occasionally precede steatosis and patients with NASH can present without much steatosis, suggesting that inflammation can occur first, indicating that many of these hits can occur in different orders or in parallel at the same time. Contributing factors to these hits include intestinal dysbiosis, dietary factors, changes to intestinal permeability, as well as endoplasmic reticulum stress and activation of additional signalling pathways.
  • PNPLA3 patatin-like phospholipase 3
  • Tight junction proteins such as zonula occludens, normally seal the junction between intestinal endothelial cells at their apical aspect and thus have a vital role in preventing translocation of harmful substances from the gut into the portal system.
  • these tight junctions are disrupted, increasing mucosal permeability and exposing both the gut mucosal cells and the liver to potentially pro-inflammatory bacterial products.
  • Translocated microbial products might contribute to the pathogenesis of fatty liver disease by several mechanisms, including stimulating pro-inflammatory and profibrotic pathways via a range of cytokines.
  • one strategy in the treatment, prevention, and/or management of NASH may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti- inflammatory effectors.
  • leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites.
  • butyrate and other SCFA e.g., derived from the microbiota
  • SCFA e.g., derived from the microbiota
  • 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- ⁇ .
  • 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.
  • Colonic propionate delivery has also been shown to reduce intrahepatocellular lipid content in NASH patients, including improvements in weight gain and intra-abdominal fat deposition (see, for example, Chambers et al, Gut, gutjnl-2014), and GLP-1 administration has been shown to reduce the degree of lipotoxic metabolites and pro -inflammatory substrates, both of which have been shown to speed NASH development, as well as reduce hepatic lipid deposition (see, for example, Bernsmeier et al., PLoS One, 9(l):e87488, 2014 and Armstrong et al., J. Hepatol., 2015).
  • trptophan metabolites including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehhyde, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown ot 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.
  • 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.
  • 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
  • the genetically engineered bacteria are useful for the prevention, treatment, and/or management of NAFLD and/or NASH.
  • the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety.
  • the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g. , butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP- 1.
  • the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g. , butyrate and/or propionate for the treatment of NAFLD and/or NASH.
  • the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g. , in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g. , in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Table 12 and elsewhere herein, in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in FIG. 17.
  • the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 12 and elsewhere herein, in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in FIG. 16.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 12, and elsewhere herein, in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g. , in the serum and/or in the gut.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g. , in FIG. 16.
  • the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 12 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 12 and elsewhere herein.
  • the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios.
  • the genetically engineered bacteria comprise a gene cassette which comprises a heterologous gene encoding a bile salt hydrolase (BSH) enzyme and is capable of processing and reducing levels of bile salts in low-oxygen environments, e.g., the gut.
  • BSH bile salt hydrolase
  • compositions comprising the genes, gene cassettes disclosed herein may be used to convert excess bile salts into non-toxic molecules in order to treat and/or prevent disorders associated with bile salts, such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection.
  • disorders associated with bile salts such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection.
  • one or more of these circuits may be combined for the treatment of NASH and/or NAFLD.
  • butyrate producing, GLP-1 secreting , and tryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of NASH and/or NAFLD.
  • the present disclosure provides engineered bacterial cells, pharmaceutical compositions thereof, and methods of modulating and treating nonalcoholic steatohepatitis (NASH).
  • the engineered bacteria disclosed herein have been constructed to comprise genetic circuits composed of, for example, one or more butyrate cassette(s), one or more propionate cassette(s), and/or one or more GLP-1 nucleic acid sequence(s), to treat the disease, as well as other optional circuitry to ensure the safety and non-colonization of the subject that is administered the engineered bacteria, such as auxotrophies, kill switches, etc.
  • These engineered bacteria are safe and well tolerated and augment the innate activities of the subject's microbiome to achieve a therapeutic effect.
  • a bacterial cell disclosed herein has been genetically engineered to comprise one or more bio synthetic circuits selected from a propionate gene cassette; a butyrate gene cassette; a GLP-1 gene, and combinations thereof, and is capable of producing propionate, butyrate, and/or GLP-1 in low-oxygen or anaerobic environments, e.g., the gut.
  • the genetically engineered bacterial cells and pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to produce propionate, butyrate, and/or GLP-1, in order to treat and/or prevent liver disease, such as nonalcoholic
  • NASH steatohepatitis
  • engineered bacterial cell refers to a bacterial cell or bacteria that have been genetically modified from their native state.
  • an engineered 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.
  • Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids.
  • engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.
  • a "programmed bacterial cell” or “programmed engineered bacterial cell” is an engineered bacterial cell 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.
  • 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.
  • a 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.
  • “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 a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell.
  • a heterologous gene may also include a native gene, or fragment thereof, introduced into a non- native host cell.
  • a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
  • the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.
  • 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 0 2) ).
  • 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 mmHg 0 2 (0-60 torr 0 2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg 0 2 ), including any and all incremental fraction(s) thereof (e.g.
  • low oxygen refers to about 60 mmHg 0 2 or less (e.g.
  • low oxygen may also refer to a range of 0 2 levels, amounts, or concentrations between 0-60 rnmHg 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., Gastroenterology, 147(5): 1055- 1063 (2014); Bergofsky et al., J Clin.
  • the term "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (0 2 ) found in a mammalian organ or tissue other than the gut, e.g. , urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g. , at a hypoxic or anoxic level.
  • "low oxygen” is meant to refer to the level, amount, or concentration of oxygen (0 2 ) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions.
  • Table A summarizes the amount of oxygen present in various organs and tissues.
  • DO amount of dissolved oxygen
  • the term "low oxygen” is meant to refer to a level, amount, or concentration of oxygen (0 2 ) that is about 6.0 mg/L DO or less, e.g. , 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g.
  • the level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (0 2 ) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium).
  • Well-aerated solutions e.g. , solutions subjected to mixing and/or stirring
  • the term "low oxygen” is meant to refer to 40% air saturation or less, e.g. , 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof ⁇ e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.
  • 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.
  • 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 ant i- 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. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.
  • a "gene cassette” or “operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce a molecule, e.g., propionate or butyrate.
  • the gene cassette or operon may also comprise additional transcription and translation elements, e.g., a ribosome binding site.
  • butyrate gene cassette and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway.
  • Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema.
  • the genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • a butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate 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.
  • butyrate biosynthesis gene refers to a gene present in a butyrate gene cassette and which performs an enzymatic function in the production of butyrate in a butyrate bio synthetic pathway.
  • a "propionate gene cassette” or "propionate operon” refers to a set of genes capable of producing propionate in a bio synthetic pathway.
  • Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola.
  • the genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and
  • Dehydration of (K)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC).
  • Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC).
  • EtfA acrolyl-CoA reductase
  • the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC are replaced by the acul gene from R. sphaeroides.
  • This gene product catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides; Asao 2013).
  • the propionate cassette comprises pet, IcdA, IcdB, IcdC, and acul.
  • the homo log of Acul in E coli, YhdH is used (see. e.g., Structure of Escherichia coli YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004).
  • This the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH.
  • the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide
  • 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).
  • 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 ah, 1994).
  • Several bacteria such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or C0 2 + H 2 into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012).
  • the acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate.
  • One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • Each gene or gene cassette may be present on a plasmid or bacterial chromosome.
  • multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein.
  • the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions.
  • 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).
  • Treating the diseases described herein may encompass increasing levels of propionate, increasing levels of butyrate, and increasing GLP- 1, and/or modulating levels of tryptophan and/or its metabolites (e.g. , kynurenine), and does not necessarily encompass the elimination of the underlying disease.
  • tryptophan and/or its metabolites e.g. , kynurenine
  • 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.
  • 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 antiinflammatory or gut barrier enhancer molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the anti- inflammatory or gut barrier enhancer molecule. In other words, the regulatory sequence acts in cis.
  • a gene may be "directly linked" to a regulatory sequence in a manner which allows expression of the gene.
  • a gene may be
  • 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, BBa_J23100, a constitutive Escherichia coli ⁇ 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.
  • a constitutive Escherichia coli ⁇ promoter e.g., an os
  • coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110
  • BBa_M13110 Bacillus subtilis ⁇ ⁇ promoter
  • promoter veg a constitutive Bacillus subtilis ⁇ ⁇ promoter
  • BBa_K823002 P veg (BBa_K823003)
  • a constitutive Bacillus subtilis ⁇ promoter e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)
  • a Salmonella promoter e.g., Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)
  • a bacteriophage T7 promoter e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_Kl 13010; BBa_Kl 13011 ; BBa_Kl 13012; BBa_R0085; BBa_R0180; BBa_R0181 ; BBa_R0182; BBa_R0183; BBa_Z0251; BB
  • 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 or gene cassette(s), that is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the no n- native genetic material is retained, expressed, and propagated.
  • non-native genetic material e.g., a gene or gene cassette(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 or gene cassette, in which the plasmid or chromosome carrying the gene or gene cassette is stably maintained in the bacterium, such that the gene or gene cassette can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.
  • copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.
  • the term "expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.
  • plasmid or "vector” refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell's genome.
  • Plasmids are usually circular and capable of autonomous replication. Plasmids may be low- copy, medium-copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell.
  • a plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding an ant i- inflammatory or gut barrier enhancer molecule.
  • a plasmid may comprise a nucleic acid sequence encoding a heterologous gene or gene cassette.
  • 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, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not.
  • Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising a gene or gene cassette 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.
  • 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.
  • the term "genetic mutation" is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene.
  • a genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene's polypeptide product.
  • a genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.
  • the term "transporter” is meant to refer to a mechanism, e.g., protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di- peptide, tri-peptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu.
  • 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 endogenous to a mammalian body, but external or exogenous to an intact microorganism cell.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the small intestine of a mammal.
  • the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut.
  • exogenous 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. In some embodiments, the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.
  • An "oxygen level-dependent promoter" or “oxygen level-dependent regulatory region” refers to a nucleic acid sequence to which one or more oxygen level- sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.
  • 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
  • FNR transcriptional regulator
  • a "tunable regulatory region” refers to a nucleic acid sequence under direct or indirect control of a transcription factor and which is capable of activating, repressing, derepressing, or otherwise controlling gene expression relative to levels of an inducer.
  • the tunable regulatory region comprises a promoter sequence.
  • the inducer may be RNS, or other inducer described herein, and the tunable regulatory region may be a RNS -responsive regulatory region or other responsive regulatory region described herein.
  • the tunable regulatory region may be operatively linked to a gene sequence(s) or gene cassette for the production of one or more payloads, e.g., a butyrogenic or other gene cassette or gene sequence(s).
  • the tunable regulatory region is a RNS-derepressible regulatory region, and when RNS is present, a RNS-sensing transcription factor no longer binds to and/or represses the regulatory region, thereby permitting expression of the operatively linked gene or gene cassette.
  • the tunable regulatory region derepresses gene or gene cassette expression relative to RNS levels.
  • Each gene or gene cassette may be operatively linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one RNS.
  • the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS).
  • exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut.
  • the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules.
  • the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter.
  • the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal.
  • the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example,
  • the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising an engineered 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
  • the promoter for example, an aerobic environment outside the gut.
  • Geck 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.
  • Microorganism refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, and protozoa.
  • the microorganism is engineered ("engineered microorganism") to produce one or more therapeutic molecules.
  • the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut.
  • the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment.
  • the engineered microorganism is an engineered bacterium.
  • the engineered microorganism is an engineered virus.
  • Non-pathogenic bacteria refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus,
  • Escherichia coli Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii,
  • Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
  • Probiotic is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism.
  • the host organism is a mammal.
  • the host organism is a human.
  • Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria.
  • probiotic bacteria examples include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent 6,835,376).
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
  • Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Nonpathogenic 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 disease, as well as those at risk of having, or who may ultimately acquire the disease.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease.
  • Liver disease e.g., nonalcoholic steatohepatitis (NASH)
  • NASH nonalcoholic steatohepatitis
  • Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection.
  • Treating nonalcoholic steatohepatitis may encompass increasing levels of propionate, increasing levels of butyrate, and increasing GLP- 1, and does not necessarily encompass the elimination of the underlying disease.
  • a "pharmaceutical composition” refers to a preparation of bacterial cells with other components such as a physiologically suitable carrier and/or excipient.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound.
  • An adjuvant is included under these phrases.
  • 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 disease, e.g., nonalcoholic
  • 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 NASH.
  • NASH nonalcoholic steatohepatitis
  • 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,
  • bactericidal refers to a molecule or protein which is capable of killing the engineered 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 engineered bacterial cell of the disclosure, or which is capable of killing the engineered bacterial cell of the disclosure.
  • the term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins.
  • the term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g. , microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases.
  • anti-toxin refers to a protein or enzyme which is capable of inhibiting the activity of a toxin.
  • antitoxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.
  • 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 bio synthetic 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.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • Recombinantly produced polypeptides and proteins expressed in host cells including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e.
  • fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments.
  • Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non- conservative amino acid substitutions, deletions or additions.
  • Polypeptides also include fusion proteins.
  • the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide.
  • the term “fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion
  • 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
  • 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.
  • n antibody generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond.
  • the recognized immunoglobulin genes include the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either ⁇ or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.
  • antibody or “antibodies” is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities.
  • antibody or “antibodies” is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies.
  • Antibodies can have more than one binding specificity, e.g., be bispecific.
  • antibody is also meant to include so-called antibody mimetics.
  • Antibody mimetics refers to small molecules, e.g., 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure.
  • Antibody mimetics include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from Sulfolobus acidocaldarius), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3 domain of Fyn), Kunitz domain peptides Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies.
  • antibody or “antibodies” is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional antibodies can for example be found in Scott et al., Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety.
  • a “single-chain antibody” or “single-chain antibodies” typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or bond, such as a disulfide bond.
  • the single-chain antibody lacks the constant Fc region found in traditional antibodies.
  • the single-chain antibody is a naturally occurring single-chain antibody, e.g., a camelid antibody.
  • the single-chain antibody is a synthetic, engineered, or modified single-chain antibody.
  • the single-chain antibody is capable of retaining
  • 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.
  • Vh and VL sequences of the scFv can be connected via the N-terminus of the VH connecting to the C-terminus of the VL or via the C- terminus of the VH connecting to the N-terminus of the VL.
  • ScFv fragments are independent folding entities that can be fused indistinctively on either end to other epitope tags or protein domains.
  • Linkers of varying length can be used to link the Vh and VL sequences, which the linkers can be glycine rich (provides flexibility) and serine or threonine rich (increases solubility).
  • Short linkers may prevent association of the two domains and can result in multimers (diabodies, tribodies, etc.). 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. Additional non- limiting examples of linkers, including other flexible linkers are described in Chen et al., 2013 (Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and
  • 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.
  • 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.
  • 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.
  • Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14 (10): 815-823 (describes optimized linker sequences for the expression of monomeric and dimeric bispecific 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 biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm.
  • the secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g.,HlyBD.
  • Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., 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.
  • 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 antinflammatory or barrier enhancer molecule(s) into 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. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated.
  • periplasmic proteases such as degS, degP or nlpl
  • 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.
  • 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, to IB, pal, degS, degP, and nlpl genes.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the disclosure provides a bacterial cell that comprises a heterologous propionate gene cassette; a heterologous butyrate gene cassette; a heterologous GLP-1 gene; a heterologous propionate gene cassette and a heterologous butyrate gene cassette; a heterologous propionate gene cassette and a heterologous GLP-1 gene; a heterologous butyrate gene cassette and a heterologous GLP-1 gene; or a heterologous propionate gene cassette, a heterologous butyrate gene cassette, and a heterologous GLP-1 gene.
  • the bacterial cell may contain more than one copy of the respective gene cassette(s) and/or gene(s).
  • the bacterial cell is a non-pathogenic bacterial cell.
  • the bacterial cell is a commensal bacterial cell.
  • the bacterial cell is a probiotic bacterial cell.
  • the bacterial cell is selected from the group consisting of a Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter formigenes bacterial cell.
  • a Bacteroides fragilis Bacteroides thetaiotaomicron
  • Bacteroides subtilis Bacteroides subtilis
  • Bifidobacterium animalis Bifidobacterium bifidum
  • Bifidobacterium infantis Bifidobacterium lactis
  • the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium animalis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one
  • the bacterial cell is a Bifidobacterium infantis bacterial cell.
  • the bacterial cell is a Bifidobacterium lactis bacterial cell. In one embodiment, the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is a Clostridium scindens bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Oxalobacter formigenes bacterial cell. In another embodiment, the bacterial cell does not include Oxalobacter formigenes.
  • the bacterial cell is a Gram positive bacterial cell. In another embodiment, the bacterial cell is a Gram negative bacterial cell.
  • the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram- negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et ah, 2007).
  • the strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et ah, 2014, emphasis added).
  • Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors ⁇ e.g., E. coli a-hemolysin, P- fimbrial adhesins) (Schultz, 2008), and E.
  • coli Nissle does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic (Sonnenborn et ah, 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. It is commonly accepted that E. coli Nissle' s therapeutic efficacy and safety have convincingly been proven (Ukena et ah, 2007). In a recent study in non-human primates, Nissle was well tolerated by female cynomolgus monkeys after 28 days of daily NG dose administration at doses up to 1 x 1012 CFU/animal. No Nissle related mortality occurred and no Nissle related effects were identified upon clinical observation, body weight, and clinical pathology assessment (see, e.g., PCT/US 16/34200).
  • the engineered bacterial cell does not colonize the subject having NASH.
  • genes from one or more different species can be introduced into one another, e.g., a gene from Lactobacillus plantarum or Methanobrevibacter smithii 3142 can be expressed in Escherichia coli.
  • the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is an engineered bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells.
  • the disclosure provides an engineered bacterial culture which comprises bacterial cells.
  • the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene or gene cassette(s) is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the genetically engineered bacteria is an auxotroph or a conditional auxotroph.
  • the genetically engineered bacteria is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil auxotroph.
  • the engineered bacteria have more than one auxo trophy, for example, they may be a AthyA and AdapA auxotroph.
  • the genetically engineered bacteria further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein.
  • the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene.
  • 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 an promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD-
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacteria is an auxotroph and further comprises a kill- switch circuit, such as any of the kill- switch circuits described herein.
  • the gene or gene cassette(s) are present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene or gene cassette(s) are present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the genetically engineered bacteria of the invention comprise a gene encoding a non-native metabolic and/or satiety effector molecule, or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector molecule.
  • the metabolic and/or satiety effector molecule is selected from the group consisting of n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP- 1, GLP-2, apo lipoprotein A- IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide
  • the genetically engineered bacteria of the invention comprise one or more gene(s) or gene cassette(s) which are capable of producing an effector, which can modulate the inflammatory status.
  • Non-limiting examples include short shain fatty acides, and tryptophan and its metabolites, as described herein.
  • the effect of the genetically engineered bacteria on the inflammatory status can be measured by methods known in the art, e.g., plasma can be drawn before and after administraton of the genetically engineered bacteria.
  • the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and plasma viscosity (PV) blood tests are commonly used to detect this increase n inflammation.
  • the genetically engineered bacteria modulate, e.g. decrease or increase, levels of inflammatory markers, eg. C-reactive protein (CRP).
  • the genetically engineered bacteria modulate, e.g. decrease, levels of inflammatory growth factors and cytokines, e.g. , IL- ⁇ , IL-6, and/or TNF- ⁇ and proinflammatory signaling, e.g. NF-kappaB signaling.
  • the genetically engineered bacteria modulate, e.g. increase, levels of anti- inflammatory growth factors and cytokines, e.g., IL4, IL- 10, IL- 13, IFN-alpha and/or transforming growth factor- beta.
  • the genetically engineered bacteria produce effectors, which bind to and stimulate the aromatic hydrocarbon receptor.
  • the genetically engineered bacteria stimulate AHR signaling in immune cell types, including T cells, B cells, NK cells, macrophages, and dendritic cells (DCs), and/or in epithelial cells.
  • the genetically engineered bacteria modulate, e.g., increase the levels of IL-22, e.g. , through stimulation of AHR.
  • the genetically engineered bacteria may reduce gut permeability. In some embodiments, the the genetically engineered bacteria may reduce the amounts of LPS and in the circulation, which are increase in metabolic disease, e.g., in NASH..
  • the genetically engineered bacteria may alter glucose metabolism, appetite, weight, and/or blood pressure in a mammal. In some embodiments, the genetically engineered bacteria may reduce liver triglyceride content. In some embodiments, the geneticially engineered bacteria may comprise one or more gene(s) and/or gene cassette(s), which, when expressed, produce metabolites or polypeptides which function to increase fatty acid oxidation, and/or decreasing lipogenesis and/or and improve hepatic glucose metabolism.
  • the geneticially engineered bacteria may comprise one or more gene(s) and/or gene cassette(s), which, when expressed, produce metabolites or polypeptides which function to stimulate glucose-dependent insulin secretion in pancreatic ⁇ - cells.
  • the geneticially engineered bacteria may comprise one or more gene(s) and/or gene cassette(s), which, when expressed, produce bile salt, which, when expressed, produce metabolites or polypeptides which function to convert excess bile salts into non-toxic molecules in order to treat and/or prevent disorders associated with bile salts, such as cardiovascular disease, metabolic disease, cirrhosis, cancer, liver disease, and C. difficile infection. .
  • the gene or gene cassette for producing the metabolic and/or satiety effector molecule and/or modulator of inflammation may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions, molecules, or metabolites specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • the gene or gene cassette for producing the metabolic and/or satiety effector and/or modulator of inflammation may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome.
  • expression from the plasmid may be useful for increasing expression of the metabolic and/or satiety effector molecule.
  • expression from the chromosome may be useful for increasing stability of expression of the metabolic and/or satiety effector molecule.
  • the gene or gene cassette for producing the metabolic and/or satiety effector molecule is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria.
  • one or more copies of the propionate biosynthesis gene cassette may be integrated into the bacterial chromosome.
  • the gene or gene cassette for producing the metabolic and/or satiety effector molecule is expressed from a plasmid in the genetically engineered bacteria.
  • the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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. 42).
  • the insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon.
  • the genetically engineered bacteria of the invention are capable of expressing a metabolic and/or satiety effector molecule that is encoded by a single gene, e.g., the molecule is GLP-1 and encoded by the GLP-1 gene.
  • genes and gene cassettes capable of producing metabolic and/or satiety effector molecules and/or modulator of inflammation are known in the art and may be expressed by the genetically engineered bacteria of the invention.
  • the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g., a ribosome binding site, to enhance expression of the therapeutic molecule.
  • the genetically engineered bacteria produce two or more metabolic and/or satiety effector molecules and/or modulator of inflammation.
  • the two or more molecules behave synergistically to ameliorate metabolic and/or liver disease, e.g., NASH and NAFLD.
  • the genetically engineered bacteria express at least one metabolic effector molecule and at least one satiety effector molecule and at least one modulator of inflammation.
  • Short-chain fatty acids primarily acetate, propionate, and butyrate
  • SCFAs Short-chain fatty acids
  • Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake.
  • rodent models of genetic or diet- induced obesity supplementation of butyrate in diet, and oral administration of acetate was shown to suppress weight gain independent of food intake suppression; Propionate was reported to inhibit food intake in humans (see, e.g.
  • SCFAs are likely efficacious in the treatment of metabolic syndrome and related disorders, and/or diabetes type2, and/or obesity.
  • SCFAs represent a major constituent of the luminal contents of the colon. Among SCFAs butyrate is believed to play an important role for epithelial homeostasis.
  • Acetate and propionate have ant i- inflammatory properties, which are comparable to those of butyrate (Tedelind et al., World J Gastroenterol. 2007 May 28; 13(20): 2826-2832.
  • strategy in the treatment, prevention, and/or management of NASH may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti- inflammatory effectors and /or gut barrier 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- ⁇ .
  • 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-i- innate lymphoid cells.
  • Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3- dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and
  • GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.
  • 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
  • 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 comprise 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. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli. In some
  • 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). Through 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 results in an increase in production of acetate for NAD recycling.
  • 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, thereby reducing or eliminating the activity of ldhA.
  • 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 thereby reducing or eliminating the activity of AdhE.
  • 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.
  • FrdA is one of two catalytic subunits in the four subunit fumarate reductase complex.
  • FrdB is the second catalytic subunit 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, thereby reducing or eliminating the activity of FrdA.
  • the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous FrdA gene. In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous FrdB, FrdC, and/or FrdD gene(s thereby reducing or eliminating the activity of FrdB, FrdC, and/or FrdD.
  • the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA and rdA genes.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA 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 ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprising one or more of these mutations also comprise a butyrate cassette.
  • 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 two-fold 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, sixfold, 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 produce lower amounts of acetate than the amounts produced by the wild type bacterium under the same conditions.
  • the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and ldhA 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, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA, 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 mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria further comprise one or more gene cassettes for the production of 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 two-fold 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, sixfold, 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 gene sequence encoding one or more polypeptides for the production of acetate is operably linked to an inducible promoter.
  • the inducible promoter is directly or indirectly induced by exogenous environmental conditions.
  • the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions.
  • the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut.
  • such molecules or metabolites are specific to certain conditions, e.g., conditions associated with NASH, such as hyperammonemia or liver damage.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hyperammonemia or liver damage, e.g., as seen in NASH.
  • the promoter is induced in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein.
  • the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture.
  • the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.
  • the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein.
  • the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
  • the gene sequence encoding one or more polypeptides and/or comprising one or more mutations or deletions in endogenous genes for the production of acetate is operably linked to a
  • the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, or under conditions present during hyperammonemia or as a consequence of liver damage or disease.
  • the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture.
  • the constitutive promoter is selected from a promoter provided in Table 27-37.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.
  • the gene sequence encoding one or more polypeptides for the production of acetate is operably linked to a RBS, enhancer or other regulatory sequence.
  • the RBS is selected from a promoter provided in Table 27-37 or is listed in Table 38-39.
  • the gene sequence encoding one or more polypeptides for the production of acetate is modified and/or mutated, e.g., to enhance stability, or increase acetate production.
  • the gene sequence encoding one or more polypeptides for the production of acetate may be codon optimized, e.g., to improve expression in the host microorganism.
  • the gene sequence encoding one or more polypeptides for the production of acetate are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences described herein for the consumption of ammonia.
  • Suitable gene sequences and circuits for the consumption of ammonia are described in Pending International Patent Application PCT/US 2015/64140 (published as WO/2017/090343) and Pending International Patent Application
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules known in the art or described herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production or catabolism of tryptophan and/or one or more of its metabolites described herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences for the secretion of IL-22.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences for the secretion of GLP2.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of acetate further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
  • a satiety effector e.g., GLP1.
  • the genetically engineered bacteria of the invention are capable of producing a metabolic and/or satiety effector molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.
  • a metabolic and/or satiety effector 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 bacterial cells described herein comprise a propionate gene cassette and are capable of producing propionate in order to treat liver disease, such as NASH.
  • the propionate gene cassette increases the level of propionate in the cell or in the subject as compared to the level of propionate in the cell or in the subject prior to expression of the propionate gene cassette.
  • the genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 2). 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 ho mo log of Acul in E coli, yhdH is used.
  • the propionate cassette comprises pet, IcdA, IcdB, IcdC, and yhdH.
  • the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA ⁇ r , 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. coli (sbm, ygfD, ygfG, ygfH).
  • 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.
  • This pathway is very similar to the oxidative propionate pathway of
  • Propionibacteria which also converts succinate to propionate.
  • Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI: 18042134).
  • mutAB methymalonyl-CoA mutase
  • mutAB methymalonyl-CoA mutase
  • GI: 18042134 methymalonyl-CoA epimerase
  • the genes may be codon-optimized, and translational and transcriptional elements may be added.
  • Tables 1-3 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette.
  • Table 4 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.
  • the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 3 (SEQ ID NO: 21- SEQ ID NO: 26) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 3 (SEQ ID NO: 21- SEQ ID NO: 26) or a functional fragment thereof.
  • genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 3 (SEQ ID NO: 21- SEQ ID NO: 26) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 3 (SEQ ID NO: 21- SEQ ID NO: 26) or a functional fragment thereof.
  • Table 4 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria.
  • the genetically engineered bacteria encode one or more polypeptide sequences of Table 4 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment or variant thereof.
  • genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of one or more polypeptide sequence of Table 4 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment thereof.
  • the bacterial cell comprises a heterologous propionate gene cassette.
  • the disclosure provides a bacterial cell that comprises a heterologous propionate gene cassette operably linked to a first promoter.
  • the first promoter is an inducible promoter.
  • the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette.
  • the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette.
  • the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.
  • a propionate gene cassette is encoded by a gene cassette derived from a bacterial species.
  • a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species.
  • a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi.
  • the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Clostridium propionicum,
  • the propionate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the propionate gene cassette has been codon-optimized for use in Lactococcus.
  • the propionate gene cassette When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions ⁇ e.g., culture or environmental conditions).
  • the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat liver disease, such as nonalcoholic steatohepatitis (NASH).
  • NASH nonalcoholic steatohepatitis
  • the present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme.
  • the term "functional fragment thereof or "functional variant thereof relates to an element having qualitative biological activity in common with the wild- type enzyme from which the fragment or variant was derived.
  • a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived.
  • the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant.
  • the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.
  • percent (%) sequence identity or “percent (%) identity,” also including “homology,” is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol.
  • the present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity,
  • hydrophobicity/hydrophilicity that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • replacing a basic amino acid with another basic amino acid e.g., replacement among Lys, Arg, His
  • an acidic amino acid with another acidic amino acid e.g., replacement among Asp and Glu
  • replacing a neutral amino acid with another neutral amino acid e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, He, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val.
  • a propionate biosynthesis enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure.
  • the gene comprising the modifications described herein may be present on a plasmid or chromosome.
  • the propionate biosynthesis gene cassette is from
  • Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionicum. In another embodiment, the propionate biosynthesis gene cassette is from a Megasphaera spp. In one embodiment, the Megasphaera spp. is Megasphaera elsdenii. In another embodiment, the propionate biosynthesis gene cassette is from Prevotella spp. In one embodiment, the Prevotella spp. is Prevotella ruminicola. Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.
  • 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 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 genes may be codon-optimized, and translational and transcriptional elements may be added.
  • the pet gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the pet gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the pet gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the pet gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the pet gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.
  • the pet 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 pet gene comprises the sequence of SEQ ID NO: 1.
  • the pet gene consists of the sequence of SEQ ID NO: 1.
  • the IcdA gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the IcdA gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the IcdA gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the IcdA gene has at least about 95% identity with SEQ ID NO:
  • the IcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the IcdA 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. In another embodiment, the IcdA gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the IcdA gene consists of the sequence of SEQ ID NO: 2.
  • the IcdB gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the IcdB gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the IcdB gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the IcdB gene has at least about 95% identity with SEQ ID NO:
  • the IcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the IcdB 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. In another embodiment, the IcdB gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the IcdB gene consists of the sequence of SEQ ID NO: 3.
  • the IcdC gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the IcdC gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the IcdC gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the IcdC gene has at least about 95% identity with SEQ ID NO:
  • the IcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the IcdA 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. In another embodiment, the IcdC gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the IcdC gene consists of the sequence of SEQ ID NO: 4.
  • the etfA gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the etfA gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 95% identity with SEQ ID NO:
  • the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Accordingly, in one embodiment, the etfA 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. In another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 5.
  • the acrB gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO:
  • the acrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the acrB 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. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 6.
  • the acrC gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO:
  • the acrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. Accordingly, in one embodiment, the acrC 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. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 7.
  • the thrA ⁇ gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the thrA ⁇ gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the thrA ⁇ gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the thrA ⁇ gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the thrA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8.
  • the thrA ⁇ 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 thrA ⁇ gene comprises the sequence of SEQ ID NO: 8.
  • the thrA ⁇ gene consists of the sequence of SEQ ID NO: 8. [0365]
  • the thrB gene has at least about 80% identity with SEQ ID NO: 9.
  • the thrB gene has at least about 85% identity with SEQ ID NO: 9.
  • the thrB gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. Accordingly, in one embodiment, the thrB 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. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 9.
  • the thrC gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10.
  • the thrC 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 thrC gene comprises the sequence of SEQ ID NO: 10.
  • the thrC gene consists of the sequence of SEQ ID NO: 10.
  • the ilvA ⁇ gene has at least about 80% identity with SEQ ID NO: 11. In another embodiment, the ilvA ⁇ gene has at least about 85% identity with SEQ ID NO: 11. In one embodiment, the ilvA ⁇ gene has at least about 90% identity with SEQ ID NO: 11. In one embodiment, the ilvA ⁇ gene has at least about 95% identity with SEQ ID NO: 11. In another embodiment, the ilvA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11.
  • the ilvA ⁇ 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: 11.
  • the ilvA ⁇ gene comprises the sequence of SEQ ID NO: 11.
  • the ilvA ⁇ gene consists of the sequence of SEQ ID NO: 11.
  • the aceE gene has at least about 80% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12.
  • the aceE 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: 12.
  • the aceE gene comprises the sequence of SEQ ID NO: 12.
  • the aceE gene consists of the sequence of SEQ ID NO: 12.
  • the aceF gene has at least about 80% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13.
  • the aceF 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: 13.
  • the aceF gene comprises the sequence of SEQ ID NO: 13.
  • the aceF gene consists of the sequence of SEQ ID NO: 13.
  • the Ipd gene has at least about 80% identity with SEQ ID NO: 14. In another embodiment, the Ipd gene has at least about 85% identity with SEQ ID NO: 14. In one embodiment, the Ipd gene has at least about 90% identity with SEQ ID NO: 14. In one embodiment, the Ipd gene has at least about 95% identity with SEQ ID NO: 14. In another embodiment, the Ipd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14.
  • the Ipd 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: 14.
  • the Ipd gene comprises the sequence of SEQ ID NO: 14.
  • the Ipd gene consists of the sequence of SEQ ID NO: 14.
  • the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15.
  • 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: 15.
  • the tesB gene comprises the sequence of SEQ ID NO: 15.
  • the tesB gene consists of the sequence of SEQ ID NO: 15.
  • the acul gene has at least about 80% identity with SEQ ID NO: 16. In another embodiment, the acul gene has at least about 85% identity with SEQ ID NO: 16. In one embodiment, the acul gene has at least about 90% identity with SEQ ID NO: 16. In one embodiment, the acul gene has at least about 95% identity with SEQ ID NO: 16. In another embodiment, the acul gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16.
  • the acul 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: 16.
  • the acul gene comprises the sequence of SEQ ID NO: 16.
  • the acul gene consists of the sequence of SEQ ID NO: 16.
  • the sbm gene has at least about 80% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.0.
  • the sbm 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: 17.
  • the sbm gene comprises the sequence of SEQ ID NO: 17.
  • the sbm gene consists of the sequence of SEQ ID NO: 17.
  • the ygfD gene has at least about 80% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18..
  • the ygfD 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: 18.
  • the ygfD gene comprises the sequence of SEQ ID NO: 18.
  • the ygfD gene consists of the sequence of SEQ ID NO: 18.
  • the ygfG gene has at least about 80% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19..
  • the ygfG 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: 19.
  • the ygfG gene comprises the sequence of SEQ ID NO: 19.
  • the ygfG gene consists of the sequence of SEQ ID NO: 19.
  • the ygfH gene has at least about 80% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20..
  • the ygfH 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: 20.
  • the ygfH gene comprises the sequence of SEQ ID NO: 20.
  • the ygfH gene consists of the sequence of SEQ ID NO: 20.
  • one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52.
  • one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 27 through SEQ ID NO: 52. In another embodiment, one or more polypeptides encoded by the propionate 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: 27 through SEQ ID NO: 52.
  • one or more polypeptides encoded by the propionate 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: 27 through SEQ ID NO: 52.
  • one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 27 through SEQ ID NO: 52.
  • one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ ID NO: 27 through SEQ ID NO: 52.
  • one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an E. coli propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. glutamicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a C. propionicum propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a R. sphaeroides propionate biosynthesis gene.
  • the propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate.
  • the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate.
  • one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production.
  • the local production of propionate reduces food intake and ameliorates metabolic disease (Lin et ah, 2012).
  • the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, e.g., as seen in NASH, 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 comprise one or more propionate cassette(s) and further comprise mutations and/or deletions in one or more of frdA, IdhA, and adhE.
  • the genetically engineered bacteria comprise one or more propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 propionate 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 sbm, ygfD, ygfG, and/or ygfH 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 sbm- ygfD-ygfG-ygfH 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 sbm, ygfD, ygfG, and/or ygfH 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 sbm-ygfD-ygfG-ygfH 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 sbm, ygfD, ygfG, and/or ygfH 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) comprising one or more sbm-ygfD-ygfG-ygfH 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 sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous IdhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) 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) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous IdhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm- ygfD-ygfG-ygfH gene cassette(s) 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) selected from sbm, ygfD, ygfG, and/or ygfH 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) comprising one or more sbm-ygfD-ygfG-ygfH 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 sbm, ygfD, ygfG, and/or ygfH 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 sbm- ygfD-ygfG-ygfH 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 two-fold 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, sixfold, 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 propionate 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 two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, fivefold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty- fold, or fifty- fold, more propionate 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, while maintaining high levels of propionate 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 propionate 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 propionate 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 propionate and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate 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 propionate and further comprise a mutation in the endogenous pta and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and ldhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate 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) encoding one or more enzymes for the production of propionate 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 propionate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta, IdhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH 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 sbm- ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand 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 sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, IdhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate 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 sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, IdhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm- ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, IdhA, 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 two-fold 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, sixfold, 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 propionate 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 two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, fivefold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty- fold, or fifty- fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate.
  • one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production.
  • the local production of propionate reduces food intake and improves gut barrier function and reduces inflammation
  • the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate 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.
  • such molecules or metabolites specific to certain conditions e.g., conditions associated with hyperammonemia, such as HE-related molecules, e.g., bilirubin, ammonia, manganese, blood coagulation factors, certain antigens and antibodies, and others described herein or known in the art, or their metabolites.
  • HE-related molecules e.g., bilirubin, ammonia, manganese, blood coagulation factors, certain antigens and antibodies, and others described herein or known in the art, or their metabolites.
  • the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to an inducible promoter.
  • the inducible promoter is directly or indirectly induced by exogenous environmental conditions.
  • the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions.
  • the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut.
  • such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, liver damage, or inflammation.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with liver damage, e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti- mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, or manganese.
  • a promoter induced by a molecule or metabolite associated with liver damage e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase
  • the promoter is induced in the presence of some other metabolite that may or may not be present in vivo, e.g., the gut, such as arabinose and other chemical inducers described herein.
  • the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture.
  • the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.
  • the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein.
  • the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
  • the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to a constitutive promoter.
  • the constitutive promoter is active under exogenous in vivo conditions, e.g., found in the gut, or under conditions present as a consequence of liver disease, e.g., NASH, e.g., hyperammonemia liver damage, or inflammation.
  • the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture.
  • the constitutive promoter is selected from a promoter provided in Table 27-37.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability.
  • the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to a RBS, enhancer or other regulatory sequence.
  • the RBS is selected from a promoter provided in Table IX or Table 27-37 or is listed in Table 38-39.
  • the gene sequence encoding one or more polypeptides for the production of propionate is modified and/or mutated, e.g., to enhance stability, or increase propionate production.
  • the gene sequence encoding one or more polypeptides for the production of propionate may be codon optimized, e.g., to improve expression in the host microorganism.
  • the gene sequence encoding one or more polypeptides for the production of propionate are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganism chromosome.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences described herein for the consumption of ammonia.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences for the production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules known in the art or described herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production or catabolism of tryptophan and/or one or more of its metabolites described herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences for the secretion of IL-22.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences for the secretion of GLP2.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of propionate further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
  • a satiety effector e.g., GLP1.
  • the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions.
  • the bacterial cells described herein may comprise a butyrate gene cassette and are capable of producing butyrate in order to treat liver disease, such as NASH.
  • the butyrate gene cassette increases the level of butyrate in the cell or in the subject as compared to the level of butyrate in the cell or in the subject prior to expression of the butyrate gene cassette.
  • the genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 3).
  • Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium,
  • 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 liver damage, e.g., as seen in NASH, , 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 5 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.
  • 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. It has been shown that a single gene from
  • Treponema denticola ⁇ ter encoding trans -2-enoynl-Co A reductase
  • 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
  • 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 liver damage, e.g., as seen in NASH, , 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 bcd2, etfB3, and etfA3, 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 liver damage, e.g., as seen in NASH, , 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.
  • thiAl, hbd, crt2, pbt, and buk e.g., from Peptoclostridium difficile
  • ter e.g., from Treponema denticola
  • 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 liver damage, e.g., as seen in NASH, , 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 bcd.2, etfB3, etfA3, thiAl, hbd, and crt2, e.g., from Peptoclostridium difficile, and tesB from E.
  • the genetically engineered bacteria comprise ter gene
  • iran5-2-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 liver damage, e.g., as seen in NASH, , 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 liver damage, e.g., as seen in NASH, , or other 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.
  • the bcd.2 gene has at least about 80% identity with SEQ ID NO: 53. In another embodiment, the bcd.2 gene has at least about 85% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the bcd.2 gene has at least about 95% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53.
  • 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: 53.
  • the bcd.2 gene comprises the sequence of SEQ ID NO: 53.
  • the bcd.2 gene consists of the sequence of SEQ ID NO: 53.
  • the etfB3 gene has at least about 80% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54.
  • 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: 54.
  • the etfB3 gene comprises the sequence of SEQ ID NO: 54.
  • the etfB3 gene consists of the sequence of SEQ ID NO: 54.
  • the etfA3 gene has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55.
  • 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: 55.
  • the etfA3 gene comprises the sequence of SEQ ID NO: 55.
  • the etfA3 gene consists of the sequence of SEQ ID NO: 55.
  • the thiAl gene has at least about 80% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene has at least about 85% identity with SEQ ID NO: 56. In one embodiment, the thiAl gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the thiAl gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the thiAl gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56.
  • 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: 56.
  • the thiAl gene comprises the sequence of SEQ ID NO: 56.
  • the thiAl gene consists of the sequence of SEQ ID NO: 56.
  • the hbd gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO:
  • the hbd gene has at least about 95% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. Accordingly, in one embodiment, 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: 57. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 57.
  • the crt2 gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO:
  • the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, 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: 58. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 58.
  • the pbt gene has at least about 80% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO:
  • the pbt gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, 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: 59. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 59.
  • the buk gene has at least about 80% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO:
  • the buk gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. Accordingly, in one embodiment, 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: 60. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 60.
  • the ter gene has at least about 80% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO:
  • the ter gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. Accordingly, in one embodiment, 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: 61. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 61.
  • the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15.
  • 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: 15.
  • the tesB gene comprises the sequence of SEQ ID NO: 15.
  • the tesB gene consists of the sequence of SEQ ID NO: 15.
  • 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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • 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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • 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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41.
  • 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.
  • 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 metabolic disease (Lin et ah, 2012).
  • 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 liver damage, e.g., as seen in NASH, , 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. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the
  • 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 bacterial cell comprises a heterologous butyrate gene cassette.
  • the disclosure provides a bacterial cell that comprises a heterologous butyrate gene cassette operably linked to a first promoter.
  • the first promoter is an inducible promoter.
  • the bacterial cell comprises a butyrate gene cassette from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises more than one copy of a native gene encoding a butyrate gene cassette.
  • the bacterial cell comprises at least one native gene encoding a butyrate gene cassette, as well as at least one copy of a butyrate gene cassette from a different organism, e.g., a different species of bacteria.
  • the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a butyrate gene cassette.
  • the bacterial cell comprises multiple copies of a gene or genes encoding a butyrate gene cassette.
  • a butyrate gene cassette is encoded by a gene cassette derived from a bacterial species.
  • a butyrate gene cassette is encoded by a gene cassette derived from a non-bacterial species.
  • a butyrate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi.
  • the gene encoding the butyrate gene cassette is derived from an organism of the genus or species that includes, but is not limited to, Peptoclostridium, Clostridium, Fusobacterium,
  • the butyrate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the butyrate gene cassette has been codon-optimized for use in Escherichia coli. In another embodiment, the butyrate gene cassette has been codon-optimized for use in Lactococcus.
  • the butyrate gene cassette When the butyrate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions).
  • the genetically engineered bacteria comprising a heterologous butyrate gene cassette may be used to generate butyrate to treat liver disease, such as nonalcoholic steatohepatitis (NASH).
  • NASH nonalcoholic steatohepatitis
  • the present disclosure also encompasses butyrate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.
  • a butyrate biosynthesis enzyme is mutagenized
  • mutants exhibiting increased activity are selected; and the mutagenized gene encoding the butyrate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure.
  • the gene comprising the modifications described herein may be present on a plasmid or chromosome.
  • the butyrate biosynthesis gene cassette is from
  • Clostridium spp. In one embodiment, the Clostridium spp. is Clostridium propionicum. In another embodiment, the butyrate biosynthesis gene cassette is from a Peptoclostridium spp. In one embodiment, the Peptoclostridium spp. is Peptoclostridium difficile. In another embodiment, the butyrate biosynthesis gene cassette is from Fusobacterium spp. In another embodiment, the butyrate biosynthesis gene cassette is from Butyrivibrio spp. In another embodiment, the butyrate biosynthesis gene cassette is from Eubacterium spp. In another embodiment, the butyrate biosynthesis gene cassette is from Treponema spp. Other butyrate biosynthesis gene cassettes are well-known to one of ordinary skill in the art.

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Abstract

La présente divulgation concerne des cellules bactériennes génétiquement modifiées comprenant un gène hétérologue codant pour une cassette génique de biosynthèse du propionate; une cassette génique de biosynthèse du butyrate; GLP-1; une cassette génique de biosynthèse du propionate et une cassette génique de biosynthèse du butyrate; une cassette génique de biosynthèse du propionate et GLP-1; une cassette génique de biosynthèse du butyrate et GLP-1; ou une cassette génique de biosynthèse du propionate, une cassette génique de biosynthèse du butyrate, and GLP- 1. Selon un autre aspect, les cellules bactériennes génétiquement modifiées comportent en outre une sécurité de type destruction des bactéries. Des compositions pharmaceutiques comprenant les bactéries génétiquement modifiées, et des méthodes de traitement d'une maladie du foie, telle que la stéatohépatite non alcoolique (SHNA), à l'aide des compositions pharmaceutiques selon la présente divulgation sont en outre décrites.
PCT/US2017/017563 2016-02-10 2017-02-10 Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna) Ceased WO2017139708A1 (fr)

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US201662293695P 2016-02-10 2016-02-10
US62/293,695 2016-02-10
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
USPCT/US2016/020530 2016-03-02
US201662336012P 2016-05-13 2016-05-13
US201662335780P 2016-05-13 2016-05-13
US62/335,780 2016-05-13
PCT/US2016/032565 WO2016183532A1 (fr) 2015-05-13 2016-05-13 Bactéries modifiées pour traiter une maladie ou un trouble
US62/336,012 2016-05-13
USPCT/US2016/032565 2016-05-13
US201662345242P 2016-06-03 2016-06-03
US62/345,242 2016-06-03
US201662347576P 2016-06-08 2016-06-08
US201662347554P 2016-06-08 2016-06-08
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US62/347,508 2016-06-08
US201662348620P 2016-06-10 2016-06-10
US201662348416P 2016-06-10 2016-06-10
US62/348,416 2016-06-10
US62/348,620 2016-06-10
US201662354681P 2016-06-24 2016-06-24
US201662354682P 2016-06-24 2016-06-24
US62/354,682 2016-06-24
PCT/US2016/039444 WO2016210384A2 (fr) 2015-06-25 2016-06-24 Bactéries manipulées pour traiter des maladies métaboliques
USPCT/US2016/039444 2016-06-24
US62/354,681 2016-06-24
US201662362954P 2016-07-15 2016-07-15
US201662362863P 2016-07-15 2016-07-15
US62/362,863 2016-07-15
US62/362,954 2016-07-15
US201662385235P 2016-09-08 2016-09-08
US15/260,319 2016-09-08
USPCT/US2016/050836 2016-09-08
US62/385,235 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
US62/423,170 2016-11-16
US201662439871P 2016-12-28 2016-12-28
US62/439,871 2016-12-28
PCT/US2016/069052 WO2017123418A1 (fr) 2016-01-11 2016-12-28 Bactéries modifiées pour traiter des maladies métaboliques
USPCT/US2016/069052 2016-12-28
PCT/US2017/013074 WO2017123676A1 (fr) 2016-01-11 2017-01-11 Bactéries recombinées modifiées pour traiter des maladies et des troubles associés à un métabolisme des acides aminés et leurs méthodes d'utilisation
PCT/US2017/012946 WO2017123592A1 (fr) 2016-01-11 2017-01-11 Bactérie manipulée pour traiter des troubles associés aux sels biliaires
USPCT/US2017/013074 2017-01-11
USPCT/US2017/012946 2017-01-11
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
USPCT/US2017/16603 2017-02-03
USPCT/US2017/016609 2017-02-03
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PCT/US2017/016609 WO2017136795A1 (fr) 2016-02-04 2017-02-03 Bactéries modifiées pour traiter des maladies associées au metabolisme du tryptophane

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CN113186140A (zh) * 2021-02-08 2021-07-30 和度生物医药(上海)有限公司 用于预防和/或治疗宿醉和肝病的基因工程细菌
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
EP3927733A4 (fr) * 2019-02-24 2022-07-06 Oncosimis Biotech Private Limited Procédé de production continue de peptide glp-1 recombinant par des bactéries
CN116478271A (zh) * 2023-06-19 2023-07-25 青岛大学 半滑舌鳎抗病基因PPARα及其编码蛋白的应用
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