WO2017139697A1 - Bacteria engineered to treat diseases associated with hyperammonemia - Google Patents

Bacteria engineered to treat diseases associated with hyperammonemia Download PDF

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WO2017139697A1
WO2017139697A1 PCT/US2017/017552 US2017017552W WO2017139697A1 WO 2017139697 A1 WO2017139697 A1 WO 2017139697A1 US 2017017552 W US2017017552 W US 2017017552W WO 2017139697 A1 WO2017139697 A1 WO 2017139697A1
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gene
bacterium
promoter
arginine
induced
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PCT/US2017/017552
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French (fr)
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WO2017139697A8 (en
WO2017139697A9 (en
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Jonathan W. Kotula
Vincent M. Isabella
Paul F. Miller
Dean Falb
Ning Li
Suman MACHINANI
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Synlogic, Inc.
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Priority claimed from PCT/US2016/020530 external-priority patent/WO2016141108A1/en
Priority claimed from PCT/US2016/032565 external-priority patent/WO2016183532A1/en
Priority claimed from US15/164,828 external-priority patent/US9688967B2/en
Priority claimed from PCT/US2016/034200 external-priority patent/WO2016200614A2/en
Priority claimed from US15/260,319 external-priority patent/US11384359B2/en
Priority claimed from PCT/US2016/050836 external-priority patent/WO2017074566A1/en
Priority claimed from PCT/US2017/016603 external-priority patent/WO2017136792A2/en
Priority claimed from PCT/US2017/016609 external-priority patent/WO2017136795A1/en
Application filed by Synlogic, Inc. filed Critical Synlogic, Inc.
Publication of WO2017139697A1 publication Critical patent/WO2017139697A1/en
Publication of WO2017139697A8 publication Critical patent/WO2017139697A8/en
Publication of WO2017139697A9 publication Critical patent/WO2017139697A9/en

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    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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

Definitions

  • Ammonia is highly toxic and generated during metabolism in all organs (Walker, 2012). In mammals, the healthy liver protects the body from ammonia by converting ammonia to non-toxic molecules, e.g., urea or glutamine, and preventing excess amounts of ammonia from entering the systemic circulation. Hyperammonemia is characterized by the decreased detoxification and/or increased production of ammonia. In mammals, the urea cycle detoxifies ammonia by enzymatically converting ammonia into urea, which is then removed in the urine.
  • Decreased ammonia detoxification may be caused by urea cycle disorders (UCDs) in which urea cycle enzymes are defective, such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency (Häberle et al., 2012).
  • UCDs urea cycle disorders
  • urea cycle disorders in which urea cycle enzymes are defective, such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency (Häberle et al., 2012).
  • Hyperammonemia can produce neurological manifestations, e.g., seizures, ataxia, stroke-like lesions, coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respiratory alkalosis, hypothermia, or death (Häberle et al., 2012; Häberle et al., 2013).
  • Ammonia is also a source of nitrogen for amino acids, which are synthesized by various biosynthesis pathways.
  • arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms.
  • Intermediate metabolites formed in the arginine biosynthesis pathway, such as citrulline, also incorporate nitrogen.
  • enhancement of arginine biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules in order to modulate or treat conditions associated with hyperammonemia.
  • histidine biosynthesis methionine biosynthesis, lysine biosynthesis, asparagine biosynthesis, glutamine biosynthesis, and tryptophan biosynthesis are also capable of incorporating excess nitrogen, and enhancement of those pathways may be used to modulate or treat conditions associated with hyperammonemia.
  • liver plays a central role in amino acid metabolism and protein synthesis and breakdown, as well as in several detoxification processes, notably those of end-products of intestinal metabolism, like ammonia.
  • Liver dysfunction resulting in hyperammonemia, may cause hepatic encephalopathy (HE), which disorder
  • liver dysfunction encompasses spectrum of potentially reversible neuropsychiatric abnormalities observed in patients with liver dysfunction (after exclusion of unrelated neurologic and/or metabolic abnormalities).
  • severe liver failure e.g., cirrhosis
  • Ammonia accumulation in the brain leads to cognitive and motor disturbances, reduced cerebral perfusion, as well as oxidative stress-mediated injury to astrocytes, the brain cells capable of metabolizing ammonia.
  • GABA ⁇ -aminobutyric acid
  • Elevated cerebral manganese concentrations and manganese deposition have also been reported in the basal ganglia of cirrhosis patients, and are suspected to contribute to the clinical presentation of HE (Cash et al., 2010; Rivera-Manc ⁇ a et al., 2012).
  • General neurological manifestations of hyperammonemia include seizures, ataxia, stroke-like lesions, Parkinsonian symptoms (such as tremors), coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respiratory alkalosis, hypothermia, or death (Häberle et al., 2012; Häberle et al., 2013).
  • SIRS systemic inflammatory response syndrome
  • TNF tumor necrosis factor
  • IL6 interleukin- 6
  • RNS reactive nitrogen species
  • ROS reactive oxygen species
  • Hyperammonemia is also a prominent feature of Huntington’s disease, an autosomal dominant disorder characterized by intranuclear/cytoplasmic aggregates and cell death in the brain (Chen et al., 2015; Chiang et al., 2007). In fact,
  • hyperammonemia is a feature of several other disorders, as discussed herein, all of which can be treated by reducing the levels of ammonia.
  • Huntington’s disease the side effects of antipsychotic drugs (e.g., haloperidol, risperidone, quetiapine) and drugs administered to suppress involuntary movements (e.g., tetrabenazine, amantadine, levetiracetam, clonazepam) may worsen muscle rigidity and cognitive decline in patients (Mayo Clinic).
  • antipsychotic drugs e.g., haloperidol, risperidone, quetiapine
  • drugs administered to suppress involuntary movements e.g., tetrabenazine, amantadine, levetiracetam, clonazepam
  • involuntary movements e.g., tetrabenazine, amantadine, levetiracetam, clonazepam
  • the disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and converting ammonia and/or nitrogen into alternate byproducts.
  • the genetically engineered bacteria reduce excess ammonia and convert ammonia and/or nitrogen into alternate byproducts.
  • the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxic ammonia. As much as 70% of excess ammonia in a hyperammonemic patient accumulates in the gastrointestinal tract.
  • Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of ammonia and/or nitrogen consumption, or production of a non-toxic byproduct, e.g., arginine or citrulline.
  • the invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with hyperammonemia, e.g., urea cycle disorders and hepatic encephalopathy.
  • the disclosure also provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules, e.g., GABA, manganese.
  • the genetically engineered bacteria reduce excess ammonia and convert ammonia and/or nitrogen into alternate byproducts.
  • the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxic ammonia.
  • the genetically engineered bacteria are capable of reducing excess ammonia and are also capable of producing one or more gut barrier enhancer molecules, e.g., one or more short chain fatty acid(s), such as butyrate and/or acetate.
  • the genetically engineered bacteria are also capable of reducing excess ammonia and other deleterious molecules, e.g., GABA, manganese.
  • compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with excess ammonia, including, for example, hepatic encephalopathy and Huntington’s disease.
  • a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette for producing butyrate, wherein the bacterium comprises an endogenous pta gene which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
  • the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, pbt, and buk genes. In some embodiments, the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, and tesb genes.
  • the bacterium comprises an endogenous adhE gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.
  • the promoter operably linked to the at least one gene or gene cassette is induced by exogenous environmental conditions. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by low-oxygen or anaerobic conditions. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by one or more molecules or metabolites indicative of liver damage.
  • the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive nitrogen species. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive oxygen species. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
  • a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette for producing butyrate, wherein the bacterium comprises at least one endogenous gene selected from frd, ldhA, and adhE, which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
  • the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; and wherein the bacterium lacks a functional ArgR.
  • each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
  • under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
  • the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; wherein the arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transc
  • the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine-mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the argininosuccinate synthase gene.
  • the operon comprising the gene encoding argininosuccinate synthase comprises a constitutively active promoter that regulates transcription of the argininosuccinate synthase gene.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by endogenous environmental conditions.
  • arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced under low oxygen conditions.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species.
  • the bacterium is a non-pathogenic bacterium. In some embodiments, the bacterium is a probiotic bacterium. In some embodiments, the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In some embodiments, the bacterium is Escherichia coli strain Nissle.
  • a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette selected from (1) a GABA metabolic gene or gene cassette (2) a GABA transport gene or gene cassette, (3) a manganese transport gene or gene cassette.
  • the at least one gene for the consumption of GABA is capable of producing a GABA catabolism enzyme.
  • the GABA catabolism enzyme is selected from GABA ⁇ -ketoglutarate transaminase (GSST) and succinate-semialdehyde dehydrogenase (SSDH).
  • the GABA transport circuit is capable of producing a GABA membrane transport protein. In some embodiments, the GABA membrane transport protein is GabP. [0026] In some embodiments, the manganese transport circuit is capable of producing a manganese membrane transport protein. In some embodiments, the manganese membrane transport protein is MntH.
  • the at least one gene or gene cassette is controlled by a promoter induced by exogenous environmental conditions. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced under low oxygen conditions. In some embodiments, the at least one gene or gene cassette is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by the presence of reactive nitrogen species.
  • the at least one gene or gene cassette is controlled by a promoter induced by the presence of reactive oxygen species.
  • the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
  • the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; and wherein the bacterium has been engineered to lack a functional ArgR.
  • each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species.
  • arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species. In some embodiments, under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine
  • biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
  • the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit is present on a plasmid in the bacterium and operably linked on the plasmid to the inducible promoter. In some embodiments, the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit, is present on a bacterial chromosome and operably linked on chromosome to the inducible promoter.
  • the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
  • the mammalian gut is a human gut.
  • composition comprising one or more of any of the bacteria disclosed herein.
  • the composition comprising the bacterium is formulated for oral or rectal
  • Disclosed herein is a method of treating a disease, disorder or condition associated with hyperammonemia, or symptom(s) thereof in a subject in need thereof comprising the step of administering to the subject any composition described herein for a period of time sufficient to lessen the severity of the disease or symptom(s).
  • the disease, disorder, or condition is hepatic encephalopathy
  • Huntington s disease, or symptom(s) thereof.
  • the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non- native component(s), which mediate one or more mechanisms of action. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted.
  • the genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression.
  • genes(s)/gene cassette(s) may be under the control of constitutive or inducible promoters.
  • exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific 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.
  • oxygen level-dependent promoters e.g., FNR-inducible promoter
  • promoters induced by HE-specific molecules or metabolites indicative of liver damage e.g., bilirubin
  • RNS inflammatory response
  • promoters induced by a metabolite may or may not be naturally present
  • the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.
  • auxotrophies such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy
  • kill switch circuits such as any of the kill-switches described herein or otherwise known in the art
  • antibiotic resistance circuits such as antibiotic resistance circuits
  • transporters for importing biological molecules or substrates
  • FIG. 1A and FIG. 1B depict the state of the arginine regulon in one embodiment of an ArgR deletion bacterium of the invention under non-inducing (FIG. 1A) and inducing (FIG. 1B) conditions.
  • FIG. 1A depicts relatively low arginine production under aerobic conditions due to arginine (“Arg” in oval) interacting with ArgA (squiggle ) to inhibit (indicated by“X”) ArgA activity, while oxygen (O 2 ) prevents (indicated by“X”) FNR (dotted boxed FNR) from dimerizing and activating f br
  • FIG. 1B depicts up-regulated arginine production under anaerobic conditions due to FNR dimerizing (two dotted boxed FNRs) and inducing FNR promoter (grey FNR box)- fbr fbr
  • ArgA mediated expression of ArgA (squiggle above argA ), which is resistant to inhibition by arginine.
  • This overcomes (curved arrow) the inhibition of the wild-type ArgA caused by arginine (“Arg” in oval) interacting with ArgA (squiggle above box depicting argA).
  • Each gene in the arginine regulon is depicted by a rectangle containing the name of the gene.
  • Each arrow adjacent to one or a cluster of rectangles depict the promoter responsible for driving transcription, in the direction of the arrow, of such gene(s).
  • Heavier lines adjacent one or a series of rectangles depict ArgR binding sites, which are not utilized because of the ArgR deletion in this bacterium. Arrows above each rectangle depict the expression product of each gene.
  • FIG. 2A and FIG. 2B depict an alternate exemplary embodiment of the present invention.
  • FIG. 2A depicts the embodiment under aerobic conditions where, in the presence of oxygen, the FNR proteins (FNR boxes) remain as monomers and are unable to bind to and activate the FNR promoter (“FNR”) which drives expression of the arginine feedback resistant argA fbr gene.
  • FNR FNR promoter
  • the wild-type ArgA protein is functional, but is susceptible to negative feedback inhibition by binding to arginine, thus keeping arginine levels at or below normal.
  • FIG. 2B depicts the same embodiment under anaerobic conditions where, in the absence of oxygen the FNR protein (FNR boxes) dimerizes and binds to and activates the FNR promoter (“FNR”).
  • FIG. 3 depicts another embodiment of the invention. In this case
  • a construct comprising an ArgR binding site (black bar) in a promoter driving expression of the Tet repressor (TetR) from the tetR gene is linked to a second promoter comprising a TetR binding site (black bar between TetR and X) that drives expression of gene X.
  • TetR Tet repressor
  • ArgR associates with arginine and binds to the ArgR binding site, thereby inhibiting expression of TetR from the tetR gene. This, in turn, removes the inhibition by TetR allowing gene X expression (black squiggle ( )).
  • FIG. 5 depicts another embodiment of the invention.
  • a construct comprising an ArgR binding site (black bar bound by the ArgR-Arg complex) in a promoter driving expression of the Tet repressor (not shown) from the tetR gene is linked to a second promoter comprising a TetR binding site (black bar) that drives expression of an auxotrophic protein necessary for host survival (“AUX”).
  • AUX auxotrophic protein necessary for host survival
  • the ArgR-arginine complex binds to the ArgR binding site, thereby inhibiting expression of TetR from the tetR gene. This, in turn, allows expression of AUX, allowing the host to survive.
  • TetR is expressed from the tetR gene and inhibits the expression of AUX, thus killing the host.
  • the construct in FIG. 5 enforces high arginine (“Arg”) production by making it necessary for host cell survival through its control of AUX expression.
  • FIG. 6 depicts a schematic diagram of the argA fbr gene under the control of an exemplary FNR promoter (fnrS) fused to a strong ribosome binding site.
  • fnrS FNR promoter
  • FIG. 7 depicts another schematic diagram of the argA fbr gene under the control of an exemplary FNR promoter (nirB) fused to a strong ribosome binding site.
  • Other regulatory elements may also be present.
  • FIG. 8 depicts a schematic diagram of the argA fbr gene under the control of an exemplary FNR promoter (nirB) fused to a weak ribosome binding site.
  • FIG. 9A and FIG. 9B depict exemplary embodiments of a FNR- responsive promoter fused to a CRP binding site.
  • FIG. 9A depicts a map of the FNR- CRP promoter region, with restriction sites shown in bold.
  • FIG. 9B depicts a schematic diagram of the argA fbr gene under the control of an exemplary FNR promoter (nirB promoter), fused to both a CRP binding site and a ribosome binding site.
  • Other regulatory elements may also be present.
  • FIG. 10A and FIG. 10B depict alternate exemplary embodiments of a FNR-responsive promoter fused to a CRP binding site.
  • FIG. 10A depicts a map of the FNR-CRP promoter region, with restriction shown in bold.
  • FIG. 10B depicts a schematic diagram of the argA fbr gene under the control of an exemplary FNR promoter (fnrS promoter), fused to both a CRP binding site and a ribosome binding site.
  • fnrS promoter exemplary FNR promoter
  • FIG. 11 depicts an exemplary embodiment of a constitutively expressed argG construct in E. coli Nissle.
  • the constitutive promoter is BBa_J23100, boxed in gray. Restriction sites for use in cloning are in bold.
  • FIG. 13 depicts a schematic diagram of an exemplary BAD promoter- fbr fbr
  • the argA gene is inserted between the fbr
  • ArgA is flanked by a ribosome binding site, a FRT site, and one or more transcription terminator sequences.
  • FIG. 15 depicts an exemplary embodiment of an engineered bacterial strain deleted for the argR and argG genes, and expressing the feedback-resistant argA fbr gene.
  • this strain further comprises one or more auxotrophic modifications on the chromosome. This strain is useful for the
  • FIG. 16 depicts an exemplary embodiment of an engineered bacterial strain which lacks ArgR binding sites and expresses the feedback-resistant argA fbr gene. In some embodiments, this strain further comprises one or more auxotrophic
  • This strain is useful for the consumption of ammonia and the production of arginine.
  • FIG. 17 depicts an exemplary embodiment of an engineered bacterial strain which lacks ArgR binding sites in all of the arginine biosynthesis operons except for argG, and expresses the feedback-resistant argA fbr gene.
  • this strain further comprises one or more auxotrophic modifications on the chromosome. This strain is useful for the consumption of ammonia and the production of citrulline.
  • FIG. 18 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. The malE/K site is circled. In some embodiments of the disclosure, FNR-ArgAfbr is inserted at the malEK locus.
  • FIG. 19 depicts three bacterial strains which constitutively express red fluorescent protein (RFP).
  • RFP red fluorescent protein
  • FIG. 20 depicts the gene organization of exemplary constructs of the disclosure.
  • strains comprising such a construct include SYN- UCD301 and SYN-UCD302.
  • SYN-UCD301 comprises ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, Wild type ThyA, and
  • FIG. 21 depicts the gene organization of an exemplary construct of the disclosure.
  • strains comprising such a construct include SYN- UCD303, SYN-UCD306, SYN-UCD307, and SYN-UCD309.
  • SYN- UCD303 comprises ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, ⁇ ThyA, and kanamycin resistance.
  • FIG. 22 depicts the gene organization of exemplary constructs of the disclosure.
  • Non-limiting examples of strains comprising such a construct include SYN- UCD304, SYN-UCD305, SYN-UCD308, and SYN-UCD310.
  • SYN- UCD304 comprises ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, wild type ThyA, and no antibiotic resistance.
  • SYN-UCD305 comprises ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, ⁇ ThyA, and no antibiotic resistance.
  • FIG. 23 depicts a bar graph of in vitro arginine levels produced by streptomycin-resistant control Nissle (SYN-UCD103), SYN-UCD201, SYN-UCD202, and SYN-UCD203 under inducing (+ATC) and non-inducing (-ATC) conditions.
  • SYN- fbr streptomycin-resistant control Nissle
  • UCD201 comprises ⁇ ArgR and no argA .
  • SYN-UCD202 comprises ⁇ ArgR and
  • SYN-UCD203 comprises
  • FIG. 24 depicts a bar graph of in vitro levels of arginine and citrulline produced by streptomycin-resistant control Nissle (SYN-UCD103), SYN-UCD104, SYN-UCD204, and SYN-UCD105 under inducing conditions.
  • SYN-UCD104 comprises wild-type ArgR, tetracycline-inducible argA fbr on a low-copy plasmid, tetracycline-inducible argG, and mutations in each ARG box for each arginine biosynthesis operon except for argG.
  • SYN-UCD204 comprises ⁇ ArgR and argA fbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid.
  • SYN-UCD105 comprises wild-type ArgR, tetracycline-inducible argA fbr on a low-copy plasmid, constitutively expressed argG (BBa_J23100 constitutive promoter), and mutations in each ARG box for each arginine biosynthesis operon except for argG.
  • FIG. 25 depicts a bar graph of in vitro arginine levels produced by streptomycin-resistant Nissle (SYN-UCD103), SYN-UCD205, and SYN-UCD204 under inducing (+ATC) and non-inducing (-ATC) conditions, in the presence (+O 2 ) or absence (-O 2 ) of oxygen.
  • SYN-UCD103 is a control Nissle construct.
  • SYN-UCD205 comprises ⁇ ArgR and argA fbr expressed under the control of a FNR-inducible promoter on a low-copy plasmid.
  • SYN204 comprises ⁇ ArgR and argA fbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid.
  • FIG. 26 depicts a bar graph of in vitro ammonia levels in culture media from SYN-UCD101, SYN-UCD102, and blank controls at baseline, two hours, and four hours. Both SYN-UCD101 and SYN-UCD102 are capable of consuming ammonia in vitro.
  • SYN-UCD101 comprises wild type ArgR, and wild type ThyA, and no ArgAFbr;
  • SYN-UCD102 comprises wild type ArgR, tetracycline-inducible argAfbr on a low copy plasmid, and wild type ThyA.
  • FIG. 27 depicts a bar graph of in vitro ammonia levels in culture media from SYN-UCD202, SYN-UCD203, and blank controls at baseline, two hours, and four hours. Both SYN-UCD202 and SYN-UCD203 are capable of consuming ammonia in vitro. SYN-UCD202 and SYN-UCD203 both comprise ⁇ ArgR, tetracycline-inducible argAfbr on a high-copy plasmid or low copy plasmid, respectively, Amp resistance, and wild type ThyA.
  • FIG. 28A, 28B, and 28C depict bar graphs of ammonia levels in hyperammonemic TAA mice.
  • FIG. 28A depicts a bar graph of ammonia levels in hyperammonemic mice treated with unmodified control Nissle or SYN-UCD202, a fbr genetically engineered strain in which the Arg repressor gene is deleted and the argA gene is under the control of a tetracycline-inducible promoter on a high-copy plasmid. A total of 96 mice were tested, and the error bars represent standard error.
  • FIG. 28B depicts a bar graph showing in vivo efficacy
  • FIG. 28C depicts a bar graph of the percent change in blood ammonia concentration between 24-48 hours post-TAA treatment.
  • FIG. 29 depicts a bar graph of ammonia levels in hyperammonemic spf ash mice. Fifty-six spf ash mice were separated into four groups. Group 1 was fed normal chow, and groups 2-4 were fed 70% protein chow following an initial blood draw. Groups were gavaged twice daily, with water, streptomycin-resistant Nissle control (SYN-UCD103), or SYN-UCD204, and blood was drawn 4 hours following the first gavage.
  • SYN-UCD103 streptomycin-resistant Nissle control
  • SYN-UCD204 SYN-UCD204
  • FIG. 30 depicts a bar graph of ammonia levels in hyperammonemic spf ash mice on a high protein diet.
  • mice were treated with SYN-UCD204 (comprising ⁇ ArgR, PfnrS-ArgAfbr on a low-copy plasmid and wild type ThyA), SYN-UCD206 (comprising ⁇ ArgR, PfnrS- ArgAfbr on a low-copy plasmid and ⁇ ThyA) or water, then switched to high protein chow after 2 days. As seen in FIG. 30, at 48 hours after switch to high protein chow ammonia levels were reduced to a similar extent in both SYN- UCD205 and SYN-UCD206, indicating that ThyA auxotrophy does not have a significant effect on efficacy.
  • FIG. 31A and 31B depict bar graphs of ammonia levels in the media at various time points post anaerobic induction.
  • FIG. 31A depicts a bar graph of the levels of arginine production of SYN-UCD205, SYN-UCD206, and SYN-UCD301 measured at 0, 30, 60, and 120 minutes.
  • FIG.31B depicts a bar graph of the levels of arginine production of SYN-UCD204 (comprising ⁇ ArgR, PfnrS-ArgAfbr on a low- copy plasmid and wild type ThyA), SYN-UCD301, SYN-UCD302, and SYN-UCD303 (all three of which comprise an integrated FNR-ArgAfbr construct; SYN UCD301 comprises ⁇ ArgR, and wtThyA; SYN 303 comprises ⁇ ArgR, and ⁇ ThyA). Results indicate that chromosomal integration of FNR ArgA fbr results in similar levels of arginine production as seen with the low copy plasmid strains expressing the same construct.
  • FIG. 32A and 32B depicts a bar graph of ammonia levels and a survival curve for hyperammonemic spf ash mice on a normal (NC) or high protein (HP) diet. Two strains with an integrated copy of FNR-ArgAfbr, one with (SYN-UCD303) and one without a ThyA deletion (SYN-UCD301) were compared.
  • FIG. 32A depicts a bar graph of ammonia levels in hyperammonemic spf ash mice on a normal (NC) or high protein (HP) diet.
  • FIG. 32B depicts a survival curve of hyperammonemic spf ash mice on a normal (NC) or high protein (HP) diet and shows that SYN-UCD301 and SYN-UCD303 displayed prolonged survival as compared to controls.
  • SYN- UCD303 comprises ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, ⁇ ThyA, and Kanamycin resistance.
  • FIG. 34A 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
  • 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. 34B 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 + 10mM thymidine at 37C. The next day, cells were diluted 1:100 in 1 mL LB + 10mM 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. 34C 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.
  • 35A depicts a bar graph of residence over time for SYN-UCD103 (streptomycin resistant Nissle).
  • FIG. 35B depicts a bar graph residence over time for SYN-UCD106, comprising ⁇ ArgR and ⁇ ThyA and no ArgAfbr.
  • FIG. 35C depicts a bar graph showing residence over time for SYN-UCD303, comprising ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, and ⁇ ThyA.
  • FIG. 36A, 36B, and 36C depict bar graphs of viable bacterial cells and arginine production.
  • Cells were either incubated with 70% isopropanol or phosphate buffered saline (PBS) as a control for 1 hour with shaking. After treatment, the cells were mixed at specific ratios in M9 media supplemented with 0.5% glucose and 3mM thymidine and incubated with shaking at 37 C for 2 hours.
  • PBS phosphate buffered saline
  • FIG. 37 Depicts the number of bacteria quantified in fecal samples collected in a non-human primate toxicity study. Pharmacokinetics and
  • FIG. 38 depicts an exemplary synthetic genetic circuit for treating hepatic encephalopathy and other disorders characterized by hyperammonemia.
  • ammonia is taken up by a bacterium (e.g., E. coli Nissle), converted to glutamate, and glutamate is subsequently metabolized to arginine.
  • a bacterium e.g., E. coli Nissle
  • Arginine ultimately exits the bacterial cell.
  • FIG. 39 depicts one embodiment of the invention.
  • the genetically engineered bacteria comprise four exemplary circuits for the treatment of hepatic encephalopathy.
  • ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
  • Arginine ultimately exits the bacterial cell.
  • the GABA membrane transport protein (GabP) is expressed by the gabP gene, and facilitates GABA transport into the cell.
  • the bacterial manganese transport protein (MntH) is expressed by the mntH gene, and facilitates manganese transport into the cell.
  • expression of a butyrate gene cassette results in the production of butyrate, and release of this gut barrier enhancer molecule outside of the cell.
  • all four circuits are each under the control of the same inducible promoter. In other embodiments, the four circuits may be under the control of different inducible promoters.
  • Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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.
  • oxygen level-dependent promoters e.g., FNR-inducible promoter
  • promoters induced by HE-specific molecules or metabolites indicative of liver damage e.g., bilirubin
  • promoters induced by inflammation or an inflammatory response e.g., can be exogenously added
  • FIG. 40 depicts one embodiment of the invention.
  • the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy.
  • ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
  • Arginine ultimately exits the bacterial cell.
  • the GABA membrane transport protein (GabP) is expressed by the gabP gene, and facilitates GABA transport into the cell.
  • both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of a different inducible promoter.
  • Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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 genetically engineered bacteria may further comprise an additional circuit for reducing the level of GABA, e.g., a circuit for metabolizing (catabolizing) GABA.
  • FIG. 41A and FIG.41B depict the catabolism of GABA following uptake into genetically engineered bacteria comprising synthetic genetic circuits.
  • GABA is converted to succinyl semialdehyde by GABA ⁇ -ketoglutarate transaminase (GSST).
  • GABA ⁇ -ketoglutarate transaminase GSST
  • Succinate-semialdehyde dehydrogenase SSDH
  • succinate becomes a substrate for the citric acid (TCA) cycle.
  • GOT glutamate oxaloacetate transaminase converts alpha-ketoglutarate to glutamate.
  • the genetically engineered bacteria of the disclosure comprise a GABA consuming circuit including, but not limited to, one or more of GSST, SSDH, and GOT.
  • FIG. 41B depicts a schematic representation of the GABA utilization pathway in E. coli Nissle.
  • FIG. 42 depicts one embodiment of the invention.
  • the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy.
  • ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
  • Arginine ultimately exits the bacterial cell.
  • the bacterial manganese transport protein (MntH) is expressed by the mntH gene, and facilitates manganese transport into the cell.
  • both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of different inducible promoter.
  • Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE- specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose.
  • FIG. 43 depicts one embodiment of the invention.
  • the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy.
  • both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of different inducible promoter.
  • Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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.
  • One or more of the butyrate cassettes described herein may be expressed by the genetically engineered bacteria comprising an arginine (and/or citrulline) producing circuit.
  • FIG. 44 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).
  • FIG. 45 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs.
  • an ammonia conversion circuit, a butyrate production circuit, a GABA transport and/or a GABA metabolic circuit, and a manganese transport circuit are inserted at four or more different chromosomal insertion sites
  • FIG. 46A and FIG.46B depict an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs.
  • an ammonia conversion circuit, a butyrate production circuit, and a GABA transport and/or GABA metabolic circuit are inserted at three different chromosomal insertion sites.
  • an ammonia conversion circuit, a GABA transport and/or GABA metabolic circuit, and a manganese transport circuit are inserted at three or more different chromosomal insertion sites.
  • FIG. 47A and FIG.47B depict exemplary schematics of the E. coli 1917 Nissle chromosome comprising multiple MoAs.
  • an ammonia conversion circuit, and a manganese transport circuit are inserted at two different chromosomal insertion sites.
  • an ammonia conversion circuit, and a GABA transport and/or GABA metabolic circuit are inserted at two or more different chromosomal insertion sites.
  • FIG. 48A depicts a schematic of a metabolic pathway for butyrate production.
  • FIG. 48B and 48C 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. 48D 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” refers 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 cassette that comprises at least the following genes: ter, thiA1, 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, thiA1, hbd, crt2, and tesB genes.
  • An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiA1, hbd, crt2, and tesB genes.
  • the tes or tesB cassette is under control of an inducible promoter other than tetracycline.
  • inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific 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.
  • oxygen level-dependent promoters e.g., FNR-inducible promoter
  • promoters induced by HE-specific molecules or metabolites indicative of liver damage e.g., bilirubin
  • RNS, ROS promoters promoters induced by inflammation or an inflammatory response
  • FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG.49E, FIG. 49F depict schematics showing the gene organization of exemplary engineered bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate.
  • FIG. 49A and 49B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.
  • FIG. 49A depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) prevents (indicated by“X”) FNR (grey boxed“FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”).
  • O2 oxygen
  • FNR grey boxed“FNR”
  • FIG. 49B 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.
  • FIG. 49C and 49D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 49C, in the absence of NO, the NsrR transcription factor (gray circle,“NsrR”) binds to and represses a
  • FIG. 49D in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG.49E and F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H2O2.
  • 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, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed.
  • the OxyR transcription factor interacts with H2O2 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.50A, FIG.50B, FIG.50C, FIG.50D, FIG.50E, and FIG.50F depict schematics showing the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate.
  • FIG. 50A and 50B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.
  • FIG. 50A depicts relatively low butyrate 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”).
  • FIG. 50B 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.
  • FIG. 50C and 50D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG.
  • the NsrR transcription factor (gray circle,“NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed.
  • the NsrR transcription factor in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 50E and 50F 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, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed.
  • FIG. 50F in the presence of H 2 O 2 , the OxyR transcription factor interacts with H 2 O 2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 50G, FIG. 50H, FIG. 50I, FIG. 50J, FIG.50K, and FIG. 50L depict schematics of the gene organization of exemplary bacteria of the disclosure.
  • FIG. 50G and FIG. 50H depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.
  • FIG. 50G depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by“X”) FNR (boxed“FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, and tesB) is expressed.
  • FIG. 50H depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed“FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.
  • the NsrR transcription factor (“NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed.
  • the NsrR in the presence of NO, the NsrR
  • FIG. 50K and FIG. 50L depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H 2 O 2 .
  • the OxyR transcription factor (circle,“OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed.
  • FIG. 50K in the absence of H 2 O 2 , the OxyR transcription factor (circle,“OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed.
  • the OxyR transcription factor interacts with H 2 O 2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.
  • FIG. 51A and FIG.51B depict graphs showing butyrate production using the circuits (SYN-UCD-503, SYN-UCD-504, SYN-UCD-505) shown in FIG. 48.
  • Cells were grown in M9 minimal media containing 0.2% glucose and induced with ATC at early log phase.
  • FIG. 51A similar amounts of butyrate were produced for each construct under aerobic vs anaerobic conditions. The ter strain produces more butyrate overall.
  • SYN-UCD503 comprises pLogic031 (bdc2 butyrate cassette under control of tet promoter on a plasmid) and SYN-UCD504 comprises pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid).
  • 51B depicts butyrate production of SYN-UCD504 (pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid)) and SYN-UCD505 (a Nissle strain comprising plasmid pLOGIC046-delta pbt.buk/tesB+, an ATC-inducible ter-comprising butyrate construct with a deletion in the pbt-buk genes and their replacement with the tesB gene).
  • the tesB construct results in greater butyrate production.
  • FIG. 52 depicts a graph of butyrate production using different butyrate- producing circuits comprising a nuoB gene deletion.
  • Strains depicted are SYN- UCD503, SYN-UCD504, SYN-UCD510 (SYN-UCD510 is the same as SYN-UCD503 except that it further comprises a nuoB deletion), and SYN-UCD511 (SYN-UCD511 is the same as SYN-UCD504 except that it further comprises a nuoB deletion).
  • the NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains.
  • NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production.
  • FIG. 53A depicts a schematic of a butyrate producing circuit under the control of an FNR promoter.
  • FIG. 53B depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes.
  • FIG. 53C depicts SYN- UCD501 in the presence and absence of glucose and oxygen in vitro.
  • SYN-UCD501 comprises pSC101 PydfZ-ter butyrate plasmid;
  • SYN-UCD500 comprises pSC101 PydfZ-bcd butyrate plasmid;
  • SYN-UCD506 comprises pSC101 nirB-bcd butyrate plasmid.
  • 53D 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 control SYN- UCD103.
  • FIG. 54A and FIG.54B depict bar graphs showing in vitro arginine (FIG. 54A) and butyrate (FIG. 54B) 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 Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)
  • SYN- UCD305 arginine producing/ammonia consuming strain comprising ⁇ ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, and ⁇ ThyA, with no antibiotic resistance
  • SYN-UCD601 butyrate producing and arginine
  • SYN-UCD601 is able to produce similar levels of arginine as SYN-UCD305 and similar levels of butyrate as SYN- UCD501 in vitro.
  • FIG. 55 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H2O, 100 mM butyrate in H20, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter ->pbt-buk butyrate plasmid.
  • H2O 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. 56 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 time points.
  • the Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.
  • FIG. 57A depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromosomally integrated butyrate copies as compared to plasmid copies.
  • Integrated butyrate strains, SYN1001 and SYN1002 both integrated at the agaI/rsml locus gave comparable butyrate production to the plasmid strain
  • FIG. 57B and FIG. 57C depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYN1001, on alkaline phosphatase activity in HT-29 cells represented in bar (FIG. 57B) and nonlinear fit (FIG. 57C) graphical formats.
  • FIG. 58A and FIG. 58B depict schematics of the gene organization of an exemplary engineered bacterium of the invention and its induction under low-oxygen conditions for the production of propionate.
  • FIG.58A 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 (pct, lcdA, lcdB, lcdC, etfA, acrB, acrC; black boxes) are expressed.
  • 58B depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • FIG. 58C and FIG. 58D depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate.
  • FIG. 58C 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, lpd; black boxes) are expressed.
  • 58D depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • FIG. 58E and FIG. 58F depict diagrams showing the gene organization of an exemplary engineered bacterium of the invention and its induction under low- oxygen conditions for the production of propionate.
  • FIG. 58E depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents
  • FIG. 58E depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
  • FIG. 59A, FIG. 59B, and FIG. 59C depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.
  • FIG. 59A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production.
  • the SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA.
  • FIG. 59B and FIG. 59C 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. 59A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production.
  • the SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA
  • FIG. 59B depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by“X”) FNR (boxed“FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfH) is expressed.
  • FIG. 59C depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed“FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.
  • propionate production is induced by NO or H 2 O 2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 49C-49F, FIG. 50C-50F, FIG. 50I-50L.
  • FIG. 59D depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 59B and FIG. 59C.
  • FIG. 60A and 60B depict diagrams of exemplary constructs which may be used to produce a positive feedback auxotroph and select for high arginine production.
  • FIG. 60A depicts a map of the astC promoter driving expression of thyA.
  • FIG. 60B depicts a schematic diagram of the thyA gene under the control of an astC promoter.
  • the regulatory region comprises binding sites for CRP, ArgR, and RNA polymerase (RNAP), and may also comprise additional regulatory elements.
  • FIG. 61 depicts another exemplary embodiment of an engineered bacterial strain to target urea cycle disorder (UCD), via the conversion of ammonia to desired products, such as citrulline or arginine.
  • the strain is deleted for the argR gene and expressing the feedback-resistant argAfbr gene.
  • this strain further comprises one or more auxotrophic modifications on the chromosome.
  • the synthetic biotic engineered to target urea cycle disorder (UCD) also has the kill-switch embodiment described in FIG. 65.
  • the Int recombinase and the Kid- Kis toxin-antitoxin system are used in a recombinant bacterial cell for treating UCD.
  • the recombinant bacterial cell is engineered to consume excess ammonia to produce beneficial byproducts to improve patient outcomes.
  • the recombinant bacterial cell also comprises a highly controllable kill switch to ensure safety.
  • the FNR promoter induces expression of the Int recombinase and also induces expression of the Kis anti-toxin.
  • the Int recombinase causes the Kid toxin gene to flip into an activated conformation, but the presence of the accumulated Kis anti-toxin suppresses the activity of the expressed Kid toxin.
  • oxygen e.g., outside the gut
  • expression of the anti-toxin is turned off. Since the toxin is constitutively expressed, it continues to accumulate and kills the bacterial cell.
  • FIG. 62 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.
  • FIG. 63 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin.
  • expression of the anti-toxin is turned off.
  • the toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
  • FIG. 64 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips at least one excision enzyme into an activated conformation.
  • the at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death.
  • the natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days.
  • the presence of multiple nested recombinases can be used to further control the timing of cell death.
  • FIG. 65 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters.
  • the recombinase then flips a second recombinase from an inverted orientation to an active conformation.
  • the activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the
  • 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. 66A depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P araBAD ), which induces expression of the Tet repressor (TetR) and an anti-toxin.
  • P araBAD ParaBAD promoter
  • TetR Tet repressor
  • FIG. 66A also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal.
  • FIG. 66B depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • FIG. 66C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal.
  • the AraC transcription factor adopts a conformation that represses transcription.
  • the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin.
  • TetR Tet repressor
  • the anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site).
  • both the anti-toxin and TetR are not expressed.
  • the araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.
  • FIG. 67A depicts the use of 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.67B-67E depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (FIG. 67B and FIG. 67C), which also contains a chromosomal component (shown in FIG. 67D and FIG. 67E).
  • the biosafety plasmid system vector comprises Kid Toxin and R6K minimal ori, dapA (FIG. 67B) and thyA (FIG. 67C) and promoter elements driving expression of these components.
  • bla is knocked out and replaced with one or more constructs described herein, in which a first protein of interest (POI1) and/or a second protein of interest, e.g., a transporter (POI2), and/or a third protein of interest (POI3) are expressed from an inducible or constitutive promoter.
  • FIG.67D and FIG. 67E depict schematics of the gene organization of the chromosomal component of a biosafety system.
  • FIG. 67D 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. 67E 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. If the plasmid containing the functional DapA is used (as shown in FIG. 67B), then the chromosomal constructs shown in FIG. 67D and FIG.
  • the chromosomal constructs shown in FIG. 67D and FIG. 67E are knocked into the ThyA locus.
  • the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid.
  • FIG. 68 depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin.
  • the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin.
  • the cell produces equal amounts of toxin and anti-toxin and is stable.
  • the cell loses the plasmid and anti-toxin begins to decay.
  • the anti-toxin decays completely, and the cell dies.
  • FIG. 69A 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. 69B 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. 69C 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. 69D 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. 70 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
  • FIG. 71A and FIG. 71B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., a gut barrier enhancer molecule, e.g., IL-22 or GLP-2, which are secreted via a diffusible outer membrane (DOM) system.
  • the therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space.
  • Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA).
  • the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpI.
  • 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. 71A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, FIG.
  • the one or more cassettes are under the control of constitutive promoters.
  • FIG. 72A, FIG. 72B, and FIG. 72C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., anti-cancer/immune modulatory effectors described herein, e.g., a gut barrier enhancer molecule, e.g., IL-22 or GLP-2, which are secreted using components of the flagellar type III secretion system.
  • a therapeutic polypeptide of interest is assembled behind a fliC-5’UTR, and is driven by the native fliC and/or fliD promoter (FIG. 72A and FIG. 72B) or a tet-inducible promoter (FIG. 72C).
  • 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. 72B and FIG. 72C.
  • FIG. 73 depicts a schematic of a polypeptide of interest displayed on the surface of the bacterium.
  • a non-limiting example of such a therapeutic protein is a scFv.
  • the polypeptide is expressed as a fusion protein, which comprises a outer membrane anchor from another protein, which was developed as part of a display system.
  • Non- limiting examples of such anchors are described herein and include LppOmpA,
  • NGIgAsig-NGIgAP NGIgAsig-NGIgAP, InaQ, Intimin, Invasin, pelB-PAL, and blcA/BAN.
  • bacterial strain which has one or more diffusible outer membrane phenotype (“leaky membrane”) mutation, e.g., as described herein.
  • FIG. 74 depicts an exemplary L-homoserine and L-methionine biosynthesis pathway. Circles indicate genes repressed by MetJ, and deletion of metJ leads to constitutive expression of these genes and activation of the pathway.
  • FIG. 75 depicts an exemplary histidine biosynthesis pathway.
  • FIG. 76 depicts an exemplary lysine biosynthesis pathway.
  • FIG. 77 depicts an exemplary asparagine biosynthesis pathway.
  • FIG. 78 depicts an exemplary glutamine biosynthesis pathway.
  • FIG. 79 depicts an exemplary tryptophan biosynthesis pathway.
  • FIG. 80 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 and sequences described herein. Different FNR-responsive promoters were used to create a library of anaerobic/low oxygen conditions inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+O 2 ) or anaerobic conditions (-O 2 ). Samples were removed at 4 hrs and the promoter activity based on ⁇ -galactosidase levels was analyzed by performing standard ⁇ -galactosidase colorimetric assays.
  • FIG. 81A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P fnrS ).
  • LacZ encodes the ⁇ -galactosidase enzyme and is a common reporter gene in bacteria.
  • FIG. 81B depicts FNR promoter activity as a function of ⁇ -galactosidase activity in 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). These data suggest that the fnrS promoter begins to drive high- level gene expression within 1 hr. under anaerobic and/or low oxygen conditions.
  • FIG. 81C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
  • FIG. 82 depicts ATC (FIG. 82A) or nitric oxide-inducible (FIG. 82B) 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 P tet -GFP reporter construct or the nitric oxide inducible P nsrR -GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units.
  • FIG. 82C depicts a schematic of the constructs.
  • FIG. 83 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. 84A 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
  • FIG. 84B depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct (SYN133) or the pLogic046- nsrR-norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001).
  • FIG. 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. To improve acetate production, while still maintaining high levels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production).
  • 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 line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG. 86A) and in 0.5% glucuronic acid MOPS (pH6.3) (FIG.86B).
  • FIG. 86C and FIG. 86D depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) (FIG.86C) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 86D).
  • Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter- pbt-buk butyrate cassettes.
  • SYN2006 comprises a FNRS ter-tesB cassette integrated at the HA1/2 locus and a deletion in the endogenous adhE gene.
  • SYN2007 comprises a FNRS ter-tesB cassette integrated at the HA1/2 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. 86E 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 HA1/2 locus of the chromosome (SYN1004) was compared with a strain comprising the same integrated cassette and additionally a deletion in the endogenous frd gene (SYN2005).
  • FIG. 86F 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 HA1/2 locus and deletions in the endogenous adhE and pta genes.
  • SYN2003 comprises FNRS- ter-pbt-buk butyrate cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene.
  • FIG. 86G and FIG. 86H depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFN ⁇ secretion in primary human PBMC cells from donor 1 (D1) (FIG.86G ) and donor 2 (D2) (FIG. 86H).
  • FIG. 87A and FIG.87B depict schematics of indole metabolite mode of action (FIG.36A) and indole biosynthesis (FIG. 87B).
  • FIG. 87A depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function.
  • PXR pregnane X receptors
  • I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production.
  • AhR aryl hydrocarbon receptor
  • Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections.
  • Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon-like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).
  • FIG. 87B depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways.
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 87A and 87B.
  • the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 87A and 87B, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde.
  • FIG. 88A and FIG. 88B depict diagrams of bacterial tryptophan metabolism pathways.
  • FIG. 88A 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 (EC1.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3);
  • FIG. 88B 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: Indole-3-pyruvic acid; IAM: Indole-3-acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3- acetonitrile; N-formyl Kyn: N-formylkynurenine;; Kyn:Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3’-Diindoly
  • Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2), EC 1.13.11.11 (Ido1); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (lad1), EC 1.2.3.7 (Aao1); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclb1, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125
  • the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 88A and 88B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 88A and 88B. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome.
  • the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • FIG. 89 depicts a schematic of the E. coli tryptophan synthesis pathway.
  • tryptophan is biosynthesized from chorismate, the principal common precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and enterobactin (enterochelin), as shown in the superpathway of chorismate metabolism.
  • Five genes encode five enzymes that catalyze tryptophan biosynthesis from chorismate.
  • the five genes trpE trpD trpC trpB trpA form a single transcription unit, the trp operon.
  • a weak internal promoter also exists within the trpD structural gene that provides low, constitutive levels of mRNA.
  • FIG. 90A, FIG. 90B, FIG. 90C, and FIG. 90D depicts schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan.
  • the genetically engineered bacteria comprise circuits for the production of tryptophan.
  • Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter.
  • the one or more cassettes are under the control of constitutive promoters.
  • Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.
  • the bacteria may also include an auxotrophy, e.g., deletion of thyA ( ⁇ thyA; thymidine dependence).
  • FIG. 90A 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. 90B, and/or FIG.90C, and/or FIG. 90D.
  • FIG. 90B 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. 90A and/or described in the description of FIG. 90A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90C, and/or FIG. 90D.
  • trpR and/or the tnaA gene are deleted to further increase levels of tryptophan produced.
  • FIG.90C depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes.
  • AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production.
  • the strain further comprises either a wild type or a feedback resistant SerA gene.
  • Escherichia coli serA-encoded 3- phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major
  • bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 90A and/or described in the description of FIG. 90A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90B, and/or FIG.90D.
  • 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. 90D 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. 90A and/or described in the description of FIG. 90A.
  • the bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90B, and/or FIG. 90C.
  • 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
  • FIG. 91A, FIG. 91B, FIG. 91D, FIG. 91D, FIG.91E, FIG. 91F, FIG. 91G, and FIG. 91H depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included.
  • FIG. 91A 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.
  • 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. 91B 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. 90A and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit for aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae) or aspC (aspartate
  • aminotransferase e.g., from E. coli, or taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp.
  • FIG. 91C 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D 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. 91D 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D 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. 91E 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan.
  • tryptophan can be imported through a transporter.
  • the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3- dioxygenase, e.g., from S.
  • FIG.91F 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. 90A and/or and/or FIG.
  • the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens) or BNA2 (indoleamine 2,3- dioxygenase, e.g., from S.
  • Afmid Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S.
  • GOT2 Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens, which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter.
  • an inducible promoter e.g., an FNR promoter.
  • FIG.91G 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. 90A and/or and/or FIG. 90B, and/or FIG.90C, and/or FIG. 90D 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.
  • FIG. 91H 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
  • FIG. 91A, FIG. 91B, FIG. 91D, FIG. 91D, FIG. 91E, FIG. 91F, FIG.91G and FIG. 91H 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. 92A, FIG. 92B, FIG. 92C, FIG. 92D, and FIG. 92E 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. In certain embodiments, 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. 90A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
  • 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 taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp.
  • trpDH Trptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-21048
  • ipdC Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae
  • iad1 Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis
  • AAO1 Indole-3- acetaldehyde oxidase, e.g., from Arabidopsis thaliana
  • an inducible promoter e.g., an FNR promoter.
  • FIG. 92B the optional circuits for tryptophan production are as depicted and described in FIG. 90A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
  • 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. coli) and or iad1 (Indole-3-acetaldehyde
  • dehydrogenase e.g., from Ustilago maydis
  • AAO1 Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana
  • an inducible promoter e.g., an FNR promoter.
  • FIG. 92C the optional circuits for tryptophan production are as depicted and described in FIG. 90A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
  • 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 taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana) or staO (L-tryptophan oxidase, e.g., from streptomyces sp.
  • trpDH Teryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-21078
  • yuc2 indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana
  • FIG. 92D the optional circuits for tryptophan production are as depicted and described in FIG. 90A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
  • 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. 92E the optional circuits for tryptophan production are as depicted and described in FIG. 90A.
  • the strain optionally comprises additional circuits as depicted and/or
  • 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 cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nit1 (Nitrilase, e.g., from Arabidopsis thaliana) and iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi), e.g., under the control of an inducible promoter e.g., an F
  • FIG. 92F the optional circuits for tryptophan production are as depicted and described in FIG. 90A.
  • the strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
  • 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 trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Ind
  • Enterobacter cloacae which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3- acetate.
  • the engineered bacterium shown in any of FIG. 92A, FIG. 92B, FIG. 92C, FIG. 92D, and FIG. 92E, FIG. 92F 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.
  • 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. 93A, FIG. 93B, and FIG. 93C depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid.
  • the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid.
  • Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter.
  • the one or more cassettes are under the control of constitutive promoters.
  • Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR- inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline.
  • the bacteria may also include an auxotrophy, e.g., deletion of thyA ( ⁇ thyA; thymidine dependence).
  • 93A 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. 90A and/or FIG. 90B and/or FIG.90C and/or FIG. 90D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.
  • FIG. 93B 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. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG. 90D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate.
  • trpDH Traptophan de
  • FIG. 93C 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. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG.90D. Additionally, the strain comprises a circuit as described in FIG. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG.90D. 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 AcuI: (indole-3-
  • the circuits further comprise fldH1 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).
  • fldH1 and/or fldH2 indole-3-lactate dehydrogenase 1 and/or 2, e.g., from Clostridium sporogenes
  • FIG. 94A and FIG.94B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production.
  • FIG. 94A and FIG.94B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production.
  • 94A 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.
  • DHQ synthase Dehydroquinate synthase
  • DHQ synthase is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids.
  • DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to dehydroquinate (DHQ).
  • DAHP 3-deoxy-D-arabino-heptulosonic acid 7-phosphate
  • DHQ dehydroquinate dehydratase 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 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.
  • AroA 3- Phosphoshikimate-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
  • AroC Chorismate synthase
  • This enzyme catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system.
  • TrpEDCAB E coli trp operon: TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L- glutamate.
  • Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5'-phosphoribosyl)-anthranilate.
  • the phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein.
  • 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 ⁇ ) functions as the ⁇ subunit of the tetrameric ( ⁇ 2- ⁇ 2) tryptophan synthase complex.
  • TrpB polypeptide functions as the ⁇ subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the ⁇ reaction.
  • TnaA Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L- tryptophan to indole, pyruvate and NH4+.
  • PheA Bifunctional chorismate mutase / prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis.
  • TyrA Bifunctional chorismate mutase / prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis.
  • TyrB, ilvE, AspC Tyrosine
  • 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
  • TyrB overlaps with the catalytic activities of branched- chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine.
  • SerA D- 3-phosphoglycerate dehydrogenase catalyzes the first committed step in the
  • SerC The serC-encoded enzyme
  • FIG. 94B 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.
  • bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEfbr, and/or TrpD, and/or TrpC, and/or TrpA, and/or TrpB.
  • endogenous TnaA is knocked out through mutation(s) and/or deletion(s).
  • bacteria are engineered to comprise one or more additional copies of SerA.
  • bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter.
  • endogenous PheA is knocked out through mutation(s) and/or deletion(s).
  • two or more of the strategies depicted in the schematic of FIG. 94B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.
  • FIG.95A and FIG. 95B and FIG. 95C depict bar graphs showing tryptophan production by various engineered bacterial strains.
  • FIG.95A 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. Additionally, using a feedback resistant trpE (trpE fbr ) has a positive effect on tryptophan production.
  • AroG fbr AroG fbr
  • 95B shows tryptophan production from a strain comprising a tet-trpE fbr DCBA, tet-aroG fbr construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically).
  • 95C depicts a bar graph showing improved tryptophan production by engineered strain comprising ⁇ trpR ⁇ tnaA, tet-trpE fbr DCBA, tet-aroG fbr through the addition of serine.
  • FIG. 96 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.
  • SYN2339 comprises ⁇ trpR ⁇ tnaA, tet-aroGfbr, tet-trpEfbrDCBA.
  • SYN2473 comprises ⁇ trpR ⁇ tnaA, tet-aroGfbr-serA, tet- trpEfbrDCBA.
  • SYN2476 comprises ⁇ trpR ⁇ tnaA, 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. 97 depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit.
  • IPA indole-3-propionic acid
  • FIG. 97 depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit.
  • IPA produced by the gut microbiota has a significant positive effect on barrier integrity.
  • IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate
  • 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
  • WAL Tryptophan ammonia lyase
  • indole-3-acrylate reductase e.g., from Clostridum botulinum
  • the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein.
  • FIG. 98 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 Nostoc punctiforme NIES-2108;
  • FldH1/FldH2 indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes
  • FldA indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes
  • FldBC indole-3-lactate dehydratase, e.g., from Clostridium sporogenes
  • FldD indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes
  • AcuI indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes
  • FldA indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes
  • acrylyl-CoA reductase e.g., from Rhodobacter sphaeroides.
  • lpdC Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae;
  • lad1 Indole-3-acetaldehyde
  • dehydrogenase e.g., from Ustilago maydis
  • Tdc Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
  • Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3- yl)pyruvate (IPyA), NH 3 , NAD(P)H and H + .
  • 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 (IPA) and indole-3-lactate-CoA.
  • Indole-3-acrylyl-CoA reductase FldD
  • acrylyl-CoA reductase AcuI
  • Indole-3-lactate dehydratase (FldBC ) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA.
  • the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 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 (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
  • FIG. 99 depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342.
  • SYN2126 comprises ⁇ trpR and ⁇ tnaA ( ⁇ trpR ⁇ tnaA).
  • SYN2339 comprises circuitry for the production of tryptophan ( ⁇ trpR ⁇ tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)).
  • SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iad1 incorporated at the end of the second construct ( ⁇ trpR ⁇ tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH- ipdC-iad1 (p15A)).
  • SYN2126 produced no tryptophan
  • SYN2339 produces increasing tryptophan over the time points measured
  • SYN2342 converts all trypophan it produces into IAA.
  • FIG. 100 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 ⁇ trpR ⁇ tnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr (p15A).
  • SYN2340 comprises ⁇ trpR ⁇ tnaA, (p15A).
  • SYN2794 comprises ⁇ trpR ⁇ tnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr - tdc Cs (p15A).
  • Results indicate that Tdc Cs from Clostridium sporogenes is more efficient the Tdc Cr from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.
  • FIG. 101A and FIG. 101B depict line graphs of ELISA results.
  • FIG. 101A 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. 101B depicts a line graph, showing an phopho- STAT3 (Tyr705) ELISA showing an antibody completion assay.
  • Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody.
  • the data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.
  • FIG. 101C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation.
  • FIG. 103A depicts a“Oxygen bypass switch” useful for aerobic pre- induction of a strain comprising one or proteins of interest (POI), e.g., one or more anti- cancer molecules or immune modulatory effectors (POI1) and a second set of one or more proteins of interest (POI2), e.g., one or more transporter(s)/importer(s) and/or exporter(s), under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture).
  • a culture vessel e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.
  • a strain with active effector molecules prior to administration. This can be done by pre-inducing the expression of these effectors as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration.
  • strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more effectors or proteins of interest.
  • FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis AJ, The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety).
  • FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions.
  • strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs.
  • This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo.
  • a LacI 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. 103B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of
  • FNRS24Y FNRS24Y.
  • the levels of Fnr S24Y 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. Bioinformatics tools for optimization of RBS are known in the art.
  • FIG. 103C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy.
  • Bioinformatics 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 P fnrS -POI constructs are maintained to allow for strong in vivo induction.
  • FIG. 104 depicts the gene organization of an exemplary construct, e.g., comprised in SYN-PKU401, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene.
  • FIG. 105 depicts the gene organization of an exemplary construct comprising LacI in reverse orientation, and a IPTG inducible promoter driving the expression of one or more POIs.
  • this construct is useful for pre- induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG.
  • inducer e.g., IPTG.
  • this construct is used alone.
  • the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs.
  • the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.
  • the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some
  • the construct is used in combination with construct expressing a second POI, e.g., a transporter, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • a second POI e.g., a transporter
  • POI2 expression may be
  • the construct is located on a plasmid, e.g., a low or high copy plasmid.
  • the construct is employed in a biosafety system, such as the system shown in FIG. 67A, FIG. 67B, FIG.67C, and FIG. 67D, FIG. 67E.
  • the construct is integrated into the genome at one or more locations described herein.
  • FIG. 106A, FIG. 106B, and FIG. 106C depict schematics of non- limiting examples of constructs for the expression of proteins of interest POI(s).
  • FIG. 106A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter.
  • the construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI.
  • the temperature sensitive CI repressor mutant, CI857 binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone.
  • the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs.
  • the construct allows pre-induction and pre-loading of a POI1 and/or a POI2 prior to in vivo 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 a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive.
  • the construct is used in combination with a POI3 expression construct.
  • a temperature sensitive system can be used to set up a conditional auxotrophy.
  • a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system.
  • the strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower).
  • FIG. 106B 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.
  • 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.
  • the construct allows pre- induction and pre-loading of POI and/or POI2 and/or POI3 prior to in vivo
  • 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.
  • the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive.
  • the construct is used in combination with a POI3 expression construct.
  • FIG. 106C depicts a schematic of a non-limiting example of the organization of a construct for the expression of protein(s) of interest POI(s) 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 POI. 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 POI1 and/or POI2 and/or POI3 prior to in vivo
  • 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.
  • the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations.
  • POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive.
  • the construct is used in combination with a POI3 expression construct.
  • FIG. 107A 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. 107B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction.
  • the Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions.
  • the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest.
  • the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions.
  • This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control.
  • this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out.
  • dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine.
  • dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine.
  • Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).
  • FIG. 108A depicts a schematic diagram of a wild-type clbA construct.
  • FIG. 108B depicts a schematic diagram of a clbA knockout construct.
  • FIG. 109 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animal disease model; 9. Assimilate into the microbiome; 10.
  • FIGs. 110A, 110B, 110C, 110D, and 110E depict a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.
  • FIG. 110A depicts the parameters for starter culture 1 (SC1): loop full– glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm.
  • FIG. 110B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C, shaking at 250 rpm.
  • SC1 starter culture 1
  • SC2 starter culture 2
  • 110C 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. 110D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1X 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS.
  • FIG. 110E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
  • FIG. 111 depicts graphs of breath versus blood in a subset of 10 subjects. Description of Embodiments
  • the invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating or treating disorders associated with hyperammonemia, e.g., urea cycle disorders, hepatic encephalopathy and other disorders associated with excess ammonia or elevated ammonia levels.
  • the genetically engineered bacteria are capable of reducing excess ammonia, particularly under certain environmental conditions, such as those in the mammalian gut.
  • the genetically engineered bacteria reduce excess ammonia by incorporating excess nitrogen in the body into non-toxic molecules, e.g., arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, or tryptophan.
  • the genetically engineered bacteria reduce excess ammonia and also reduce one or more other toxic substances, e.g., GABA and/or manganese. In some embodiments, the genetically engineered bacteria reduce excess ammonia and also reduce GABA levels, e.g., by importing GABA and/or by metabolizing GABA. In some embodiments, the genetically engineered bacteria reduce excess ammonia and also reduce manganese levels, e.g., by importing manganese.
  • the genetically engineered bacteria may additionally produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia (e.g., UCDs, HE, etc).
  • the genetically engineered bacteria may also produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia.
  • the genetically engineered bacteria produce a short chain fatty acid, e.g., butyrate, propionate, and/or acetate.
  • the engineered bacteria reduce excess ammonia and produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate.
  • the engineered bacteria reduce excess ammonia, reduce one or more other toxic substances, e.g., GABA and/or manganese, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate.
  • the genetically engineered bacteria reduce excess ammonia, reduce GABA levels, e.g., by importing GABA and/or by metabolizing GABA, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate.
  • the genetically engineered bacteria reduce excess ammonia, reduce manganese levels, e.g., by importing manganese, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate.
  • the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits.
  • auxotrophies such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy
  • kill switch circuits such as any of the kill-switches described herein or otherwise known in the art
  • antibiotic resistance circuits such as antibiotic resistance circuits
  • transporters for importing biological molecules or substrates
  • any one or more of the payload or therapeutic circuits may be regulated by a constitutive promoter.
  • the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • any one or more of the payload or therapeutic circuits may be regulated by a tissue-specific promoter.
  • the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • any one or more of the payload or therapeutic circuits may be regulated by an inducible promoter.
  • the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • any one or more of the payload or therapeutic circuits may be regulated by an inducible promoter that is responsive to environmental conditions, factors, or cues, e.g., environmental conditions, factors, or cues found in the mammalian gut.
  • Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific 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.
  • oxygen level-dependent promoters e.g., FNR-inducible promoter
  • promoters induced by HE-specific molecules or metabolites indicative of liver damage e.g., bilirubin
  • RNS inflammatory response
  • 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 tetra
  • any one or more of the payload or therapeutic circuits e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits
  • any one or more of the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • any one or more of the payload or therapeutic circuits e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits
  • any one or more of the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • the additional circuits e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits
  • Hyperammonemia is used to refer to increased concentrations of ammonia in the body. Hyperammonemia is caused by decreased detoxification and/or increased production of ammonia.
  • Decreased detoxification may result from urea cycle disorders (UCDs), such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or from bypass of the liver, e.g., open ductus hepaticus; and/or deficiencies in glutamine synthetase (Hoffman et al., 2013; Häberle et al., 2013).
  • UTDs urea cycle disorders
  • Decreased detoxification may also result from liver disorders such as hepatic encephalopathy, acute liver failure, or chronic liver failure; and neurodegenerative disorders such as Huntington’s disease (Chen et al., 2015; Chiang et al., 2007). Increased production of ammonia may result from infections, drugs, neurogenic bladder, and intestinal bacterial overgrowth (Häberle et al., 2013).
  • liver disorders such as hepatic encephalopathy, acute liver failure, or chronic liver failure
  • organic acid disorders such as isovaleric aciduria; 3- methylcrotonylglycinuria; methylmalonic acidemia; propionic aciduria; propinic acidemia; fatty acid oxidation defects; carnitine cycle defects; carnitine deficiency; ⁇ - oxidation deficiency; lysinuric protein intolerance; pyrroline-5-carboxylate synthetase deficiency; pyruvate carboxylase deficiency; ornithine aminotransferase deficiency; carbonic anhydrase deficiency; hyperinsulinism-hyperammonemia syndrome;
  • Reye's syndrome is a rare condition primarily affecting the liver and brain. The disorder most commonly develops in children ages 5 to 14 after an otherwise unremarkable viral illness. An elevated blood ammonia level characteristically occurs in patients with Reye's syndrome, leading to brain swelling and ammonia toxicity.
  • a diagnostic signal of hyperammonemia is a plasma ammonia concentration of at least about 50 ⁇ mol/L, at least about 80 ⁇ mol/L, at least about 150 ⁇ mol/L, at least about 180 ⁇ mol/L, or at least about 200 ⁇ mol/L (Leonard, 2006; Hoffman et al., 2013; Häberle et al., 2013).
  • “Ammonia” is used to refer to gaseous ammonia (NH3), ionic ammonia (NH4+), or a mixture thereof. In bodily fluids, gaseous ammonia and ionic ammonium exist in equilibrium:
  • ammonia may refer to gaseous ammonia, ionic ammonia, and/or total ammonia.
  • “Detoxification” of ammonia is used to refer to the process or processes, natural or synthetic, by which toxic ammonia is removed and/or converted into one or more non-toxic molecules, including but not limited to: arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, tryptophan, or urea.
  • the urea cycle for example, enzymatically converts ammonia into urea for removal from the body in the urine. Because ammonia is a source of nitrogen for many amino acids, which are synthesized via numerous biochemical pathways, enhancement of one or more of those amino acid biosynthesis pathways may be used to incorporate excess nitrogen into non- toxic molecules.
  • arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms, thereby incorporating excess nitrogen into non-toxic molecules.
  • glutamate which comprises one nitrogen atom
  • arginine which comprises four nitrogen atoms
  • citrulline is not reabsorbed from the large intestine, and as a result, excess citrulline in the large intestine is not considered to be harmful.
  • Arginine biosynthesis may also be modified to produce citrulline as an end product; citrulline comprises three nitrogen atoms and thus the modified pathway is also capable of incorporating excess nitrogen into non-toxic molecules.
  • “Arginine regulon,”“arginine biosynthesis regulon,” and“arg regulon” are used interchangeably to refer to the collection of operons in a given bacterial species that comprise the genes encoding the enzymes responsible for converting glutamate to arginine and/or intermediate metabolites, e.g., citrulline, in the arginine biosynthesis pathway.
  • the arginine regulon also comprises operators, promoters, ARG boxes, and/or regulatory regions associated with those operons.
  • the arginine regulon includes, but is not limited to, the operons encoding the arginine biosynthesis enzymes N- acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine
  • the arginine regulon comprises an operon encoding ornithine acetyltransferase and associated operators, promoters, ARG boxes, and/or regulatory regions, either in addition to or in lieu of N- acetylglutamate synthetase and/or N-acetylornithinase.
  • one or more operons or genes of the arginine regulon may be present on a plasmid in the bacterium.
  • a bacterium may comprise multiple copies of any gene or operon in the arginine regulon, wherein one or more copies may be mutated or otherwise altered as described herein.
  • One gene may encode one enzyme, e.g., N-acetylglutamate synthetase (argA).
  • Two or more genes may encode distinct subunits of one enzyme, e.g., subunit A and subunit B of carbamoylphosphate synthase (carA and carB).
  • two or more genes may each independently encode the same enzyme, e.g., ornithine transcarbamylase (argF and argI).
  • the arginine regulon includes, but is not limited to, argA, encoding N-acetylglutamate synthetase; argB, encoding N- acetylglutamate kinase; argC, encoding N-acetylglutamylphosphate reductase; argD, encoding acetylornithine aminotransferase; argE, encoding N-acetylornithinase; argG, encoding argininosuccinate synthase; argH, encoding argininosuccinate lyase; one or both of argF and argI, each of which independently encodes ornithine transcarbamylase; carA, encoding the small subunit of carbamoylphosphate synthase; carB, encoding the large subunit of carbamoylphosphate synthase; operons thereof; operators thereof
  • the arginine regulon comprises argJ, encoding ornithine acetyltransferase (either in addition to or in lieu of N-acetylglutamate synthetase and/or N- acetylornithinase), operons thereof, operators thereof, promoters thereof, ARG boxes thereof, and/or regulatory regions thereof.
  • “Arginine operon,”“arginine biosynthesis operon,” and“arg operon” are used interchangeably to refer to a cluster of one or more of the genes encoding arginine biosynthesis enzymes under the control of a shared regulatory region comprising at least one promoter and at least one ARG box.
  • the one or more genes are co-transcribed and/or co-translated. Any combination of the genes encoding the enzymes responsible for arginine biosynthesis may be organized, naturally or synthetically, into an operon. For example, in B.
  • subtilis the genes encoding N- acetylglutamylphosphate reductase, N-acetylglutamate kinase, N-acetylornithinase, N- acetylglutamate kinase, acetylornithine aminotransferase, carbamoylphosphate synthase, and ornithine transcarbamylase are organized in a single operon, argCAEBD-carAB- argF, under the control of a shared regulatory region comprising a promoter and ARG boxes.
  • argCAEBD-carAB- argF the genes encoding N- acetylglutamylphosphate reductase, N-acetylglutamate kinase, N-acetylornithinase, N- acetylglutamate kinase, acetylornithine aminotransferase, carbamoyl
  • the genes encoding N-acetylornithinase, N- acetylglutamylphosphate reductase, N-acetylglutamate kinase, and argininosuccinate lyase are organized in two bipolar operons, argECBH.
  • the operons encoding the enzymes responsible for arginine biosynthesis may be distributed at different loci across the chromosome. In unmodified bacteria, each operon may be repressed by arginine via ArgR.
  • arginine and/or intermediate byproduct production may be altered in the genetically engineered bacteria of the invention by modifying the expression of the enzymes encoded by the arginine biosynthesis operons as provided herein.
  • Each arginine operon may be present on a plasmid or bacterial chromosome.
  • multiple copies of any arginine operon, or a gene or regulatory region within an arginine operon may be present in the bacterium, wherein one or more copies of the operon or gene 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 product (e.g., operon or gene or regulatory region) to enhance copy number or to comprise multiple different components of an operon performing multiple different functions.
  • “ARG box consensus sequence” refers to an ARG box nucleic acid sequence, the nucleic acids of which are known to occur with high frequency in one or more of the regulatory regions of argR, argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and/or carB.
  • each arg operon comprises a regulatory region comprising at least one 18-nucleotide imperfect palindromic sequence, called an ARG box, that overlaps with the promoter and to which the repressor protein binds (Tian et al., 1992).
  • the nucleotide sequences of the ARG boxes may vary for each operon, and the consensus ARG box sequence is A/T nTGAAT A/T A/T T/A T/A ATTCAn T/A (Maas, 1994).
  • the arginine repressor binds to one or more ARG boxes to actively inhibit the transcription of the arginine biosynthesis enzyme(s) that are operably linked to that one or more ARG boxes.
  • “Mutant arginine regulon” or“mutated arginine regulon” is used to refer to an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of each of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct, e.g., citrulline, in the arginine biosynthesis pathway, such that the mutant arginine regulon produces more arginine and/or intermediate byproduct than an unmodified regulon from the same bacterial subtype under the same conditions.
  • an intermediate byproduct e.g., citrulline
  • the genetically engineered bacteria comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr, and a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
  • an arginine feedback resistant N- acetylglutamate synthase mutant e.g., argAfbr
  • a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that
  • the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis.
  • the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr, a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor.
  • an arginine feedback resistant N-acetylglutamate synthase mutant e.g., argAfbr
  • a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor.
  • the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr and a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes.
  • the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr and a mutant or deleted arginine repressor.
  • the mutant arginine regulon comprises an operon encoding wild-type N-acetylglutamate synthetase and one or more nucleic acid mutations in at least one ARG box for said operon. In some embodiments, the mutant arginine regulon comprises an operon encoding wild-type N- acetylglutamate synthetase and mutant or deleted arginine repressor.
  • the mutant arginine regulon comprises an operon encoding ornithine acetyltransferase (either in addition to or in lieu of N-acetylglutamate synthetase and/or N-acetylornithinase) and one or more nucleic acid mutations in at least one ARG box for said operon.
  • the ARG boxes overlap with the promoter in the regulatory region of each arginine biosynthesis operon.
  • the regulatory region of one or more arginine biosynthesis operons is sufficiently mutated to disrupt the palindromic ARG box sequence and reduce ArgR binding, but still comprises sufficiently high homology to the promoter of the non-mutant regulatory region to be recognized as the native operon-specific promoter.
  • the operon comprises at least one nucleic acid mutation in at least one ARG box such that ArgR binding to the ARG box and to the regulatory region of the operon is reduced or eliminated.
  • bases that are protected from DNA methylation and bases that are protected from hydroxyl radical attack during ArgR binding are the primary targets for mutations to disrupt ArgR binding (see, e.g., Table 3).
  • the promoter of the mutated regulatory region retains sufficiently high homology to the promoter of the non-mutant regulatory region such that RNA polymerase binds to it with sufficient affinity to promote transcription of the operably linked arginine biosynthesis enzyme(s).
  • the G/C:A/T ratio of the promoter of the mutant differs by no more than 10% from the G/C:A/T ratio of the wild-type promoter.
  • more than one ARG box may be present in a single operon.
  • At least one of the ARG boxes in an operon is altered to produce the requisite reduced ArgR binding to the regulatory region of the operon. In an alternate aspect of these embodiments, each of the ARG boxes in an operon is altered to produce the requisite reduced ArgR binding to the regulatory region of the operon.
  • ArgR binding is used to refer to a reduction in repressor binding to an ARG box in an operon or a reduction in the total repressor binding to the regulatory region of said operon, as compared to repressor binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions.
  • ArgR binding to a mutant ARG box and regulatory region of an operon is at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, at least about 90% lower, or at least about 95% lower than ArgR binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions.
  • reduced ArgR binding to a mutant ARG box and regulatory region results in at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold increased mRNA expression of the one or more genes in the operon.
  • “ArgR” or“arginine repressor” is used to refer to a protein that is capable of suppressing arginine biosynthesis by regulating the transcription of arginine biosynthesis genes in the arginine regulon.
  • argR arginine repressor protein
  • expression of the gene that encodes for the arginine repressor protein (“argR”) is increased in a wild-type bacterium, arginine biosynthesis is decreased.
  • expression of argR is decreased in a wild-type bacterium, or if argR is deleted or mutated to inactivate arginine repressor function, arginine biosynthesis is increased.
  • Bacteria that“lack any functional ArgR” and“ArgR deletion bacteria” are used to refer to bacteria in which each arginine repressor has significantly reduced or eliminated activity as compared to unmodified arginine repressor from bacteria of the same subtype under the same conditions. Reduced or eliminated arginine repressor activity can result in, for example, increased transcription of the arginine biosynthesis genes and/or increased concentrations of arginine and/or intermediate byproducts, e.g., citrulline. Bacteria in which arginine repressor activity is reduced or eliminated can be generated by modifying the bacterial argR gene or by modifying the transcription of the argR gene. For example, the chromosomal argR gene can be deleted, can be mutated, or the argR gene can be replaced with an argR gene that does not exhibit wild-type repressor activity.
  • “Operably linked” refers a nucleic acid sequence, e.g., a gene encoding feedback resistant ArgA, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis.
  • a regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • 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.
  • the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage.
  • the metabolites may be gut specific.
  • the metabolite may be associated with hepatic encephalopathy, e.g., bilirubin.
  • hepatic encephalopathy e.g., bilirubin.
  • molecules or metabolites include, 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, and manganese in their blood and intestines.
  • Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein.
  • the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter.
  • the genetically engineered bacteria comprise a promoter 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.
  • “Exogenous environmental condition(s)” refer to setting(s) or circumstance(s) under which the promoter described herein is induced.
  • the phrase “exogenous environmental conditions” is meant to refer to the environmental conditions external to the engineered micororganism, but endogenous or native to the host subject environment.
  • “exogenous” and“endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are
  • the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous
  • exogenous environmental conditions are specific to the small intestine of a mammal.
  • exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease state (e.g., HE).
  • 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). In some embodiments, the exogenous
  • the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter.
  • the genetically engineered microorganism of the diclosure comprise an oxygen level-dependent promoter.
  • bacteria have evolved transcription factors that are capable of sensing oxygen levels. 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, ANR, and DNR.
  • FNR-responsive promoters include, but are not limited to, FNR, ANR, and DNR.
  • ANR-responsive promoters include, but are not limited to, ANR, and DNR.
  • DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.
  • a promoter was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010).
  • the PfnrS promoter is activated under anaerobic conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression.
  • PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA.
  • PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS.
  • “Gut barrier function enhancer molecules” include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF- ⁇ 1, TGF- ⁇ 2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, and kynurenine.
  • a gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene.
  • a gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. These molecules may also be referred to as therapeutic molecules.
  • a“gene cassette” or“operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce a gut barrier function enhancer molecule, e.g., butyrate, propionate.
  • the gene cassette or operon may also comprise additional transcription and translation elements, e.g., a ribosome binding site.
  • A“butyrogenic gene cassette,”“butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway.
  • Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio,
  • 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, thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013).
  • One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk.
  • a butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 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 thiA1, 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, thiA1, hbd, crt2, and tesB.
  • a“propionate gene cassette” or“propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway.
  • Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum,
  • the genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, which encode propionate CoA- transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyzes the reduction of lactate to propionate. Dehydration of (R)-lactoy
  • 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 acuI 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
  • the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI.
  • the homolog of AcuI 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 pct, lcdA, lcdB, lcdC, 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,
  • 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. by the genetically engineered bacteria of the disclosure.
  • 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 converts succinyl CoA to L- methylmalonylCoA
  • YgfD is a Sbm-interacting protein kinase with GTPase activity
  • ygfG methylmalonylCoA decarboxylase
  • PropionylCoA, and ygfH converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009).
  • 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).
  • 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.
  • the genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria.
  • Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994).
  • Several bacteria such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or CO 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.
  • “GABA” and“ ⁇ -aminobutyric acid” are used to refer to the predominant inhibitory neurotransmitter (C 4 H 9 NO 2 ) in the mammalian central nervous system. In humans, GABA is also directly responsible for regulating muscle tone. GABA is capable of activating the GABA A receptor, which is part of a ligand-gated ion channel complex, as well as the GABA B metabotropic G protein-coupled receptor. Neurons that produce GABA are known as“GABAergic” neurons, and activation of GABA receptors is described as GABAergic tone (i.e., increased activation of GABA receptors refers to increased GABAergic tone).
  • “GABA transporter” and“GabP” are used to refer to a membrane transport protein that is capable of transporting GABA into bacterial cells (see, e.g., Li et al., 2001).
  • the gabP gene encodes a high-affinity GABA permease responsible for GABA transport (Li et al., 2001).
  • the GABA transporter is encoded by a gabP gene derived from a bacterial species, including but not limited to, Bacillus subtilis and Escherichia coli.
  • These endogenous GABA transporter genes may be a source of genes for the genetically engineered bacteria of the invention. Any suitable gene(s) encoding a GABA transporter may be used.
  • “Manganese” refers to a chemical element with the symbol“Mn” and atomic number 25.
  • manganese is an essential trace metal and plays an important role in enzyme-mediated catalysis, but can also have deleterious effects.
  • Cells maintain manganese under tight homeostatic control in order to avoid toxicity.
  • Some disorders associated with hyperammonemia may also be characterized by elevated levels of manganese; manganese may contribute to disease pathogenesis (e.g., hepatic encephalopathy) (Rivera-Manc ⁇ a et al., 2012).
  • “Manganese transporter” and“MntH” refer to a membrane transport protein that is capable of transporting manganese into bacterial cells (see, e.g., Jensen and Jensen, 2014).
  • the mntH gene encodes a proton-stimulated, divalent metal cation uptake system involved in manganese transport (Porcheron et al., 2013).
  • the manganese transporter is encoded by a mntH gene derived from a bacterial species, including but not limited to, Salmonella typhimurium, Shigella flexneri, Yersinia pestis, and Escherichia coli.
  • These endogenous manganese transporter genes may be a source of genes for the genetically engineered bacteria of the invention. Any suitable gene(s) encoding a manganese transporter may be used.
  • a“non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype.
  • the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013).
  • the non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette.
  • “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
  • the non- native nucleic acid sequence e.g., gene or gene cassette, may be present on a plasmid or bacterial chromosome.
  • the genetically engineered bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR- responsive promoter operably linked to a butyrogenic gene cassette, or an arginine production cassette.
  • a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR- responsive promoter operably linked to a butyrogenic gene cassette, or an arginine production cassette.
  • multiple copies of the gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies may be mutated or otherwise altered as described herein.
  • the genetically engineered bacteria are engineered to comprise multiple copies of the same non-native nucleic acid sequence, e.g., 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.
  • non-native nucleic acid sequence e.g., gene, gene cassette, or regulatory region
  • “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked.
  • Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive
  • Escherichia coli ⁇ S promoter e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)
  • a constitutive Escherichia coli ⁇ 32 promoter e.g., htpG heat shock promoter (BBa_J45504)
  • a constitutive Escherichia coli ⁇ 70 promoter e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter
  • BBa_K119000 BBa_K119001
  • M13K07 gene I promoter BBa_M13101
  • M13K07 gene II promoter BBa_M13102
  • M13K07 gene III promoter BBa_M13103
  • M13K07 gene IV promoter BBa_M13104
  • M13K07 gene V promoter BBa_M13105
  • M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter
  • BBa_M13108 M13110 (BBa_M13110)
  • a constitutive Bacillus subtilis ⁇ A promoter e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P liaG (BBa_K823000), P lepA (BBa_K823002), P veg (BBa_K823003)
  • a constitutive Bacillus subtilis ⁇ B promoter e.g., promoter ctc (BBa_K143010), promoter gsiB
  • BBa_K143011 a Salmonella promoter (e.g., Pspv2 from Salmonella
  • BBa_K112706 Pspv from Salmonella (BBa_K112707)
  • a bacteriophage T7 promoter e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997;
  • BBa_K113010 BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180;
  • SP6 promoter e.g., SP6 promoter (BBa_J64998)
  • genetically engineered bacteria that“overproduce” arginine or an intermediate byproduct refer to bacteria that comprise a mutant arginine regulon.
  • the engineered bacteria may comprise a feedback resistant form of ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or intermediate byproduct than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria may alternatively or further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes.
  • the genetically engineered bacteria may alternatively or further comprise a mutant or deleted arginine repressor.
  • the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more arginine than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more citrulline or other intermediate byproduct than unmodified bacteria of the same subtype under the same conditions.
  • the mRNA transcript levels of one or more of the arginine biosynthesis genes in the genetically engineered bacteria are at least about 1.5-fold, at least about 2- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold higher than the mRNA transcript levels in unmodified bacteria of the same subtype under the same conditions.
  • the unmodified bacteria will not have detectable levels of arginine, intermediate byproduct, and/or transcription of the gene(s) in such operons.
  • protein and/or transcription levels of arginine and/or intermediate byproduct will be detectable in the corresponding genetically engineered bacterium having the mutant arginine regulon. Transcription levels may be detected by directly measuring mRNA levels of the genes. Methods of measuring arginine and/or intermediate byproduct levels, as well as the levels of transcript expressed from the arginine biosynthesis genes, are known in the art. Arginine and citrulline, for example, may be measured by mass spectrometry.
  • “Gut” refers to the organs, glands, tracts, and systems that are
  • the gut comprises the gastrointestinal tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine.
  • the gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas.
  • the upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine.
  • the lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal.
  • Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.
  • the term“low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O 2 ) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., ⁇ 21% O 2; ⁇ 160 torr O 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 (O 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.
  • O 2 oxygen
  • the term “low oxygen” is meant to refer to a level, amount, or concentration of O 2 that is 0-60 mmHg O 2 (0-60 torr O 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 O 2 ), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O 2 , 0.75 mmHg O 2 , 1.25 mmHg O 2 , 2.175 mmHg O 2 , 3.45 mmHg O 2 , 3.75 mmHg O 2 , 4.5 mmHg O 2 , 6.8 mmHg O 2
  • “low oxygen” refers to about 60 mmHg O 2 or less (e.g., 0 to about 60 mmHg O 2) .
  • the term“low oxygen” may also refer to a range of O 2 levels, amounts, or concentrations between 0-60 mmHg O 2 (inclusive), e.g., 0-5 mmHg O2, ⁇ 1.5 mmHg O2, 6-10 mmHg, ⁇ 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way.
  • the term“low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O 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 (O 2 ) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions.
  • O 2 oxygen
  • Table 2 summarizes the amount of oxygen present in various organs and tissues.
  • DO dissolved oxygen
  • the term“low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O 2 ) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way.
  • the level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (O2) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium).
  • Well-aerated solutions e.g., solutions subjected to mixing and/or stirring
  • oxygen producers or consumers are 100% air saturated.
  • the term“low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%.
  • any range of air saturation levels between 0-40%, inclusive e.g., 0-5%, 0.05– 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5– 2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.
  • the exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
  • the term“low oxygen” is meant to refer to 9% O2 saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O2 saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%.
  • any range of O2 saturation levels between 0-9%, inclusive e.g., 0-5%, 0.05– 0.1%, 0.1-0.2%, 0.1- 0.5%, 0.5– 2.0%, 0-8%, 5-7%, 0.3-4.2% O2, etc.
  • the exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
  • the term“gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence.
  • the gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence.
  • the gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence.
  • Microorganism refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell.
  • microorganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, 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.
  • 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,
  • Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
  • “payload” refers to one or more polynucleotides and/or polypeptides of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus.
  • the payload is encoded by a gene or multiple genes or an operon.
  • the one or more genes and/or operon(s) comprising the payload are endogenous to the microorganism.
  • the one or more elements of the payload is derived from a different microorganism and/or organism.
  • the payload is a therapeutic payload.
  • the payload is encoded by genes for the biosynthesis of a molecule.
  • the payload is encoded by genes for the metabolism, catabolism, or degradation of a molecule. In some embodiments, the payload is encoded by genes for the importation of a molecule. In some embodiments, the payload is encoded by genes for the exportation of a molecule. In some embodiments, the payload is a regulatory molecule(s), e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism.
  • the genetically engineered microorganism comprises two or more payloads.
  • Non-limiting examples of payload(s) include one or more of the following: (1) ArgAfbr, (2) mutated Arg Boxes, (3) mutated ArgR, (4) mutated ArgG, (5) butyrate biosynthetic cassette, (6) proprionate biosynthetic cassette, (7) acetate biosynthetic cassette; (8) GABA-metabolizing cassette, (9) GABA-transporter, (10) Mn-transporter (11) tryptophan or any of its metabolites, e.g., kynurenine, kynurenic acid, and indole metabolites described herein (11) secreted or surface displayed polypeptides, e.g., GLP-2 or IL-22.
  • Payloads include IL-10, IL-27, TGF- ⁇ 1, TGF- ⁇ 2, elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2,. Payloads also include mutated endogenous genes, which allow the production of certain metabolites or polypeptides or improve the production of certain metabolites or polypeptides. Other exemplary payloads include mutated sequence(s) that result in an auxotrophy, e.g., thyA auxotrophy, kill switch circuit, antibiotic resistance circuits, transporter sequence for importing biological molecules or substrates, secretion circuit.
  • auxotrophy e.g., thyA auxotrophy
  • kill switch circuit e.g., antibiotic resistance circuits
  • transporter sequence for importing biological molecules or substrates, secretion circuit.
  • “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
  • the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria 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
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
  • Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • “stably maintained” or“stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a feedback resistant argA gene, mutant arginine repressor, and/or other mutant arginine regulon that is incorporated into the host genome or propagated on a self-replicating extra- chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated.
  • the stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • the stable bacterium may be a genetically engineered bacterium comprising an argAfbr gene, in which the plasmid or chromosome carrying the argAfbr gene is stably maintained in the bacterium, such that argAfbr can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.
  • the terms“modulate” and“treat” and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof.
  • “modulate” and“treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient.
  • “modulate” and“treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.
  • “modulate” and“treat” refer to slowing the
  • prevention and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.
  • Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder.
  • Primary hyperammonemia is caused by UCDs, which are autosomal recessive or X-linked inborn errors of metabolism for which there are no known cures.
  • Hyperammonemia can also be secondary to other disruptions of the urea cycle, e.g., toxic metabolites, infections, and/or substrate deficiencies. Hyperammonemia can also contribute to other pathologies.
  • Huntington’s disease is an autosomal dominant disorder for which there are no known cures.
  • Urea cycle abnormalities characterized by hyperammonemia, high blood citrulline, and suppression of urea cycle enzymes may contribute to the pathology of Huntington’s disease, an autosomal dominant disorder for which there are no known cures.
  • Treating hyperammonemia may encompass reducing or eliminating excess ammonia and/or associated symptoms, and does not necessarily encompass the elimination of the underlying hyperammonemia- associated disorder.
  • composition refers to a preparation of genetically engineered bacteria of the invention 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
  • compositions to further facilitate administration of an active ingredient.
  • examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.
  • terapéuticaally effective dose and“therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g.,
  • a therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder associated with elevated ammonia concentrations.
  • a therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.
  • polypeptide includes“polypeptide” as well as “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • polypeptides “peptides,”“dipeptides,”“tripeptides,“oligopeptides,”“protein,”“amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of“polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation,
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention.
  • a polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure.
  • polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded.
  • the term“peptide” or“polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.
  • An“isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • Recombinantly produced polypeptides and proteins expressed in host cells including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e.
  • fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments.
  • Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.
  • Polypeptides also include fusion proteins.
  • the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide.
  • the term“fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques.
  • Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins.
  • “Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.“Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution.
  • amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.
  • the term“sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity.
  • amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 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.
  • linker refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains.
  • synthetic refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.
  • the term“codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism.
  • 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.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • 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.
  • 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 polypeptide from the engineered bacteria.
  • the N-terminal peptide secretion tag is removed upon translocation of the“passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu.
  • an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu.
  • 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, tolB, pal, degS, degP, and nlpl.
  • Lpp functions as the primary‘staple’ of the bacterial cell wall to the peptidoglycan.
  • TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype.
  • the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes.
  • the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl.
  • the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.
  • the term“transporter” is meant to refer to a mechanism, e.g., protein or proteins, for importing a molecule, e.g., amino acid, toxin, metabolite, substrate, etc. into the microorganism from the extracellular milieu.
  • phrase“and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present.
  • “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C.
  • the phrase“and/or” may be used interchangeably with“at least one of” or“one or more of” the elements in a list.
  • the genetically engineered bacteria disclosed herein are capable of reducing excess ammonia and converting ammonia and/or nitrogen into alternate byproducts.
  • the genetically engineered bacteria are naturally non-pathogenic bacteria.
  • the genetically engineered bacteria are commensal bacteria.
  • the genetically engineered bacteria are probiotic bacteria.
  • the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity.
  • Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli,
  • Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii.
  • Bacillus coagulans Bacillus subtilis
  • the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.
  • the genetically engineered bacteria are any suitable bacteria.
  • 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 al., 2007).
  • the strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added).
  • Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli ⁇ -hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E.
  • E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E.
  • coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn’s disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle’s therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007).
  • arginine-mediated regulation is remarkably well conserved in very divergent bacteria, i.e., Gram-negative bacteria, such as E. coli, Salmonella enterica serovar Typhimurium, Thermotoga, and Moritella profunda, and Gram-positive bacteria, such as B. subtilis, Geobacillus
  • arginine repressor is universally conserved in bacterial genomes and that its recognition signal (the ARG box), a weak palindrome, is also conserved between genomes (Makarova et al., 2001).
  • Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum
  • the residence time of bacteria in vivo can be determined using the methods described herein. In some embodiments, the residence time is calculated for a human subject. A non-limiting example using a streptomycin-resistant E. coli Nissle comprising a wild-type ArgR and a wild-type arginine regulon is provided herein. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention.
  • the arginine biosynthesis pathway is capable of converting glutamate to arginine in an eight-step enzymatic process involving the enzymes N-acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase (Cunin et al., 1986).
  • the first five steps involve N-acetylation to generate an ornithine precursor.
  • ornithine transcarbamylase also known as ornithine carbamoyltransferase
  • the final two steps involve carbamoylphosphate utilization to generate arginine from citrulline.
  • the first and fifth steps in arginine biosynthesis may be catalyzed by the bifunctional enzyme ornithine acetyltransferase.
  • This bifunctionality was initially identified when ornithine acetyltransferase (argJ) was shown to complement both N- acetylglutamate synthetase (argA) and N-acetylornithinase (argE) auxotrophic gene mutations in E. coli (Mountain et al., 1984; Crabeel et al., 1997).
  • ArgA encodes N-acetylglutamate synthetase
  • argB encodes N- acetylglutamate kinase
  • argC encodes N-acetylglutamylphosphate reductase
  • argD encodes acetylornithine aminotransferase
  • argE encodes N-acetylornithinase
  • argF encodes ornithine transcarbamylase
  • argI also encodes ornithine transcarbamylase
  • argG encodes argininosuccinate synthase
  • argH encodes argininosuccinate lyase
  • argJ encodes ornithine acetyltransferase.
  • CarA encodes the small A subunit of carbamoylphosphate synthase having glutaminase activity
  • carB encodes the large B subunit of carbamoylphosphate synthase that catalyzes carbamoylphosphate synthesis from ammonia.
  • Different combinations of one or more of these arginine biosynthesis genes i.e., argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB
  • the regulatory region of each operon contains at least one ARG box, and the number of ARG boxes per regulatory region may vary between operons and bacteria.
  • the genes that regulate arginine biosynthesis in bacteria are scattered across the chromosome and organized into multiple operons that are controlled by a single repressor, which Maas and Clark (1964) termed a“regulon.” Each operon is regulated by a regulatory region comprising at least one 18-nucleotide imperfect palindromic sequence, called an ARG box, that overlaps with the promoter and to which the repressor protein binds (Tian et al., 1992; Tian et al., 1994).
  • the argR gene encodes the repressor protein, which binds to one or more ARG boxes (Lim et al., 1987).
  • Arginine functions as a corepressor that activates the arginine repressor.
  • the ARG boxes that regulate each operon may be non-identical, and the consensus ARG box sequence is (Maas, 1994). In addition, the
  • argR argin-1 binds to argin-1 binds to argin-1 binds to argin-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR-1 binds to argR.
  • the genetically engineered bacteria comprise a mutant arginine regulon and produce more arginine and/or an intermediate byproduct, e.g., citrulline, than unmodified bacteria of the same subtype under the same conditions.
  • the mutant arginine regulon comprises one or more nucleic acid mutations that reduce or prevent arginine-mediated repression– via ArgR binding to ARG boxes and/or arginine binding to N-acetylglutamate synthetase– of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine in the arginine biosynthesis pathway, thereby enhancing arginine and/or intermediate byproduct biosynthesis.
  • the bacteria are genetically engineered to consume excess ammonia via another metabolic pathway, e.g., a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, and a tryptophan biosynthesis pathway.
  • another metabolic pathway e.g., a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, and a tryptophan biosynthesis pathway.
  • an ammonia conversion circuit refers to a metabolic pathway by which excess ammonia may be consumed and/or reduced.
  • Histidine biosynthesis is carried out by eight genes located within a single operon in E. coli.
  • Three of the eight genes of the operon (hisD, hisB, and hisI) encode bifunctional enzymes, and two (hisH and hisF) encode polypeptide chains which together form one enzyme to catalyze a single step, for a total of 10 enzymatic reactions (Alifano et al., 1996).
  • the product of the hisG gene, ATP phosphoribosyltransferase is inhibited at the protein level by histidine.
  • the genetically engineered bacteria of the invention comprise a feedback- resistant hisG.
  • Bacteria may be mutagenized and/or screened for feedback-resistant hisG mutants using techniques known in the art. Bacteria engineered to comprise a feedback-resistant hisG would have elevated levels of histidine production, thus increasing ammonia consumption and reducing hyperammonemia. Alternatively, one or more genes required for histidine biosynthesis could be placed under the control of an inducible promoter, such as a FNR-inducible promoter, and allow for increased production of rate-limiting enzymes. Any other suitable modification(s) to the histidine biosynthesis pathway may be used to increase ammonia consumption.
  • an inducible promoter such as a FNR-inducible promoter
  • the bacterial methionine regulon controls the three-step synthesis of methionine from homoserine (i.e., acylation, sulfurylation, and methylation).
  • the metJ gene encodes a regulatory protein that, when combined with methionine or a derivative thereof, causes repression of genes within the methionine regulon at the transcriptional level (Saint-Girons et al., 1984; Shoeman et al., 1985).
  • the genetically engineered bacteria of the invention comprise deleted, disrupted, or mutated metJ. Bacteria engineered to delete, disrupt, or mutate metJ would have elevated levels of methionine production, thus increasing ammonia consumption and reducing hyperammonemia. Any other suitable modification(s) to the methionine biosynthesis pathway may be used to increase ammonia consumption. Lysine Biosynthesis Pathway
  • Microorganisms synthesize lysine by one of two pathways.
  • the diaminopimelate (DAP) pathway is used to synthesize lysine from aspartate and pyruvate (Dogovski et al., 2012), and the aminoadipic acid pathway is used to synthesize lysine from alpha-ketoglutarate and acetyl coenzyme A.
  • DAP diaminopimelate
  • aminoadipic acid pathway is used to synthesize lysine from alpha-ketoglutarate and acetyl coenzyme A.
  • DHDPS dihydrodipicolinate synthase
  • the genetically engineered bacteria of the invention comprise a feedback-resistant DHDPS.
  • Bacteria engineered to comprise a feedback- resistant DHDPS would have elevated levels of histidine production, thus increasing ammonia consumption and reducing hyperammonemia.
  • lysine production could be optimized by placing one or more genes required for lysine biosynthesis under the control of an inducible promoter, such as a FNR-inducible promoter. Any other suitable modification(s) to the lysine biosynthesis pathway may be used to increase ammonia consumption.
  • Asparagine is synthesized directly from oxaloacetate and aspartic acid via the oxaloacetate transaminase and asparagine synthetase enzymes, respectively.
  • either L-glutamine or ammonia serves as the amino group donor.
  • the genetically engineered bacteria of the invention overproduce asparagine as compared to unmodified bacteria of the same subtype under the same conditions, thereby consuming excess ammonia and reducing hyperammonemia.
  • asparagine synthesis may be optimized by placing one or both of these genes under the control of an inducible promoter, such as a FNR- inducible promoter. Any other suitable modification(s) to the asparagine biosynthesis pathway may be used to increase ammonia consumption.
  • oxoglutarate is tightly regulated by three enzymes.
  • Glutamate dehydrogenase catalyzes the reductive amination of oxoglutarate to yield glutamate in a single step.
  • Glutamine synthetase catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine (Lodeiro et al., 2008).
  • Glutamine synthetase also acts with glutamine– oxoglutarate amino transferase (also known as glutamate synthase) in a cyclic reaction to produce glutamate from glutamine and oxoglutarate.
  • the genetically engineered bacteria of the invention express glutamine synthetase at elevated levels as compared to unmodified bacteria of the same subtype under the same conditions.
  • Bacteria engineered to have increased expression of glutamine synthetase would have elevated levels of glutamine production, thus increasing ammonia consumption and reducing hyperammonemia.
  • expression of glutamate dehydrogenase and/or glutamine–oxoglutarate amino transferase could be modified to favor the consumption of ammonia.
  • glutamine synthetase Since the production of glutamine synthetase is regulated at the transcriptional level by nitrogen (Feng et al., 1992; van Heeswijk et al., 2013), placing the glutamine synthetase gene under the control of different inducible promoter, such as a FNR-inducible promoter, may also be used to improve glutamine production. Any other suitable modification(s) to the glutamine and glutamate biosynthesis pathway may be used to increase ammonia consumption.
  • the genes required for the synthesis of tryptophan from a chorismate precursor are organized as a single transcriptional unit, the trp operon.
  • the trp operon is under the control of a single promoter that is inhibited by the tryptophan repressor (TrpR) when high levels of tryptophan are present. Transcription of the trp operon may also be terminated in the presence of high levels of charged tryptophan tRNA.
  • the genetically engineered bacteria of the invention comprise a deleted, disrupted, or mutated trpR gene.
  • the deletion, disruption, or mutation of the trpR gene, and consequent inactivation of TrpR function, would result in elevated levels of both tryptophan production and ammonia consumption.
  • one or more enzymes required for tryptophan biosynthesis could be placed under the control of an inducible promoter, such as a FNR-inducible promoter. Any other suitable modification(s) to the tryptophan biosynthesis pathway may be used to increase ammonia consumption.
  • the genetically engineered bacteria comprise an arginine biosynthesis pathway and are capable of reducing excess ammonia.
  • the genetically engineered bacteria comprise a mutant arginine regulon in which one or more operons encoding arginine biosynthesis enzyme(s) is derepressed to produce more arginine or an intermediate byproduct, e.g., citrulline, than unmodified bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria overproduce arginine.
  • the genetically engineered bacteria overproduce citrulline; this may be additionally beneficial, because citrulline is currently used as a therapeutic for particular urea cycle disorders (National Urea Cycle Disorders Foundation).
  • the genetically engineered bacteria overproduce an alternate intermediate byproduct in the arginine biosynthesis pathway, such as any of the intermediates described herein.
  • the genetically engineered bacterium consumes excess ammonia by producing more arginine, citrulline, and/or other intermediate byproduct than an unmodified bacterium of the same bacterial subtype under the same conditions.
  • Enhancement of arginine and/or intermediate byproduct biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules in order to treat conditions associated with hyperammonemia, including urea cycle disorders and hepatic encephalopathy.
  • arginine biosynthesis genes within an operon varies across species, strains, and subtypes of bacteria, e.g., bipolar argECBH in E. coli K12, argCAEBD-carAB-argF in B. subtilis, and bipolar carAB-argCJBDF in L. plantarum.
  • operon organization from different bacteria are shown in Table 3 (in some instances, the genes are putative and/or identified by sequence homology to known sequences in Escherichia coli; in some instances, not all of the genes in the arginine regulon are known and/or shown below).
  • the arginine biosynthesis enzymes vary across species, strains, and subtypes of bacteria.
  • Each operon is regulated by a regulatory region comprising at least one promoter and at least one ARG box, which control repression and expression of the arginine biosynthesis genes in said operon.
  • the genetically engineered bacteria of the invention comprise an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct in the arginine biosynthesis pathway.
  • Reducing or eliminating arginine-mediated repression may be achieved by reducing or eliminating ArgR repressor binding (e.g., by mutating or deleting the arginine repressor or by mutating at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes) and/or arginine binding to N-acetylglutamate synthetase (e.g., by mutating the N-acetylglutamate synthetase to produce an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argA fbr ).
  • the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, and carbamoylphosphate synthase, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis.
  • the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransfer
  • the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis.
  • the genetically engineered bacteria may further comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr , a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor.
  • an arginine feedback resistant N-acetylglutamate synthase mutant e.g., argA fbr
  • a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor.
  • the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for one or more of the operons that encode N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
  • argininosuccinate lyase carbamoylphosphate synthase, and wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis.
  • the ARG boxes for the operon encoding argininosuccinate synthase (argG) maintain the ability to bind to ArgR, thereby driving citrulline biosynthesis.
  • the regulatory region of the operon encoding argininosuccinate synthase (argG) may be a constitutive, thereby driving arginine biosynthesis.
  • the regulatory region of one or more alternate operons may be constitutive.
  • genes encoding multiple enzymes may be organized in bipolar operons or under the control of a shared regulatory region; in these instances, the regulatory regions may need to be
  • argE and argCBH are organized in two bipolar operons, argECBH, and those regulatory regions may be deconvoluted in order to generate constitutive versions of argE and/or argCBH.
  • all ARG boxes in one or more operons that comprise an arginine biosynthesis gene are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in one or more operons that encode an arginine biosynthesis enzyme are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in each operon that comprises an arginine biosynthesis gene are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in each operon that encodes an arginine biosynthesis enzyme are mutated to reduce or eliminate ArgR binding.
  • the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase, argininosuccinate synthase driven by a ArgR-repressible regulatory region, and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for each of the operons that encode N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, carbamoylphosphate synthase, and optionally, wild-type N-acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and
  • the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase, argininosuccinate synthase driven by a constitutive promoter, and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for each of the operons that encode N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate lyase, carbamoylphosphate synthase, and optionally, wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and enhancing arginine biosynthesis.
  • the genetically engineered bacteria comprise a mutant arginine regulon and a feedback resistant ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or an intermediate byproduct than unmodified bacteria of the same subtype under the same conditions.
  • the mutant arginine regulon and/or a feedback resistant ArgA may be integrated into the bacterial chromosome at one or more integration sites or may be present on one or more plasmids.
  • the genetically engineered bacteria additionally comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase,
  • acetylornithine aminotransferase N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, and carbamoylphosphate synthase, such that the arginine regulon is derepressed and biosynthesis of arginine and/or an intermediate byproduct, e.g., citrulline, is enhanced.
  • an intermediate byproduct e.g., citrulline
  • the mutant arginine regulon comprises an operon encoding ornithine acetyltransferase and one or more nucleic acid mutations in at least one ARG box for said operon.
  • the one or more nucleic acid mutations results in the disruption of the palindromic ARG box sequence, such that ArgR binding to that ARG box and to the regulatory region of the operon is reduced or eliminated, as compared to ArgR binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions.
  • nucleic acids that are protected from DNA methylation and hydroxyl radical attack during ArgR binding are the primary targets for mutations to disrupt ArgR binding.
  • the mutant arginine regulon comprises at least three nucleic acid mutations in one or more ARG boxes for each of the operons that encode the arginine biosynthesis enzymes described above.
  • the ARG box overlaps with the promoter, and in the mutant arginine regulon, the G/C:A/T ratio of the mutant promoter region differs by no more than 10% from the G/C:A/T ratio of the wild-type promoter region (Table 4).
  • the promoter retains sufficiently high homology to the non-mutant promoter such that RNA polymerase binds with sufficient affinity to promote transcription.
  • the wild-type genomic sequences comprising ARG boxes and mutants thereof for each arginine biosynthesis operon in E.
  • coli Nissle are shown in Table 4.
  • the ARG boxes are indicated in italics, and the start codon of each gene is boxed.
  • the RNA polymerase binding sites are underlined (Cunin, 1983; Maas, 1994). In some embodiments, the underlined sequences are not altered. Bases that are protected from DNA methylation during ArgR binding are highlighted, and bases that are protected from hydroxyl radical attack during ArgR binding are bolded (Charlier et al., 1992). The highlighted and bolded bases are the primary targets for mutations to disrupt ArgR binding.
  • more than one ARG box may be present in a single operon.
  • at least one of the ARG boxes in an operon is mutated to produce the requisite reduced ArgR binding to the regulatory region of the operon.
  • each of the ARG boxes in an operon is mutated to produce the requisite reduced ArgR binding to the regulatory region of the operon.
  • the number of ARG boxes per regulatory region may vary across bacteria, and the nucleotide sequences of the ARG boxes may vary for each operon.
  • the carAB operon in E. coli Nissle comprises two ARG boxes, and one or both ARG box sequences may be mutated.
  • the argG operon in E. coli Nissle comprises three ARG boxes, and one, two, or three ARG box sequences may be mutated, disrupted, or deleted. In some embodiments, all three ARG box sequences are mutated, disrupted, or deleted, and a constitutive promoter, e.g., BBa_J23100, is inserted in the regulatory region of the argG operon.
  • a constitutive promoter e.g., BBa_J23100
  • Table 5 depicts the wild-type genomic sequence of the regulatory region and 5’ portion of the argG gene in E. coli Nissle, and a constitutive mutant thereof.
  • the promoter region of each sequence is underlined, and a 5’ portion of the argG gene is boxed.
  • ArgR binding sites are in uppercase and underlined.
  • the mutant sequence the 5’ untranslated region is in uppercase and underlined.
  • Bacteria expressing argG under the control of the constitutive promoter are capable of producing arginine.
  • Bacteria expressing argG under the control of the wild-type, ArgR-repressible promoter are capable of producing citrulline.
  • a map of the wild-type argG operon E. coli Nissle and a constitutively expressing mutant thereof is shown in FIG. 12.
  • the ArgR binding affinity to a mutant ARG box or regulatory region of an operon is at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, at least about 90% lower, or at least about 95% lower than the ArgR binding affinity to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions.
  • the reduced ArgR binding to a mutant ARG box and regulatory region increases mRNA expression of the gene(s) in the associated operon by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold.
  • a fluorophore is added to a sample reaction mixture that may contain arg mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore.
  • the reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles.
  • the accumulating amplicon is quantified after each cycle of the qPCR.
  • the number of cycles at which fluorescence exceeds the threshold is the threshold cycle (C T ).
  • At least one C T result for each sample is generated, and the C T result(s) may be used to determine mRNA expression levels of the arginine biosynthesis genes.
  • the genetically engineered bacteria comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
  • argininosuccinate lyase, and carbamoylphosphate synthase additionally comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise a feedback resistant form of ArgA, as well as one or more nucleic acid mutations in each ARG box of one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, ornithine acetyltransferase, and carbamoylphosphate synthase.
  • the genetically engineered bacteria comprise a feedback resistant form of ArgA, argininosuccinate synthase driven by a ArgR- repressible regulatory region, as well as one or more nucleic acid mutations in each ARG box of each of the operons that encode the arginine biosynthesis enzymes N- acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate lyase, ornithine acetyltransferase, and carbamoylphosphate synthase.
  • the bacteria are capable of producing citrulline.
  • the genetically engineered bacteria comprise a feedback resistant form of ArgA, argininosuccinate synthase expressed from a constitutive promoter, as well as one or more nucleic acid mutations in each ARG box of each of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
  • the bacteria are capable of producing arginine.
  • Table 6 shows examples of mutant constructs in which one or more nucleic acid mutations reduce or eliminate arginine-mediated repression of each of the arginine operons.
  • the mutant constructs comprise feedback resistant form of ArgA driven by an oxygen level-dependent promoter, e.g., a FNR promoter.
  • Each mutant arginine regulon comprises one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
  • argininosuccinate lyase carbamoylphosphate synthase, and wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby enhancing arginine and/or intermediate byproduct biosynthesis.
  • mutant arginine regulon constructs are shown in Table 6.
  • the mutations may be present on a plasmid or chromosome.
  • the arginine regulon is regulated by a single repressor protein.
  • the arginine regulon may be regulated by two putative repressors (Nicoloff et al., 2004).
  • the arginine regulon of the invention is regulated by more than one repressor protein.
  • the mutant arginine regulon is expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the mutant arginine regulon is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.
  • the genetically engineered bacteria of the invention comprise an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct in the arginine biosynthesis pathway.
  • the reduction or elimination of arginine-mediated repression may be achieved by reducing or eliminating ArgR repressor binding, e.g., by mutating at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes (as discussed above) or by mutating or deleting the arginine repressor (discussed here) and/or by reducing or eliminating arginine binding to N-acetylglutamate synthetase (e.g., by mutating the N- acetylglutamate synthetase to produce an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr ).
  • the genetically engineered bacteria lack a functional ArgR repressor and therefore ArgR repressor-mediated transcriptional repression of each of the arginine biosynthesis operons is reduced or eliminated.
  • the engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive.
  • the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis.
  • each copy of a functional argR gene normally present in a corresponding wild-type bacterium is independently deleted or rendered inactive by one or more nucleotide deletions, insertions, or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium is deleted.
  • the arginine regulon is regulated by a single repressor protein.
  • the arginine regulon may be regulated by two distinct putative repressors (Nicoloff et al., 2004).
  • two distinct ArgR proteins each comprising a different amino acid sequence are mutated or deleted in the genetically engineered bacteria.
  • the genetically modified bacteria comprising a mutant or deleted arginine repressor additionally comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise a feedback resistant form of ArgA, lack any functional arginine repressor, and are capable of producing arginine.
  • the genetically engineered bacteria further lack functional ArgG and are capable of producing citrulline.
  • the argR gene is deleted in the genetically engineered bacteria.
  • the argR gene is mutated to inactivate ArgR function.
  • the argG gene is deleted in the genetically engineered bacteria. In some embodiments, the argG gene is mutated to inactivate ArgR function. In some embodiments, the genetically engineered bacteria comprise argA fbr and deleted ArgR. In some embodiments, the genetically engineered bacteria comprise argA fbr , deleted ArgR, and deleted argG. In some embodiments, the deleted ArgR and/or the deleted argG is deleted from the bacterial genome and the argA fbr is present in a plasmid. In some embodiments, the deleted ArgR and/or the deleted argG is deleted from the bacterial genome and the argA fbr is chromosomally integrated. In one specific embodiment, the genetically modified bacteria comprise chromosomally integrated argA fbr , deleted genomic ArgR, and deleted genomic argG. In another specific embodiment, the genetically modified bacteria comprise
  • argA fbr present on a plasmid, deleted genomic ArgR, and deleted genomic argG.
  • citrulline rather than arginine is produced.
  • the genetically engineered bacteria of the invention produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more arginine, citrulline, other intermediate byproduct, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions.
  • qPCR quantitative PCR
  • Primers specific for arginine biosynthesis genes e.g., argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB, may be designed and used to detect mRNA in a sample according to methods known in the art (Fraga et al., 2008).
  • a fluorophore is added to a sample reaction mixture that may contain arg mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore.
  • the reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles.
  • the accumulating amplicon is quantified after each cycle of the qPCR.
  • the number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT).
  • CT threshold cycle
  • At least one C T result for each sample is generated, and the C T result(s) may be used to determine mRNA expression levels of the arginine biosynthesis genes.
  • the mutant ArgR and/or a feedback resistant ArgA may be integrated into the bacterial
  • chromosome at one or more integration sites or may be present on one or more plasmids.
  • the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argA fbr .
  • the genetically engineered bacteria comprise a mutant arginine regulon comprising an arginine feedback resistant ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or an intermediate byproduct than unmodified bacteria of the same subtype under the same conditions.
  • the arginine feedback resistant N-acetylglutamate synthetase protein (argA fbr ) is significantly less sensitive to L-arginine than the enzyme from the feedback sensitive parent strain (see, e.g., Eckhardt et al., 1975; Rajagopal et al., 1998).
  • the feedback resistant argA gene can be present on a plasmid or chromosome.
  • expression from the plasmid may be useful for increasing argA fbr expression.
  • expression from the chromosome may be useful for increasing stability of argA fbr expression.
  • any of the described mutant sequences involved in the arginine biosynthetic pathway are integrated into the bacterial chromosome at one or more integration sites.
  • one or more copies of the sequence encoding the arginine feedback resistant N- acetylglutamate synthase may be integrated into the bacterial chromosome. Having multiple copies of the arginine feedback resistant N-acetylglutamate synthase integrated into the chromosome allows for greater production of the N-acetylglutamate synthase and also permits fine-tuning of the level of expression.
  • the argA fbr gene is expressed under the control of a constitutive promoter. In some embodiments, the argA fbr gene is expressed under the control of a promoter that is induced by exogenous environmental conditions.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut in a healthy or disease state, e.g., propionate.
  • 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.
  • the exogenous environmental conditions are low- oxygen or anaerobic conditions, such as the environment of the mammalian gut.
  • nucleic acid sequence of an exemplary sequence is shown in Table 7.
  • polypeptide sequence of an exemplary sequence is shown in Table 8.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 30 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 SEQ ID NO: 30 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 SEQ ID NO: 30 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 30 or a functional fragment thereof.
  • the genetically engineered bacteria encode a polypeptide sequence of SEQ ID NO: 31 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria encode a polypeptide sequence encodes a polypeptide, which contains one or more conservative amino acid
  • genetically engineered bacteria encode 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 DNA sequence of SEQ ID NO: 31 or a functional fragment thereof.
  • arginine feedback inhibition of N-acetylglutamate synthetase is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in the genetically engineered bacteria when the arginine feedback resistant N-acetylglutamate synthetase is active, as compared to a wild-type N-acetylglutamate synthetase from bacteria of the same subtype under the same conditions.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the argA fbr gene, such that argA fbr can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • a bacterium may comprise multiple copies of the feedback resistant argA gene.
  • the feedback resistant argA gene is expressed on a low-copy plasmid.
  • the low-copy plasmid may be useful for increasing stability of expression.
  • the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions.
  • the feedback resistant argA gene is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing argA fbr expression.
  • the feedback resistant argA gene is expressed on a chromosome.
  • the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product or circuits performing multiple different functions.
  • MOAs mechanisms of action
  • insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 18.
  • the genetically engineered bacteria may include four copies of argA fbr inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ.
  • the genetically engineered bacteria may include three copies of argA fbr inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three mutant arginine regulons, e.g., two producing citrulline and one producing arginine, inserted at three different insertion sites dapA, cea, and araC/BAD.
  • three different insertion sites e.g., malE/K, insB/I, and lacZ
  • three mutant arginine regulons e.g., two producing citrulline and one producing arginine, inserted at three different insertion sites dapA, cea, and araC/BAD.
  • the plasmid or chromosome also comprises wild- type ArgR binding sites, e.g., ARG boxes.
  • ARG boxes wild- type ArgR binding sites
  • the presence and/or build- up of functional ArgR may result in off-target binding at sites other than the ARG boxes, which may cause off-target changes in gene expression.
  • a plasmid or chromosome that further comprises functional ARG boxes may be used to reduce or eliminate off-target ArgR binding, i.e., by acting as an ArgR sink.
  • the plasmid or chromosome does not comprise functional ArgR binding sites, e.g., the plasmid or chromosome comprises modified ARG boxes or does not comprise ARG boxes.
  • the feedback resistant argA gene is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the feedback resistant argA gene is present in the chromosome and operably linked to a promoter that is induced under low- oxygen or anaerobic conditions. In some embodiments, the feedback resistant argA gene is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut.
  • the feedback resistant argA gene is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the feedback resistant argA gene is present on a
  • the genetically engineered bacteria comprise a variant or mutated oxygen level-dependent transcriptional regulator, e.g., FNR, ANR, or DNR, in addition to the corresponding oxygen level-dependent promoter.
  • the variant or mutated oxygen level-dependent transcriptional regulator increases the transcription of operably linked genes in a low-oxygen or anaerobic environment.
  • the corresponding wild-type transcriptional regulator retains wild-type activity.
  • the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.
  • the mutant oxygen level-dependent transcriptional regulator is a FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., 2006).
  • the genetically engineered bacteria comprise an oxygen level-dependent transcriptional regulator from a different bacterial species that reduces and/or consumes ammonia in low-oxygen or anaerobic environments.
  • the mutant oxygen level-dependent transcriptional regulator is a FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011).
  • the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.
  • the genetically engineered bacteria comprise argA fbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter, as well as wild-type argA expressed under the control of a mutant regulatory region comprising one or more ARG box mutations as discussed above.
  • the genetically engineered bacteria comprise argA fbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter and do not comprise wild-type argA.
  • the mutant arginine regulon comprises argA fbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter, and further comprises wild-type argA without any ARG box mutations.
  • an oxygen level-dependent promoter e.g., a FNR promoter
  • the genetically engineered bacteria express argA fbr from a plasmid and/or chromosome.
  • the argA fbr gene is expressed under the control of a constitutive promoter.
  • the argA fbr gene is expressed under the control of an inducible promoter.
  • argA fbr is expressed under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, e.g., a FNR fbr promoter.
  • fbr sequence is shown in Table 9.
  • the FNR promoter sequence is bolded and the argA sequence is boxed.
  • the nucleic acid sequence of a FNR promoter-driven argA fbr plasmid is shown in Table 10, with the FNR promoter sequence bolded and argA fbr sequence boxed.
  • Table 11 shows the nucleic acid sequence of an exemplary pSC101 plasmid. Any suitable FNR promoter(s) may be combined with any suitable feedback- resistant ArgA. Non-limiting FNR promoter sequences are provided in Table 6.
  • the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 16, SEQ ID NO: 17, nirB1 promoter (SEQ ID NO: 18), nirB2 promoter (SEQ ID NO: 19), nirB3 promoter (SEQ ID NO: 20), ydfZ promoter (SEQ ID NO: 21), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 22), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 23), fnrS, an
  • fnrS1 promoter SEQ ID NO: 24 or fnrS2 promoter SEQ ID NO: 25 anaerobically induced small RNA gene
  • fnrB promoter fused to a crp binding site SEQ ID NO: 26
  • fnrS fused to a crp binding site SEQ ID NO: 27.
  • Table 12 depicts the nucleic acid f br
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 32 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 SEQ ID NO: 32. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 32, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 32.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 33 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 SEQ ID NO: 33. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 33, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 33.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 35 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 SEQ ID NO: 35. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 35, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 35.
  • the genetically engineered bacteria comprise argA fbr integrated into the chromosome.
  • the integrated fbrArgA is under the control of the fnrS promoter.
  • an antibiotic resistance cassette is also present at the same site. In some embodiments, no antibiotic resistance cassette is present.
  • the antibiotic resistance is chloramphenicol. In some embodiments, the antibiotic resistance is kanamycin.
  • the genetically engineered bacteria comprising argA fbr integrated into the chromosome is a thyA auxotroph.
  • the genetically engineered bacteria comprise argA fbr integrated into the chromosome and also comprise an ArgR mutation or have ArgR deleted.
  • the genetically engineered bacteria comprise argA fbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, and comprise a thyA auxotrophy.
  • the genetically engineered bacteria comprise argA fbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, comprise a thyA auxotrophy, and comprise an antibiotic resistance cassette.
  • the genetically engineered bacteria comprise argAfbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, comprise a thyA auxotrophy, and comprise a kanamycin resistance cassette.
  • the genetically engineered bacteria is SYN- UCD305. In another specific embodiment, the genetically engineered bacteria is SYN_UCD303.
  • Table 13 shows non-limiting examples of FNRS-fbrArgA constructs which are integrated into the chromosome.
  • SEQ ID NO: 36 comprises FNRS-fbrArgA and chloramphenicol resistance, e.g., as comprised in SYN-UCD301, SYN-UCD302.
  • SEQ ID NO: 37 comprises FNRS-fbrArgA and kanamycin resistance, e.g., as comprised inSYN- UCD303, SYN-UCD306, SYN-UCD307, and SYN-UCD309.
  • SEQ ID NO: 38 FNRS- fbrArgA and no antibiotic resistance, e.g., as comprised in SYN-UCD305 SYN- UCD304, SYN-UCD308, SYNUCD310.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 36 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 SEQ ID NO: 36 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 SEQ ID NO: 36 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 37 or a functional fragment thereof.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 37 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 SEQ ID NO: 37 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 SEQ ID NO: 37 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 37 or a functional fragment thereof.
  • the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 38 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 SEQ ID NO: 38 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 SEQ ID NO: 38 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 38 or a functional fragment thereof.
  • the gene sequence encoding one or more polypeptides for the reduction of ammonia levels 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, such as HE-related molecules.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
  • 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 reduction of ammonia levels 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 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 IX and X.
  • 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 reduction of ammonia levels e.g., ArgAfbr
  • the RBS is selected from a promoter provided in Table IX or
  • the gene sequence encoding one or more polypeptides for the reduction of ammonia levels is modified and/or mutated, e.g., to enhance stability, or increase ammonia catalysis.
  • the gene sequence encoding one or more polypeptides for the reduction of ammonia levels may be codon optimized, e.g., to improve expression in the host microorganism.
  • the gene sequence encoding one or more polypeptides for the reduction of ammonia levels 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 reduction of ammonia levels, e.g., ArgAfbr, 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 reduction of ammonia levels, e.g., ArgAfbr, 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 reduction of ammonia levels, e.g., ArgAfbr, 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 reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the reduction of ammonia levels, e.g., ArgAfbr, and/or one or more of its metabolites described herein.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr 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 reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels e.g., ArgAfbr
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels further comprise a GABA transport circuit and/or a GABA metabolic circuit.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
  • the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene.
  • the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene.
  • a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
  • An important consideration in practicing the invention is to ensure that ammonia is not overproduced as a byproduct of arginine and/or citrulline catabolism.
  • arginase catalyzes the hydrolytic cleavage of arginine into ornithine and urea (Cunin et al., 1986).
  • Urease which may be produced by gut bacteria, catalyzes the cleavage of urea into carbon dioxide and ammonia (Summerskill, 1966; Aoyagi et al., 1966; Cunin et al., 1986).
  • urease activity may generate ammonia that can be toxic for human tissue (Konieczna et al., 2012).
  • the gene arcD encodes an arginine/ornithine antiporter, which may also liberate ammonia (Vander Wauven et al., 1984; Gamper et al., 1991; Meng et al., 1992).
  • the genetically engineered bacteria comprising a mutant arginine regulon additionally includes mutations that reduce or eliminate arginine catabolism, thereby reducing or eliminating further ammonia production.
  • the genetically engineered bacteria also comprise mutations that reduce or eliminate ArcD activity. In certain embodiments, ArcD is deleted.
  • the genetically engineered bacteria also comprise mutations that reduce or eliminate AstA activity. In certain embodiments, AstA is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate SpeA activity. In certain embodiments, SpeA is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate arginase activity. In certain embodiments, arginase is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate urease activity. In certain embodiments, urease is deleted. In some embodiments, one or more other genes involved in arginine catabolism are mutated or deleted.
  • Hepatic encephalopathy is characterized by neurocognitive changes in patients and biochemical derangements have been implicated in pathogenesis.
  • elevated ammonia levels are suspected to partly contribute to disease pathophysiology.
  • elevated levels of cerebral GABA and manganese levels have been noted and suspected to contribute to clinical presentation.
  • the disclosure provides genetically engineered microorganisms, e.g., bacteria and virus, pharmaceutical compositions thereof, and methods of modulating or treating diseases or disorders associated with
  • the genetically engineered bacteria are capable of reducing excess ammonia in a mammal.
  • the genetically engineered bacteria reduce excess ammonia by incorporating excess nitrogen in the body into non-toxic molecules, e.g., arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, or tryptophan.
  • the genetically engineered bacteria further comprise one or more circuits (genetic sequence) to reduce the levels of other toxic or deleterious molecule(s), e.g., GABA, manganese.
  • the genetically engineered bacteria further comprise one or more circuits to produce a gut barrier enhancer molecule, e.g., a short chain fatty acid such as butyrate, propionate, and acetate.
  • a gut barrier enhancer molecule e.g., a short chain fatty acid such as butyrate, propionate, and acetate.
  • This disclosure also provides compositions and therapeutic methods for reducing excess ammonia and other deleterious molecules, e.g., GABA and manganese.
  • the disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules.
  • the disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules and further producing one or more therapeutic molecules, such as a gut barrier function enhancer molecule, e.g., butyrate.
  • the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia in which the circuits are under the control of an inducible promoter. In some embodiments, the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia and one or more circuits for reducing other deleterious molecules in which one or more of the circuits are under the control of an inducible promoter.
  • the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia and one or more circuits for reducing other deleterious molecules and further producing one or more therapeutic molecules, such as a gut barrier function enhancer molecule, e.g., butyrate in which one or more of the circuits and/or therapeutic molecule(s) are under the control of an inducible promoter.
  • the compositions and methods disclosed herein may be used for treating a disease or disorder associated with excess ammonia, for example, hepatic encephalopathy or Huntington’s disease, and/or one or more symptoms associated with disease or disorder associated with excess ammonia, such as hepatic encephalopathy or Huntington’s disease.
  • GABA ⁇ -Aminobutyric acid
  • GABA activates the post-synaptic GABA A receptor, which is part of a ligand-gated chloride- specific ion channel complex. Activation of this complex on a post-synaptic neuron allows chloride ions to enter the neuron and exert an inhibitory effect. Alterations of such GABAergic neurotransmission have been implicated in the pathophysiology of several neurological disorders, including epilepsy (Jones-Davis and MacDonald, 2003), Huntington’s disease (Krogsgaard-Larsen, 1992), and hepatic encephalopathy (Jones and Basile, 1997).
  • GABAergic tone prevents neuronal firing until a sufficiently potent stimulatory stimulus is received, or until the inhibitory tone is otherwise released.
  • Increased GABAergic tone in hepatic encephalopathy was initially described in the early 1980s, based on a report of similar visual response patterns in rabbits with galactosamine-induced liver failure and rabbits treated with allosteric modulators of the GABA A receptor (e.g., pentobarbital, diazepam) (Jones and Basile, 1997). Clinical improvements in HE patients treated with a highly selective
  • Increased GABAergic tone in HE has since been proposed as a consequence of one or more of the following: (1) increased GABA concentrations in the brain, (2) altered integrity of the GABA A receptor, and/or (3) increased concentrations of endogenous modulators of the GABA A receptor (Ahboucha and Butterworth, 2004).
  • GabP is a member of the amino acid/polymaine/organocation (APC) transporter superfamily, one of the two largest families of secondary active transporters (Jack et al., 2000).
  • GabP protein, encoded by the gabP gene consists of 466 amino acids and 12 transmembrane alpha- helices, wherein both N- and C- termini face the cytosol (Hu and King, 1998a).
  • the GabP residue sequence also includes a consensus amphipathic region (CAR), which is conserved between members of the APC family from bacteria to mammals (Hu and King, 1998b).
  • GABA succinyl semialdehyde
  • SSA succinyl semialdehyde
  • GSST GABA ⁇ -ketoglutarate transaminase
  • Succinate-semialdehyde dehydrogenase (SSDH) then catalyzes the second and only other specific step in GABA catabolism, the oxidation of succinyl semialdehyde to succinate (Dover and Halpern, 1972).
  • succinate becomes a substrate for the citric acid (TCA) cycle.
  • the bacteria are genetically engineered to consume excess ammonia via a metabolic pathway, e.g., an arginine biosynthesis pathway, a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, or a tryptophan biosynthesis pathway as described herein (an“ammonia conversion circuit”).
  • a metabolic pathway e.g., an arginine biosynthesis pathway, a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, or a tryptophan biosynthesis pathway as described herein (an“ammonia conversion circuit”).
  • the genetically engineered bacteria comprise an arginine biosynthesis pathway and are capable of reducing excess ammonia.
  • the ammonia conversion circuit is under the control of an inducible promoter.
  • the ammonia conversion circuit is under the control of an oxygen level-dependent promoter, e.g., an FNR-inducible promoter. In some embodiments, the ammonia conversion circuit is under the control of a promoter induced by a molecule or metabolite associated with hepatic
  • encephalopathy e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, ,transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, or manganese
  • the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA membrane transport protein(s), e.g., GabP, and are capable of transporting GABA into the cell (a“GABA transport circuit”) (FIG. 41).
  • GABA membrane transport protein(s) e.g., GabP
  • the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA catabolism enzyme(s), e.g., GSST, SSDH, and/or COT (a“GABA metabolic circuit”) (FIG. 41).
  • the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA membrane transport protein(s), e.g., GabP, and one or more circuits for producing one or more GABA catabolism enzyme(s), e.g., GSST, SSDH, and/or COT (a“GABA metabolic circuit”) (FIG. 41).
  • the genetically engineered bacteria comprise an ammonia conversion circuit, a GABA transport circuit, and a GABA metabolic circuit.
  • the ammonia conversion circuit, GABA transport circuit, and GABA metabolic circuit are under the control of the same promoter.
  • the ammonia conversion circuit, GABA transport circuit, and GABA metabolic circuit are under the control of different promoters.
  • Exemplary promoters include any of the promoters disclosed herein.
  • the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage.
  • a promoter induced by a molecule or metabolite for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage.
  • molecules or metabolites include, e.g., bilirubin, aspartate
  • the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter.
  • the genetically engineered bacteria comprise a promoter 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 amino acid sequence of an exemplary GabP transporter is shown in Table 48.
  • the genetically engineered bacteria comprise the amino acid sequence of SEQ ID NO: 105 or a functional fragment thereof.
  • the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 105 or a functional fragment thereof.
  • genetically engineered bacteria comprise an amino 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 amino acid sequence of SEQ ID NO: 105 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 105 or a functional fragment thereof.
  • a non-limiting example of a polynucleotide sequence is shown in Table 49 (SEQ ID NO: 106).
  • the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA 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, such as HE-related molecules.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
  • 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 uptake and/or catabolism of GABA 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 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 IX or Table X.
  • 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 uptake and/or catabolism of GABA is operably linked to a RBS, enhancer or other regulatory sequence.
  • the RBS is selected from a promoter provided in
  • the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA is modified and/or mutated, e.g., to enhance stability, or increase GABA catalysis.
  • the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA may be codon optimized, e.g., to improve expression in the host microorganism.
  • the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA 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 uptake and/or catabolism of GABA further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”)
  • a manganese membrane transport protein e.g., MntH
  • the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene.
  • the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene.
  • a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
  • Manganese is a biologically important trace metal and is required for the survival of most living organisms. In mammals, manganese is excreted in the bile, but its disposal is affected by the impaired flow of bile from the liver to the duodenum (i.e., cholestasis) that accompanies liver failure. Similar to ammonia, elevated concentrations of manganese play a role in the development of hepatic encephalopathy (Rivera-Manc ⁇ a et al., 2012).
  • Nramp Natural resistance-associated macrophage protein
  • Nramp1 transporter for manganese has been shown in metal accumulation studies, wherein overexpression of Staphylococcus aureus mntH resulted in increased levels of cell- associated manganese, but no accumulation of calcium, copper, iron, magnesium, or zinc (Horsburgh et al., 2002). Additionally, Bacillus subtilis strains comprising a mutation in the mntH gene exhibited impaired growth in metal-free medium that was rescued by the addition of manganese (Que and Helmann, 2000). The amino acid sequence of an exemplary MntH transporter is shown in Table 50. In some embodiment
  • the genetically engineered bacteria comprise the amino acid sequence of SEQ ID NO: 107 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 SEQ ID NO: 107 or a functional fragment thereof.
  • genetically engineered bacteria comprise an amino 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 amino acid sequence of SEQ ID NO: 107 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 107 or a functional fragment thereof.
  • a non-limiting example of a polynucleotide sequence is shown in Table 51 (SEQ ID NO: 108).
  • High-affinity manganese uptake may also be mediated by ABC (ATP- binding cassette) transporters.
  • ABC ATP- binding cassette
  • Members of this transporter superfamily utilize the hydrolysis of ATP to fuel the import or export of diverse substrates, ranging from ions to macromolecules, and are well characterized for their role in multi-drug resistance in both prokaryotic and eukaryotic cells.
  • Non-limiting examples of bacterial ABC transporters involved in manganese import include MntABCD (Bacilis subtilis, Staphylococcus aureus), SitABCD (Salmonella typhimurium, Shigella flexneri), PsaABCD (Streptococcus pneumoniae), and YfeABCD (Yersinia pestis) (Bearden and Perry, 1999; Kehres et al., 2002; McAllister et al., 2004; Zhou et al., 1999).
  • the MntABCD transporter complex consists of three subunits, wherein MntC and MntD are integral membrane proteins that comprise the permease subunit mediate cation transport, MntB is the ATPase, and MntA binds and delivers manganese to the permease submit.
  • Other ABC transporter operons such as sitABCD, psaABCD, and yfeABCD, exhibit similar subunit organization and function (Higgins, 1992; Rees et al., 2009).
  • the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”) (FIG. 42).
  • a manganese membrane transport protein e.g., MntH
  • the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, and a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, and a GABA transport circuit. In some embodiments, the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, a GABA transport circuit, and a GABA metabolic circuit. In some embodiments, the circuits are under the control of the same promoter. In alternate embodiments, the circuits are under the control of different promoters.
  • the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage.
  • Non-limiting examples of molecules or metabolites include, 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, and manganese in their blood and intestines.
  • the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter.
  • the genetically engineered bacteria comprise a promoter 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 gene sequence encoding one or more polypeptides for the uptake of manganese 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.
  • molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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 hepatic encephalopathy e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphata
  • 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 uptake of manganese 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 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 XI or Table X.
  • 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 uptake of manganese is operably linked to a RBS, enhancer or other regulatory sequence.
  • the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI.
  • the gene sequence encoding one or more polypeptides for the uptake of manganese is modified and/or mutated, e.g., to enhance stability, or increase manganese uptake or catalysis.
  • the gene sequence encoding one or more polypeptides for the uptake of manganese may be codon optimized, e.g., to improve expression in the host microorganism.
  • the gene sequence encoding one or more polypeptides for the uptake of manganese 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 microorganism chromosome
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese 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 uptake of manganese 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 uptake of manganese 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 uptake of manganese 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 uptake of manganese 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 uptake of manganese 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 uptake of manganese further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese 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 uptake of manganese further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
  • the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise a GABA transport circuit and/or a GABA metabolic circuit.
  • the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene.
  • the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene.
  • a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
  • One strategy in the treatment, prevention, and/or management of certain diseases, disorders, or conditions associated with hyperammonemia, including HE, 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.
  • one approach in the treatment, prevention, and/or management of gut barrier health e.g., in the context of hyperammonemia in HE, may be to provide a treatment which contains one or more of such metabolites.
  • butyrate and other SCFA e.g., derived from the
  • microbiota are known to promote maintaining 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 anti-inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, Il-6, and IFN-gamma), and inhibition of NF- ⁇ B.
  • 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 Th17 cell activity, and the maintenance of intraepithelial lymphocytes and ROR ⁇ t+ innate lymphoid cells.
  • Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3-dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis.
  • transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3-dioxygenase (IDO), which is linked to suppression of T cell
  • indole metabolites e.g., indole 3-propionic acid (IPA)
  • PXR Pregnane X receptor
  • indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.
  • the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites.
  • the genetically engineered bacteria of the invention comprise an acetate gene cassette and are capable of producing acetate.
  • the genetically engineered bacteria may include any suitable set of acetate biosynthesis genes.
  • the bacteria 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.
  • the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced.
  • the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from Escherichia coli.
  • the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from
  • Acetitomaculum Acetoanaerobium, Acetohalobium, Acetonema, Balutia,
  • Thermoacetogenium may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, 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. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions. In some embodiments, 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 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 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 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, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle.
  • 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.
  • the acetate formation from 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.,
  • 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 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, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the 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.
  • molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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 hepatic encephalopathy e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphata
  • 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 constitutive promoter.
  • 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 IX or Table X.
  • 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 IX or Table X or is listed in Table XI.
  • 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 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.
  • 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. In any of the embodiments described above and elsewhere 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 GLP2. In any of the embodiments described above and elsewhere 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 a satiety effector, e.g., GLP1.
  • a satiety effector e.g., GLP1.
  • the genetically engineered bacteria comprising one or more gene sequences for the production of acetate further comprise a GABA transport circuit and/or a GABA metabolic circuit.
  • the genetically engineered bacteria comprising one or more gene sequences for the production of acetate further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
  • the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene.
  • the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene.
  • a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
  • the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions.
  • the genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 2 and Table 3).
  • Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema.
  • 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, thiA1, 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, thiA1, 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 bcd2, etfB3, etfA3, and thiA1 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 thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola.
  • a butyrate gene cassette In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli).
  • a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB.
  • the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • additional genes may be mutated or knocked out, to further increase the levels of butyrate production.
  • Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization.
  • Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
  • the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
  • Table 14 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.
  • the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile.
  • the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • the genetically engineered bacteria of the invention comprise thiA1, 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 inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • the gene products of pbt and buk convert butyrylCoA to Butyrate.
  • the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA.
  • the genetically engineered bacteria comprise bcd2, etfB3, etfA3, thiA1, hbd, and crt2, e.g., from
  • the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E.
  • Coli and produce butyrate in low-oxygen conditions, in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells.
  • the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis.
  • local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders.
  • the bcd2 gene has at least about 80% identity with SEQ ID NO: 39. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 39. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 39. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 39. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39.
  • the bcd2 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: 39.
  • the bcd2 gene comprises the sequence of SEQ ID NO: 39.
  • the bcd2 gene consists of the sequence of SEQ ID NO: 39.
  • the etfB3 gene has at least about 80% identity with SEQ ID NO: 40. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 40. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 40. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 40. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40.
  • 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: 40.
  • the etfB3 gene comprises the sequence of SEQ ID NO: 40.
  • the etfB3 gene consists of the sequence of SEQ ID NO: 40.
  • the etfA3 gene has at least about 80% identity with SEQ ID NO: 41. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 41. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 41. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 41. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41.
  • 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: 41.
  • the etfA3 gene comprises the sequence of SEQ ID NO: 41.
  • the etfA3 gene consists of the sequence of SEQ ID NO: 41.
  • the thiA1 gene has at least about 80% identity with SEQ ID NO: 42. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 42. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 42. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 42. In another embodiment, the thiA1 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42.
  • the thiA1 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: 42.
  • the thiA1 gene comprises the sequence of SEQ ID NO: 42.
  • the thiA1 gene consists of the sequence of SEQ ID NO: 42.
  • the hbd gene has at least about 80% identity with SEQ ID NO: 43. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 43. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 43. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 43. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43.
  • 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: 43.
  • the hbd gene comprises the sequence of SEQ ID NO: 43.
  • the hbd gene consists of the sequence of SEQ ID NO: 43.
  • the crt2 gene has at least about 80% identity with SEQ ID NO: 44. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 44. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 44. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 44. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44.
  • 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: 44.
  • the crt2 gene comprises the sequence of SEQ ID NO: 44.
  • the crt2 gene consists of the sequence of SEQ ID NO: 44.
  • the pbt gene has at least about 80% identity with SEQ ID NO: 45. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 45. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 45. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 45. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45.
  • 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: 45.
  • the pbt gene comprises the sequence of SEQ ID NO: 45.
  • the pbt gene consists of the sequence of SEQ ID NO: 45.
  • the buk gene has at least about 80% identity with SEQ ID NO: 46. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 46. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 46. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 46. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46.
  • 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: 46.
  • the buk gene comprises the sequence of SEQ ID NO: 46.
  • the buk gene consists of the sequence of SEQ ID NO: 46.
  • the ter gene has at least about 80% identity with SEQ ID NO: 47. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 47. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 47. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 47. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47.
  • 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: 47.
  • the ter gene comprises the sequence of SEQ ID NO: 47.
  • the ter gene consists of the sequence of SEQ ID NO: 47.
  • the tesB gene has at least about 80% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48.
  • 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: 48.
  • the tesB gene comprises the sequence of SEQ ID NO: 48.
  • the tesB gene consists of the sequence of SEQ ID NO: 48.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding Bcd2.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 164.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 164.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 164.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 164.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 164.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 164.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding etfB3.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 165.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 165.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 165.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 165.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 165.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 165.
  • the genetically engineered bacteria comprise one or more gene sequence(s) etfA3.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 166.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 166.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 166.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 166.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 166.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 166.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding Ter.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 167.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 167.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 167.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 167.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 167.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding ThiA.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 168.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 168.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 168.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 168.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 168.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 168.
  • the genetically engineered bacteria comprise one or more gene sequence(s) Hbd.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 169.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 169.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 169.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 169.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 169.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding Crt2: Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 170.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 170.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 170.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 170.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 170.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 170.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding Pbt.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 171.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 171.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 171.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 171.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 171.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding Buk.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 172. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 172. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 172. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 172.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 172. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 172.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding TesB.
  • the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 173.
  • the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 173.
  • the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 173.
  • the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 173.
  • the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 173.
  • the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 173.
  • one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Treponema denticola butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a C. glutamicum butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a Peptoclostridicum difficile butyrate biosynthesis gene.
  • the butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate.
  • one or more targeted deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production).
  • Non-limiting examples of such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol).
  • Deletions which may be introduced therefore include deletion of adhE, ldh, and frd.
  • the genetically engineered bacteria comprise one or more butyrate- producing cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more butyrate producing cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt- buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd- crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
  • the genetically engineered bacteria produce 0% to 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, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2- fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the need may arise to prevent and/or reduce acetate production of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of butyrate production.
  • an engineered or naturally occurring strain e.g., E. coli Nissle
  • one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate.
  • a non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate.
  • Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for butyrate production.
  • one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for butyrate synthesis.
  • Such mutations and/or deletions include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and ldhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt- buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and ldhA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd- crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
  • the genetically engineered bacteria produce 0% to 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, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2- fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate, alone or in combination with various mutations in genes of the mixed acid fermentation pathway, as described herein.
  • one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production.
  • the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation.
  • 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.
  • the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
  • the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the butyrate gene cassette in nature.
  • the gene sequence encoding one or more polypeptides for the production of butyrate 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.
  • molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules.
  • the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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 hepatic encephalopathy e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphata
  • 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 butyrate 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 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 IX or Table X.
  • 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 butyrate is operably linked to a RBS, enhancer or other regulatory sequence.
  • the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI.
  • the gene sequence encoding one or more polypeptides for the production of butyrate is modified and/or mutated, e.g., to enhance stability, or increase butyrate production.
  • the gene sequence encoding one or more polypeptides for the production of butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 butyrate 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 comprising one or more gene sequences for the production of butyrate further comprise a GABA transport circuit and/or a GABA metabolic circuit.
  • the genetically engineered bacteria comprising one or more gene sequences for the production of butyrate further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
  • the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene.
  • the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene.
  • a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
  • the genetically engineered bacteria of the invention are capable of producing an anti-inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.
  • an anti-inflammatory or gut barrier enhancer molecule e.g., propionate
  • the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions.
  • the genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 16A, Table 16B, and Table 16C).
  • 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 pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA.
  • the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI.
  • the homolog of AcuI in E coli, yhdH is used.
  • the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH.
  • the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd, 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.
  • carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl- CoA to propionyl-CoA.
  • the genes may be codon-optimized, and translational and transcriptional elements may be added.
  • Table 16A, B, and C lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette.
  • Table 16D lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes.

Abstract

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating disorders associated with hyperammonemia are disclosed.

Description

Bacteria Engineered to Treat Diseases Associated with Hyperammonemia
Related Applications [0001] This application claims the benefit of PCT Application No.
PCT/US2016/020530, filed March 2, 2016; PCT Application No. PCT/US2016/050836, filed September 8, 2016, U.S. Application No.15/260,319, filed September 8, 2016; U.S. Provisional Application No.62/293,749, filed February 10, 2016; U.S. Provisional Application No. 62/347,567, filed June 8, 2016; U.S. Provisional Application No.
62/348,699, filed June 10, 2016; PCT Application No. PCT/US2016/34200, filed May 25, 2016; U.S. Application No.15/164,828, filed May 25, 2016; U.S. Provisional Application No. 62/347,508, filed June 8, 2016; U.S. Provisional Application No.
62/354,682, filed June 24, 2016; U.S. Provisional Application No. 62/362,954, filed July 15, 2016; U.S. Provisional Application No.62/385,235, filed September 8, 2016; U.S. Provisional Application No.62/423,170, filed November 16, 2016; U.S.
Provisional Application No. 62/439,871, filed December 28, 2016; PCT Application No. PCT/US2016/032565, filed May 13, 2016; PCT Application No.
PCT/US2017/016603, filed February 3, 2017; and PCT Application No.
PCT/US2017/016609, filed February 3, 2017. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties to provide continuity of disclosure. Background
[0002] Ammonia is highly toxic and generated during metabolism in all organs (Walker, 2012). In mammals, the healthy liver protects the body from ammonia by converting ammonia to non-toxic molecules, e.g., urea or glutamine, and preventing excess amounts of ammonia from entering the systemic circulation. Hyperammonemia is characterized by the decreased detoxification and/or increased production of ammonia. In mammals, the urea cycle detoxifies ammonia by enzymatically converting ammonia into urea, which is then removed in the urine. Decreased ammonia detoxification may be caused by urea cycle disorders (UCDs) in which urea cycle enzymes are defective, such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency (Häberle et al., 2012). The National Urea Cycle Disorders Foundation estimates that the prevalence of UCDs is 1 in 8,500 births. In addition, several non-UCD disorders, such as hepatic
encephalopathy, portosystemic shunting, and organic acid disorders, can also cause hyperammonemia. Hyperammonemia can produce neurological manifestations, e.g., seizures, ataxia, stroke-like lesions, coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respiratory alkalosis, hypothermia, or death (Häberle et al., 2012; Häberle et al., 2013).
[0003] Ammonia is also a source of nitrogen for amino acids, which are synthesized by various biosynthesis pathways. For example, arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms. Intermediate metabolites formed in the arginine biosynthesis pathway, such as citrulline, also incorporate nitrogen. Thus, enhancement of arginine biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules in order to modulate or treat conditions associated with hyperammonemia. Likewise, histidine biosynthesis, methionine biosynthesis, lysine biosynthesis, asparagine biosynthesis, glutamine biosynthesis, and tryptophan biosynthesis are also capable of incorporating excess nitrogen, and enhancement of those pathways may be used to modulate or treat conditions associated with hyperammonemia.
[0004] Current therapies for hyperammonemia and UCDs aim to reduce ammonia excess, but are widely regarded as suboptimal (Nagamani et al., 2012;
Hoffmann et al., 2013; Torres-Vega et al., 2014). Most UCD patients require substantially modified diets consisting of protein restriction. However, a low-protein diet must be carefully monitored; when protein intake is too restrictive, the body breaks down muscle and consequently produces ammonia. In addition, many patients require supplementation with ammonia scavenging drugs, such as sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate, and one or more of these drugs must be administered three to four times per day (Leonard, 2006; Diaz et al., 2013). Side effects of these drugs include nausea, vomiting, irritability, anorexia, and menstrual disturbance in females (Leonard, 2006). In children, the delivery of food and medication may require a gastrostomy tube surgically implanted in the stomach or a nasogastric tube manually inserted through the nose into the stomach. When these treatment options fail, a liver transplant may be required (National Urea Cycle Disorders Foundation). Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for disorders associated with hyperammonemia, including urea cycle disorders.
[0005] The liver plays a central role in amino acid metabolism and protein synthesis and breakdown, as well as in several detoxification processes, notably those of end-products of intestinal metabolism, like ammonia. Liver dysfunction, resulting in hyperammonemia, may cause hepatic encephalopathy (HE), which disorder
encompasses spectrum of potentially reversible neuropsychiatric abnormalities observed in patients with liver dysfunction (after exclusion of unrelated neurologic and/or metabolic abnormalities). In HE, severe liver failure (e.g., cirrhosis) and/or
portosystemic shunting of blood around the liver permit elevated arterial levels of ammonia to permeate the blood-brain barrier (Williams, 2006), resulting in altered brain function.
[0006] Ammonia accumulation in the brain leads to cognitive and motor disturbances, reduced cerebral perfusion, as well as oxidative stress-mediated injury to astrocytes, the brain cells capable of metabolizing ammonia. There is evidence to suggest that excess ammonia in the brain disrupts neurotransmission by altering levels of the predominant inhibitory neurotransmitter, γ-aminobutyric acid (GABA)
(Ahboucha and Butterworth, 2004). Elevated cerebral manganese concentrations and manganese deposition have also been reported in the basal ganglia of cirrhosis patients, and are suspected to contribute to the clinical presentation of HE (Cash et al., 2010; Rivera-Mancía et al., 2012). General neurological manifestations of hyperammonemia include seizures, ataxia, stroke-like lesions, Parkinsonian symptoms (such as tremors), coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respiratory alkalosis, hypothermia, or death (Häberle et al., 2012; Häberle et al., 2013).
[0007] Ammonia dysmetabolism cannot solely explain all the neurological changes that are seen in patients with HE. Sepsis is a well-known precipitating factor for HE. The systemic inflammatory response syndrome (SIRS) results from the release and circulation of proinflammatory cytokines and mediators. In patients with cirrhosis, SIRS may exacerbate the symptoms of HE, both in patients with minimal and overt HE in a process likely mediated by tumor necrosis factor (TNF) and interleukin- 6 (IL6). Notably, enhanced production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) occurs in cultured astrocytes that are exposed to ammonia, inflammatory cytokines, hyponatremia or benzodiazepines.
[0008] Hyperammonemia is also a prominent feature of Huntington’s disease, an autosomal dominant disorder characterized by intranuclear/cytoplasmic aggregates and cell death in the brain (Chen et al., 2015; Chiang et al., 2007). In fact,
hyperammonemia is a feature of several other disorders, as discussed herein, all of which can be treated by reducing the levels of ammonia.
[0009] Current therapies for hepatic encephalopathy, Huntington’s disease, and other diseases and disorders associated with excess ammonia levels, are insufficient (Cash et al., 2010; Córdoba and Mínguez, 2008; Shannon and Fraint, 2015). In
Huntington’s disease, the side effects of antipsychotic drugs (e.g., haloperidol, risperidone, quetiapine) and drugs administered to suppress involuntary movements (e.g., tetrabenazine, amantadine, levetiracetam, clonazepam) may worsen muscle rigidity and cognitive decline in patients (Mayo Clinic). Antibiotics directed to urease- producing bacteria were shown to have severe secondary effects, such as
nephrotoxicity, especially if administered for long periods (Blanc et al., 1992; Berk and Chalmers, 1970). Protein restriction is also no longer a mainstay therapy, as it can favor protein degradation and poor nutritional status, and has been associated with increased mortality (Kondrup and Müller, 1997; Vaqero et al., 2003). Protein restriction is only appropriate for one third of cirrhotic patients with HE (Nguyen and Morgan, 2014). Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for hepatic encephalopathy and Huntington’s disease. Summary
[0010] The disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and converting ammonia and/or nitrogen into alternate byproducts. In certain embodiments, the genetically engineered bacteria reduce excess ammonia and convert ammonia and/or nitrogen into alternate byproducts. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxic ammonia. As much as 70% of excess ammonia in a hyperammonemic patient accumulates in the gastrointestinal tract.
Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of ammonia and/or nitrogen consumption, or production of a non-toxic byproduct, e.g., arginine or citrulline. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with hyperammonemia, e.g., urea cycle disorders and hepatic encephalopathy.
[0011] The disclosure also provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules, e.g., GABA, manganese. In certain embodiments, the genetically engineered bacteria reduce excess ammonia and convert ammonia and/or nitrogen into alternate byproducts. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to reduce toxic ammonia. In some embodiments, the genetically engineered bacteria are capable of reducing excess ammonia and are also capable of producing one or more gut barrier enhancer molecules, e.g., one or more short chain fatty acid(s), such as butyrate and/or acetate. In certain embodiments, the genetically engineered bacteria are also capable of reducing excess ammonia and other deleterious molecules, e.g., GABA, manganese.
[0012] The disclosure also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with excess ammonia, including, for example, hepatic encephalopathy and Huntington’s disease.
[0013] Disclosed herein is a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette for producing butyrate, wherein the bacterium comprises an endogenous pta gene which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
[0014] In some embodiments, the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, pbt, and buk genes. In some embodiments, the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, and tesb genes.
[0015] In some embodiments, the bacterium comprises an endogenous adhE gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.
[0016] In some embodiments, the promoter operably linked to the at least one gene or gene cassette is induced by exogenous environmental conditions. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by low-oxygen or anaerobic conditions. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive nitrogen species. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive oxygen species. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
[0017] Disclosed herein is a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette for producing butyrate, wherein the bacterium comprises at least one endogenous gene selected from frd, ldhA, and adhE, which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
[0018] In some embodiments, the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; and wherein the bacterium lacks a functional ArgR. In some embodiments, each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted. In some embodiments, under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
[0019] In some embodiments, the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; wherein the arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase; and wherein each operon except the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine-mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the genes in the operon. In some embodiments, the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine-mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the argininosuccinate synthase gene.
[0020] In some embodiments, the operon comprising the gene encoding argininosuccinate synthase comprises a constitutively active promoter that regulates transcription of the argininosuccinate synthase gene. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by endogenous environmental conditions. In some embodiments, arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced under low oxygen conditions. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species.
[0021] In some embodiments, the bacterium is a non-pathogenic bacterium. In some embodiments, the bacterium is a probiotic bacterium. In some embodiments, the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In some embodiments, the bacterium is Escherichia coli strain Nissle.
[0022] In some embodiments, the at least one gene or gene cassette for producing a butyrate is present on a plasmid in the bacterium and operably linked on the plasmid to the inducible promoter. In some embodiments, the at least one gene or gene cassette for producing butyrate is present on a bacterial chromosome and operably linked on chromosome to the inducible promoter.
[0023] Disclosed herein is a bacterium comprising at least one gene or gene cassette for the consumption of ammonia and at least one gene or gene cassette selected from (1) a GABA metabolic gene or gene cassette (2) a GABA transport gene or gene cassette, (3) a manganese transport gene or gene cassette.
[0024] In some embodiments, the at least one gene for the consumption of GABA is capable of producing a GABA catabolism enzyme. In some embodiments, the GABA catabolism enzyme is selected from GABA α-ketoglutarate transaminase (GSST) and succinate-semialdehyde dehydrogenase (SSDH).
[0025] In some embodiments, the GABA transport circuit is capable of producing a GABA membrane transport protein. In some embodiments, the GABA membrane transport protein is GabP. [0026] In some embodiments, the manganese transport circuit is capable of producing a manganese membrane transport protein. In some embodiments, the manganese membrane transport protein is MntH.
[0027] In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by exogenous environmental conditions. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced under low oxygen conditions. In some embodiments, the at least one gene or gene cassette is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by the presence of reactive nitrogen species. In some embodiments, the at least one gene or gene cassette is controlled by a promoter induced by the presence of reactive oxygen species. In some embodiments, the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
[0028] In some embodiments, the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; and wherein the bacterium has been engineered to lack a functional ArgR. In some embodiments, each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
[0029] In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species. In some embodiments, arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species. In some embodiments, under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine
biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
[0030] In some embodiments, the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit, is present on a plasmid in the bacterium and operably linked on the plasmid to the inducible promoter. In some embodiments, the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit, is present on a bacterial chromosome and operably linked on chromosome to the inducible promoter.
[0031] In some embodiments, the bacterium is an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut. In some
embodiments, the mammalian gut is a human gut.
[0032] Disclosed herein is a pharmaceutically acceptable composition comprising one or more of any of the bacteria disclosed herein. In some embodiments, the composition comprising the bacterium is formulated for oral or rectal
administration.
[0033] Disclosed herein is a method of treating a disease, disorder or condition associated with hyperammonemia, or symptom(s) thereof in a subject in need thereof comprising the step of administering to the subject any composition described herein for a period of time sufficient to lessen the severity of the disease or symptom(s). In some embodiments, the disease, disorder, or condition is hepatic encephalopathy,
Huntington’s disease, or symptom(s) thereof.
[0034] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non- native component(s), which mediate one or more mechanisms of action. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted. The genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression.
[0035] These gene(s)/gene cassette(s) may be under the control of constitutive or inducible promoters. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific 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.
[0036] In addition, the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Brief Description of the Figures
[0037] FIG. 1A and FIG. 1B depict the state of the arginine regulon in one embodiment of an ArgR deletion bacterium of the invention under non-inducing (FIG. 1A) and inducing (FIG. 1B) conditions. FIG. 1A depicts relatively low arginine production under aerobic conditions due to arginine (“Arg” in oval) interacting with ArgA (squiggle ) to inhibit (indicated by“X”) ArgA activity, while oxygen (O2) prevents (indicated by“X”) FNR (dotted boxed FNR) from dimerizing and activating fbr
the FNR promoter (grey FNR box) and the argA gene under its control. FIG. 1B depicts up-regulated arginine production under anaerobic conditions due to FNR dimerizing (two dotted boxed FNRs) and inducing FNR promoter (grey FNR box)- fbr fbr
mediated expression of ArgA (squiggle above argA ), which is resistant to inhibition by arginine. This overcomes (curved arrow) the inhibition of the wild-type ArgA caused by arginine (“Arg” in oval) interacting with ArgA (squiggle above box depicting argA). Each gene in the arginine regulon is depicted by a rectangle containing the name of the gene. Each arrow adjacent to one or a cluster of rectangles depict the promoter responsible for driving transcription, in the direction of the arrow, of such gene(s). Heavier lines adjacent one or a series of rectangles depict ArgR binding sites, which are not utilized because of the ArgR deletion in this bacterium. Arrows above each rectangle depict the expression product of each gene.
[0038] FIG. 2A and FIG. 2B depict an alternate exemplary embodiment of the present invention. FIG. 2A depicts the embodiment under aerobic conditions where, in the presence of oxygen, the FNR proteins (FNR boxes) remain as monomers and are unable to bind to and activate the FNR promoter (“FNR”) which drives expression of the arginine feedback resistant argAfbr gene. The wild-type ArgA protein is functional, but is susceptible to negative feedback inhibition by binding to arginine, thus keeping arginine levels at or below normal. All of the arginine repressor (ArgR) binding sites in the promoter regions of each arginine biosynthesis gene (argA, argE, argC, argB, argH, argD, argI, argG, carA, and carB) have been mutated (black bars; black“X”) to reduce or eliminate binding to ArgR. FIG. 2B depicts the same embodiment under anaerobic conditions where, in the absence of oxygen the FNR protein (FNR boxes) dimerizes and binds to and activates the FNR promoter (“FNR”). This drives expression of the arginine feedback resistant argAfbr gene (black squiggle ( ) = argAfbr gene expression product), which is resistant to feedback inhibition by arginine (“Arg” in ovals). All of the arginine repressor (ArgR) binding sites in the promoter regions of each arginine biosynthetic gene (argA, argE, argC, argB, argH, argD, argI, argG, carA, and carB) have been mutated (black bars) to reduce or eliminate binding to ArgR (black“X”), thus preventing inhibition by an arginine-ArgR complex. This allows high level production of arginine.
[0039] FIG. 3 depicts another embodiment of the invention. In this
embodiment, a construct comprising an ArgR binding site (black bar) in a promoter driving expression of the Tet repressor (TetR) from the tetR gene is linked to a second promoter comprising a TetR binding site (black bar between TetR and X) that drives expression of gene X. Under low arginine concentrations, TetR is expressed and inhibits the expression of gene X. At high arginine concentrations, ArgR associates with arginine and binds to the ArgR binding site, thereby inhibiting expression of TetR from the tetR gene. This, in turn, removes the inhibition by TetR allowing gene X expression (black squiggle ( )).
[0040] FIG. 4 depicts another embodiment of the invention. In this embodiment, a construct comprising an ArgR binding site (black bar) in a promoter driving expression of the Tet repressor (TetR) from the tetR gene is linked to a second promoter comprising a TetR binding site (black bar bound to TetR oval) that drives expression of green fluorescent protein (“GFP”). Under low arginine concentrations, TetR is expressed and inhibits the expression of GFP. At high arginine concentrations, ArgR associates with arginine and binds to the ArgR binding site, thereby inhibiting expression of TetR from the tetR gene. This, in turn, removes the inhibition by TetR allowing GFP expression. By mutating a host containing this construct, high arginine producers can be selected on the basis of GFP expression using fluorescence-activated cell sorting (“FACS”).
[0041] FIG. 5 depicts another embodiment of the invention. In this embodiment, a construct comprising an ArgR binding site (black bar bound by the ArgR-Arg complex) in a promoter driving expression of the Tet repressor (not shown) from the tetR gene is linked to a second promoter comprising a TetR binding site (black bar) that drives expression of an auxotrophic protein necessary for host survival (“AUX”). Under high arginine concentrations, the ArgR-arginine complex binds to the ArgR binding site, thereby inhibiting expression of TetR from the tetR gene. This, in turn, allows expression of AUX, allowing the host to survive. Under low arginine concentrations, TetR is expressed from the tetR gene and inhibits the expression of AUX, thus killing the host. The construct in FIG. 5 enforces high arginine (“Arg”) production by making it necessary for host cell survival through its control of AUX expression.
[0042] FIG. 6 depicts a schematic diagram of the argAfbr gene under the control of an exemplary FNR promoter (fnrS) fused to a strong ribosome binding site.
[0043] FIG. 7 depicts another schematic diagram of the argAfbr gene under the control of an exemplary FNR promoter (nirB) fused to a strong ribosome binding site. Other regulatory elements may also be present.
[0044] FIG. 8 depicts a schematic diagram of the argAfbr gene under the control of an exemplary FNR promoter (nirB) fused to a weak ribosome binding site. [0045] FIG. 9A and FIG. 9B depict exemplary embodiments of a FNR- responsive promoter fused to a CRP binding site. FIG. 9A depicts a map of the FNR- CRP promoter region, with restriction sites shown in bold. FIG. 9B depicts a schematic diagram of the argAfbr gene under the control of an exemplary FNR promoter (nirB promoter), fused to both a CRP binding site and a ribosome binding site. Other regulatory elements may also be present.
[0046] FIG. 10A and FIG. 10B depict alternate exemplary embodiments of a FNR-responsive promoter fused to a CRP binding site. FIG. 10A depicts a map of the FNR-CRP promoter region, with restriction shown in bold. FIG. 10B depicts a schematic diagram of the argAfbr gene under the control of an exemplary FNR promoter (fnrS promoter), fused to both a CRP binding site and a ribosome binding site.
[0047] FIG. 11 depicts an exemplary embodiment of a constitutively expressed argG construct in E. coli Nissle. The constitutive promoter is BBa_J23100, boxed in gray. Restriction sites for use in cloning are in bold.
[0048] FIG. 12 depicts a map of the wild-type argG operon E. coli Nissle, and a constitutively expressing mutant thereof. ARG boxes are present in the wild-type operon, but absent from the mutant. ArgG is constitutively expressed under the control of the BBa_J23100 promoter.
[0049] FIG. 13 depicts a schematic diagram of an exemplary BAD promoter- fbr fbr
driven argA construct. In this embodiment, the argA gene is inserted between the fbr
araC and araD genes. ArgA is flanked by a ribosome binding site, a FRT site, and one or more transcription terminator sequences.
[0050] FIG. 14 depicts an exemplary embodiment of an engineered bacterial strain deleted for the argR gene and expressing the feedback-resistant argAfbr gene. In some embodiments, this strain further comprises one or more auxotrophic modifications on the chromosome. This strain is useful for the consumption of ammonia and the production of arginine.
[0051] FIG. 15 depicts an exemplary embodiment of an engineered bacterial strain deleted for the argR and argG genes, and expressing the feedback-resistant argAfbr gene. In some embodiments, this strain further comprises one or more auxotrophic modifications on the chromosome. This strain is useful for the
consumption of ammonia and the production of citrulline. [0052] FIG. 16 depicts an exemplary embodiment of an engineered bacterial strain which lacks ArgR binding sites and expresses the feedback-resistant argAfbr gene. In some embodiments, this strain further comprises one or more auxotrophic
modifications on the chromosome. This strain is useful for the consumption of ammonia and the production of arginine.
[0053] FIG. 17 depicts an exemplary embodiment of an engineered bacterial strain which lacks ArgR binding sites in all of the arginine biosynthesis operons except for argG, and expresses the feedback-resistant argAfbr gene. In some embodiments, this strain further comprises one or more auxotrophic modifications on the chromosome. This strain is useful for the consumption of ammonia and the production of citrulline.
[0054] FIG. 18 depicts a map of exemplary integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. The malE/K site is circled. In some embodiments of the disclosure, FNR-ArgAfbr is inserted at the malEK locus.
[0055] FIG. 19 depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) is non-fluorescent.
[0056] FIG. 20 depicts the gene organization of exemplary constructs of the disclosure. Non-limiting examples of strains comprising such a construct include SYN- UCD301 and SYN-UCD302. SYN-UCD301 comprises∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, Wild type ThyA, and
Chloramphenicol resistance.
[0057] FIG. 21 depicts the gene organization of an exemplary construct of the disclosure. Non-limiting examples of strains comprising such a construct include SYN- UCD303, SYN-UCD306, SYN-UCD307, and SYN-UCD309. For example, SYN- UCD303 comprises∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and kanamycin resistance. [0058] FIG. 22 depicts the gene organization of exemplary constructs of the disclosure. Non-limiting examples of strains comprising such a construct include SYN- UCD304, SYN-UCD305, SYN-UCD308, and SYN-UCD310. For example, SYN- UCD304 comprises∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, wild type ThyA, and no antibiotic resistance. SYN-UCD305 comprises ∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and no antibiotic resistance.
[0059] FIG. 23 depicts a bar graph of in vitro arginine levels produced by streptomycin-resistant control Nissle (SYN-UCD103), SYN-UCD201, SYN-UCD202, and SYN-UCD203 under inducing (+ATC) and non-inducing (-ATC) conditions. SYN- fbr
UCD201 comprises∆ArgR and no argA . SYN-UCD202 comprises∆ArgR and
fbr
tetracycline-inducible argA on a high-copy plasmid. SYN-UCD203 comprises
fbr
∆ArgR and tetracycline-driven argA on a low-copy plasmid.
[0060] FIG. 24 depicts a bar graph of in vitro levels of arginine and citrulline produced by streptomycin-resistant control Nissle (SYN-UCD103), SYN-UCD104, SYN-UCD204, and SYN-UCD105 under inducing conditions. SYN-UCD104 comprises wild-type ArgR, tetracycline-inducible argAfbr on a low-copy plasmid, tetracycline-inducible argG, and mutations in each ARG box for each arginine biosynthesis operon except for argG. SYN-UCD204 comprises∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid. SYN-UCD105 comprises wild-type ArgR, tetracycline-inducible argAfbr on a low-copy plasmid, constitutively expressed argG (BBa_J23100 constitutive promoter), and mutations in each ARG box for each arginine biosynthesis operon except for argG.
[0061] FIG. 25 depicts a bar graph of in vitro arginine levels produced by streptomycin-resistant Nissle (SYN-UCD103), SYN-UCD205, and SYN-UCD204 under inducing (+ATC) and non-inducing (-ATC) conditions, in the presence (+O2) or absence (-O2) of oxygen. SYN-UCD103 is a control Nissle construct. SYN-UCD205 comprises∆ArgR and argAfbr expressed under the control of a FNR-inducible promoter on a low-copy plasmid. SYN204 comprises∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid.
[0062] FIG. 26 depicts a bar graph of in vitro ammonia levels in culture media from SYN-UCD101, SYN-UCD102, and blank controls at baseline, two hours, and four hours. Both SYN-UCD101 and SYN-UCD102 are capable of consuming ammonia in vitro. SYN-UCD101 comprises wild type ArgR, and wild type ThyA, and no ArgAFbr; SYN-UCD102 comprises wild type ArgR, tetracycline-inducible argAfbr on a low copy plasmid, and wild type ThyA.
[0063] FIG. 27 depicts a bar graph of in vitro ammonia levels in culture media from SYN-UCD202, SYN-UCD203, and blank controls at baseline, two hours, and four hours. Both SYN-UCD202 and SYN-UCD203 are capable of consuming ammonia in vitro. SYN-UCD202 and SYN-UCD203 both comprise∆ArgR, tetracycline-inducible argAfbr on a high-copy plasmid or low copy plasmid, respectively, Amp resistance, and wild type ThyA.
[0064] FIG. 28A, 28B, and 28C depict bar graphs of ammonia levels in hyperammonemic TAA mice. FIG. 28A depicts a bar graph of ammonia levels in hyperammonemic mice treated with unmodified control Nissle or SYN-UCD202, a fbr genetically engineered strain in which the Arg repressor gene is deleted and the argA gene is under the control of a tetracycline-inducible promoter on a high-copy plasmid. A total of 96 mice were tested, and the error bars represent standard error. Blood ammonia (BA) levels in mice treated with SYN-UCD202 are lower than ammonia levels in mice treated with unmodified control Nissle at day 4 and day 5 (Nissle, BA = 220 mM; SYN-UCD202, BA = 105 mM; BANissle - BASYN-UCD202 = 115 mM; average blood volume = 1.5 mL. FIG. 28B depicts a bar graph showing in vivo efficacy
(ammonia consumption) of SYN-UCD204 in the TAA mouse model, relative to streptomycin-resistant control Nissle (SYN-UCD103) and vehicle-only controls. FIG. 28C depicts a bar graph of the percent change in blood ammonia concentration between 24-48 hours post-TAA treatment.
[0065] FIG. 29 depicts a bar graph of ammonia levels in hyperammonemic spfash mice. Fifty-six spfash mice were separated into four groups. Group 1 was fed normal chow, and groups 2-4 were fed 70% protein chow following an initial blood draw. Groups were gavaged twice daily, with water, streptomycin-resistant Nissle control (SYN-UCD103), or SYN-UCD204, and blood was drawn 4 hours following the first gavage. SYN-UCD204, comprising∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid, significantly reduced blood ammonia to levels below the hyperammonemia threshold. [0066] FIG. 30 depicts a bar graph of ammonia levels in hyperammonemic spfash mice on a high protein diet. Mice were treated with SYN-UCD204 (comprising ∆ArgR, PfnrS-ArgAfbr on a low-copy plasmid and wild type ThyA), SYN-UCD206 (comprising∆ArgR, PfnrS- ArgAfbr on a low-copy plasmid and∆ThyA) or water, then switched to high protein chow after 2 days. As seen in FIG. 30, at 48 hours after switch to high protein chow ammonia levels were reduced to a similar extent in both SYN- UCD205 and SYN-UCD206, indicating that ThyA auxotrophy does not have a significant effect on efficacy.
[0067] FIG. 31A and 31B depict bar graphs of ammonia levels in the media at various time points post anaerobic induction. FIG. 31A depicts a bar graph of the levels of arginine production of SYN-UCD205, SYN-UCD206, and SYN-UCD301 measured at 0, 30, 60, and 120 minutes. FIG.31B depicts a bar graph of the levels of arginine production of SYN-UCD204 (comprising∆ArgR, PfnrS-ArgAfbr on a low- copy plasmid and wild type ThyA), SYN-UCD301, SYN-UCD302, and SYN-UCD303 (all three of which comprise an integrated FNR-ArgAfbr construct; SYN UCD301 comprises∆ArgR, and wtThyA; SYN 303 comprises∆ArgR, and∆ThyA). Results indicate that chromosomal integration of FNR ArgA fbr results in similar levels of arginine production as seen with the low copy plasmid strains expressing the same construct.
[0068] FIG. 32A and 32B depicts a bar graph of ammonia levels and a survival curve for hyperammonemic spfash mice on a normal (NC) or high protein (HP) diet. Two strains with an integrated copy of FNR-ArgAfbr, one with (SYN-UCD303) and one without a ThyA deletion (SYN-UCD301) were compared. FIG. 32A depicts a bar graph of ammonia levels in hyperammonemic spfash mice on a normal (NC) or high protein (HP) diet. Ammonia levels of spf-ash mice in a high protein diet were reduced in the SYN-UCD301 and SYN-UCD303 groups as compared to the H2O high protein diet control group. The observed reduction in ammonia levels was similar in both SYN- UCD301 and SYN-UCD303, indicating that ThyA auxotrophy does not have a significant effect on efficacy of SYN-UCD303. FIG. 32B depicts a survival curve of hyperammonemic spfash mice on a normal (NC) or high protein (HP) diet and shows that SYN-UCD301 and SYN-UCD303 displayed prolonged survival as compared to controls. [0069] FIG. 33 depicts a graph of blood ammonia levels in an hyperammonemic spfash mice on a normal (NC) or high protein (HP) diet. For SYN- UCD303, doses of 1 X107, 1X108, 1X109, and 1 X1010 cells were administered daily over a time course of 12 days. Blood ammonia levels were measured on day 5. Both doses of 1X108 and 1X109 were sufficient to result in a significant reduction of blood ammonia levels in this model. SYN-UCD303 comprises∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Kanamycin resistance.
[0070] FIG. 34A 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.
[0071] FIG. 34B depicts a graph showing bacterial cell growth of a Nissle thyA auxotroph strain (thyA knock-out) in various concentrations of thymidine. A
chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in LB + 10mM thymidine at 37C. The next day, cells were diluted 1:100 in 1 mL LB + 10mM 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.
[0072] FIG. 34C 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. [0073] FIG. 35A, 35B, and 35C depict bar graphs of bacterial residence in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each time point, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating. FIG. 35A depicts a bar graph of residence over time for SYN-UCD103 (streptomycin resistant Nissle). FIG. 35B depicts a bar graph residence over time for SYN-UCD106, comprising∆ArgR and∆ThyA and no ArgAfbr. FIG. 35C depicts a bar graph showing residence over time for SYN-UCD303, comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, and∆ThyA.
[0074] FIG. 36A, 36B, and 36C depict bar graphs of viable bacterial cells and arginine production. Cells were either incubated with 70% isopropanol or phosphate buffered saline (PBS) as a control for 1 hour with shaking. After treatment, the cells were mixed at specific ratios in M9 media supplemented with 0.5% glucose and 3mM thymidine and incubated with shaking at 37 C for 2 hours. As seen in FIG. 36A and 36B., a greater ratio of isopropanol treated cells to untreated in a culture results in fewer CFUs as determined by plating, and lower levels of arginine production. Arginine production relative to amount of bacteria present remained constant across the various cultures (FIG. 87C). These results indicate that only viable bacteria are contributing to arginine production.
[0075] FIG. 37. Depicts the number of bacteria quantified in fecal samples collected in a non-human primate toxicity study. Pharmacokinetics and
pharmacodynamics resulting from administration of SYN-UCD107 (a kanamycin resistant Nissle) and SYN-UCD303 (comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Kanamycin resistance) was compared over 50 days. Results indicate that under these dosing conditions, similar amounts of bacteria were recovered with the auxotroph SYN-UCD303 as control kanamycin resistant Nissle in the feces. Similar results have been observed in mice.
[0076] FIG. 38 depicts an exemplary synthetic genetic circuit for treating hepatic encephalopathy and other disorders characterized by hyperammonemia. In the ammonia conversion circuit, ammonia is taken up by a bacterium (e.g., E. coli Nissle), converted to glutamate, and glutamate is subsequently metabolized to arginine.
Arginine ultimately exits the bacterial cell.
[0077] FIG. 39 depicts one embodiment of the invention. In this embodiment, the genetically engineered bacteria comprise four exemplary circuits for the treatment of hepatic encephalopathy. In one circuit, ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
Arginine ultimately exits the bacterial cell. In a second circuit, the GABA membrane transport protein (GabP) is expressed by the gabP gene, and facilitates GABA transport into the cell. In a third circuit, the bacterial manganese transport protein (MntH) is expressed by the mntH gene, and facilitates manganese transport into the cell. In a fourth circuit, expression of a butyrate gene cassette results in the production of butyrate, and release of this gut barrier enhancer molecule outside of the cell. In some embodiments, all four circuits are each under the control of the same inducible promoter. In other embodiments, the four circuits may be under the control of different inducible promoters. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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.
[0078] FIG. 40 depicts one embodiment of the invention. In this embodiment, the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy. In one circuit, ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
Arginine ultimately exits the bacterial cell. In a second circuit, the GABA membrane transport protein (GabP) is expressed by the gabP gene, and facilitates GABA transport into the cell. In some embodiments, both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of a different inducible promoter. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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. In other embodiments, the genetically engineered bacteria may further comprise an additional circuit for reducing the level of GABA, e.g., a circuit for metabolizing (catabolizing) GABA.
[0079] FIG. 41A and FIG.41B depict the catabolism of GABA following uptake into genetically engineered bacteria comprising synthetic genetic circuits. In FIG. 41A, upon entry into the cell, GABA is converted to succinyl semialdehyde by GABA α-ketoglutarate transaminase (GSST). Succinate-semialdehyde dehydrogenase (SSDH) then catalyzes the second and only other specific step in GABA catabolism, the oxidation of succinyl semialdehyde to succinate. Ultimately, succinate becomes a substrate for the citric acid (TCA) cycle. GOT (glutamate oxaloacetate transaminase) converts alpha-ketoglutarate to glutamate. In certain embodiments, the genetically engineered bacteria of the disclosure comprise a GABA consuming circuit including, but not limited to, one or more of GSST, SSDH, and GOT. FIG. 41B depicts a schematic representation of the GABA utilization pathway in E. coli Nissle.
[0080] FIG. 42 depicts one embodiment of the invention. In this embodiment, the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy. In one circuit, ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine.
Arginine ultimately exits the bacterial cell. In a second circuit, the bacterial manganese transport protein (MntH) is expressed by the mntH gene, and facilitates manganese transport into the cell. In some embodiments, both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of different inducible promoter. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE- specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. FIG. 43 depicts one embodiment of the invention. In this embodiment, the genetically engineered bacteria comprise two exemplary circuits for the treatment of hepatic encephalopathy. In one circuit, ammonia is taken up by the bacterium, converted to glutamate, and glutamate is subsequently metabolized to arginine. Arginine ultimately exits the bacterial cell. In a second circuit, expression of a butyrate gene cassette results in the production of butyrate, and release of this gut barrier enhancer molecule outside of the cell. In some embodiments, both circuits are under the control of the same inducible promoter. In other embodiments, the two circuits may each be under the control of different inducible promoter. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), promoters induced by inflammation or an inflammatory response, 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. One or more of the butyrate cassettes described herein may be expressed by the genetically engineered bacteria comprising an arginine (and/or citrulline) producing circuit. FIG. 44 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple mechanisms of action (MoAs).
[0081] FIG. 45 depicts an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs. In some embodiments, an ammonia conversion circuit, a butyrate production circuit, a GABA transport and/or a GABA metabolic circuit, and a manganese transport circuit are inserted at four or more different chromosomal insertion sites
[0082] FIG. 46A and FIG.46B depict an exemplary schematic of the E. coli 1917 Nissle chromosome comprising multiple MoAs. In FIG. 46A, an ammonia conversion circuit, a butyrate production circuit, and a GABA transport and/or GABA metabolic circuit are inserted at three different chromosomal insertion sites. In FIG. 46B, an ammonia conversion circuit, a GABA transport and/or GABA metabolic circuit, and a manganese transport circuit are inserted at three or more different chromosomal insertion sites.
[0083] FIG. 47A and FIG.47B depict exemplary schematics of the E. coli 1917 Nissle chromosome comprising multiple MoAs. In FIG. 47A, an ammonia conversion circuit, and a manganese transport circuit are inserted at two different chromosomal insertion sites. In FIG. 47B, an ammonia conversion circuit, and a GABA transport and/or GABA metabolic circuit are inserted at two or more different chromosomal insertion sites.
[0084] FIG. 48A depicts a schematic of a metabolic pathway for butyrate production. FIG. 48B and 48C 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. 48D 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” refers 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 cassette that comprises at least the following genes: ter, thiA1, 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, thiA1, hbd, crt2, and tesB genes. An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiA1, hbd, crt2, and tesB genes. In some embodiments, 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 HE-specific 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.
[0085] FIG. 49A, FIG. 49B, FIG. 49C, FIG. 49D, FIG.49E, FIG. 49F depict schematics showing the gene organization of exemplary engineered bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIG. 49A and 49B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 49A depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) 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, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 49B 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. FIG. 49C and 49D depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In FIG. 49C, in the absence of NO, the NsrR transcription factor (gray circle,“NsrR”) binds to and represses a
corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 49D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIG.49E and F depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H2O2. In FIG. 49E, in the absence of H2O2, 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, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 49F, in the presence of H2O2, the OxyR transcription factor interacts with H2O2 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.
[0086] FIG.50A, FIG.50B, FIG.50C, FIG.50D, FIG.50E, and FIG.50F depict schematics showing the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate. FIG. 50A and 50B depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 50A depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) 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, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed. FIG. 50B 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. FIG. 50C and 50D depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 50C, in the absence 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, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 50D, in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. FIG. 50E and 50F depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H2O2. In FIG. 50E, in the absence of H2O2, the OxyR transcription factor (gray circle,“OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) is expressed. In FIG. 50F, in the presence of H2O2, the OxyR transcription factor interacts with H2O2 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.
[0087] FIG. 50G, FIG. 50H, FIG. 50I, FIG. 50J, FIG.50K, and FIG. 50L depict schematics of the gene organization of exemplary bacteria of the disclosure. FIG. 50G and FIG. 50H depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions. FIG. 50G depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) prevents (indicated by“X”) FNR (boxed“FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, and tesB) is expressed. FIG. 50H depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed“FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate. FIG. 50I and FIG. 50J depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In FIG. 50I, in the absence of NO, the NsrR transcription factor (“NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed. In FIG. 50J, in the presence of NO, the NsrR
transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. FIG. 50K and FIG. 50L depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H2O2. In FIG. 50K, in the absence of H2O2, the OxyR transcription factor (circle,“OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed. In FIG. 50L, in the presence of H2O2, the OxyR transcription factor interacts with H2O2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.
[0088] FIG. 51A and FIG.51B depict graphs showing butyrate production using the circuits (SYN-UCD-503, SYN-UCD-504, SYN-UCD-505) shown in FIG. 48. Cells were grown in M9 minimal media containing 0.2% glucose and induced with ATC at early log phase. As seen in FIG. 51A, similar amounts of butyrate were produced for each construct under aerobic vs anaerobic conditions. The ter strain produces more butyrate overall. SYN-UCD503 comprises pLogic031 (bdc2 butyrate cassette under control of tet promoter on a plasmid) and SYN-UCD504 comprises pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid). FIG. 51B depicts butyrate production of SYN-UCD504 (pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid)) and SYN-UCD505 (a Nissle strain comprising plasmid pLOGIC046-delta pbt.buk/tesB+, an ATC-inducible ter-comprising butyrate construct with a deletion in the pbt-buk genes and their replacement with the tesB gene). The tesB construct results in greater butyrate production.
[0089] FIG. 52 depicts a graph of butyrate production using different butyrate- producing circuits comprising a nuoB gene deletion. Strains depicted are SYN- UCD503, SYN-UCD504, SYN-UCD510 (SYN-UCD510 is the same as SYN-UCD503 except that it further comprises a nuoB deletion), and SYN-UCD511 (SYN-UCD511 is the same as SYN-UCD504 except that it further comprises a nuoB deletion). The NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production. [0090] FIG. 53A depicts a schematic of a butyrate producing circuit under the control of an FNR promoter. FIG. 53B depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd 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. 53C depicts SYN- UCD501 in the presence and absence of glucose and oxygen in vitro. SYN-UCD501 comprises pSC101 PydfZ-ter butyrate plasmid; SYN-UCD500 comprises pSC101 PydfZ-bcd butyrate plasmid; SYN-UCD506 comprises pSC101 nirB-bcd butyrate plasmid. FIG. 53D 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 control SYN- UCD103.
[0091] FIG. 54A and FIG.54B depict bar graphs showing in vitro arginine (FIG. 54A) and butyrate (FIG. 54B) 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 Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)), and SYN- UCD305 (arginine producing/ammonia consuming strain comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus, and∆ThyA, with no antibiotic resistance), and SYN-UCD601 (butyrate producing and arginine
producing/ammonia consuming strain comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Logic156 (pSC101 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.
[0092] FIG. 55 depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H2O, 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.
[0093] FIG. 56 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 time points. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to Clostridium spp. in RCM media.
[0094] FIG. 57A depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromosomally integrated butyrate copies as compared to plasmid copies. Integrated butyrate strains, SYN1001 and SYN1002 (both integrated at the agaI/rsml locus) gave comparable butyrate production to the plasmid strain
SYN501.
[0095] FIG. 57B and FIG. 57C depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYN1001, on alkaline phosphatase activity in HT-29 cells represented in bar (FIG. 57B) and nonlinear fit (FIG. 57C) graphical formats.
[0096] FIG. 58A and FIG. 58B depict schematics of the gene organization of an exemplary engineered bacterium of the invention and its induction under low-oxygen conditions for the production of propionate. FIG.58A depicts relatively low propionate production under aerobic conditions in which oxygen (O2) 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 (pct, lcdA, lcdB, lcdC, etfA, acrB, acrC; black boxes) are expressed. FIG. 58B depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
[0097] FIG. 58C and FIG. 58D, depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate. FIG. 58C depicts relatively low propionate production under aerobic conditions in which oxygen (O2) 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, lpd; black boxes) are expressed. FIG. 58D depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
[0098] FIG. 58E and FIG. 58F depict diagrams showing the gene organization of an exemplary engineered bacterium of the invention and its induction under low- oxygen conditions for the production of propionate. FIG. 58E depicts relatively low propionate production under aerobic conditions in which oxygen (O2) 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, lpd, tesB; black boxes) are expressed. FIG. 58E depicts increased propionate 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 propionate biosynthesis enzymes, which leads to the production of propionate.
[0099] FIG. 59A, FIG. 59B, and FIG. 59C depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure. FIG. 59A depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from E. coli for propionate production. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. FIG. 59B and FIG. 59C 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. 59B depicts relatively low propionate production under aerobic conditions in which oxygen (O2) prevents (indicated by“X”) FNR (boxed“FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfH) is expressed. FIG. 59C depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed“FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H2O2 as depicted and described for the butyrate cassette(s) in the preceding FIG. 49C-49F, FIG. 50C-50F, FIG. 50I-50L.
[0100] FIG. 59D depicts a bar graph of proprionate concentrations produced in vitro by the wild type E coli BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in FIG. 59B and FIG. 59C.
[0101] FIG. 60A and 60B depict diagrams of exemplary constructs which may be used to produce a positive feedback auxotroph and select for high arginine production. FIG. 60A depicts a map of the astC promoter driving expression of thyA. FIG. 60B depicts a schematic diagram of the thyA gene under the control of an astC promoter. The regulatory region comprises binding sites for CRP, ArgR, and RNA polymerase (RNAP), and may also comprise additional regulatory elements.
[0102] FIG. 61 depicts another exemplary embodiment of an engineered bacterial strain to target urea cycle disorder (UCD), via the conversion of ammonia to desired products, such as citrulline or arginine. The strain is deleted for the argR gene and expressing the feedback-resistant argAfbr gene. In some embodiments, this strain further comprises one or more auxotrophic modifications on the chromosome. The synthetic biotic engineered to target urea cycle disorder (UCD) also has the kill-switch embodiment described in FIG. 65. In this example, the Int recombinase and the Kid- Kis toxin-antitoxin system are used in a recombinant bacterial cell for treating UCD. The recombinant bacterial cell is engineered to consume excess ammonia to produce beneficial byproducts to improve patient outcomes. The recombinant bacterial cell also comprises a highly controllable kill switch to ensure safety. In response to a low oxygen environment (e.g., such as that found in the gut), the FNR promoter induces expression of the Int recombinase and also induces expression of the Kis anti-toxin. The Int recombinase causes the Kid toxin gene to flip into an activated conformation, but the presence of the accumulated Kis anti-toxin suppresses the activity of the expressed Kid toxin. In the presence of oxygen (e.g., outside the gut), expression of the anti-toxin is turned off. Since the toxin is constitutively expressed, it continues to accumulate and kills the bacterial cell.
[01] FIG. 62 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.
[02] FIG. 63 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell.
[03] FIG. 64 depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death.
[0103] FIG. 65 depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the
recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell.
[0104] Hyperammonemia can also contribute to other pathologies. FIG. 66A depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (ParaBAD), which induces expression of the Tet repressor (TetR) and an anti-toxin. 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). However, when 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. FIG. 66A 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. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell. FIG. 66B depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit. FIG. 66C depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. 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). However, when 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 either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit.
[0105] FIG. 67A depicts the use of 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.67B-67E depict schematics of non- limiting examples of the gene organization of plasmids, which function as a component of a biosafety system (FIG. 67B and FIG. 67C), which also contains a chromosomal component (shown in FIG. 67D and FIG. 67E). The biosafety plasmid system vector comprises Kid Toxin and R6K minimal ori, dapA (FIG. 67B) and thyA (FIG. 67C) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, in which a first protein of interest (POI1) and/or a second protein of interest, e.g., a transporter (POI2), and/or a third protein of interest (POI3) are expressed from an inducible or constitutive promoter. FIG.67D and FIG. 67E depict schematics of the gene organization of the chromosomal component of a biosafety system. FIG. 67D 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. 67E 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. If the plasmid containing the functional DapA is used (as shown in FIG. 67B), then the chromosomal constructs shown in FIG. 67D and FIG. 67E are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in FIG. 67C), then the chromosomal constructs shown in FIG. 67D and FIG. 67E are knocked into the ThyA locus. In this system, 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.
[0106] FIG. 68 depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies.
[0107] FIG. 69A 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.
[0108] FIG. 69B 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. In this system, 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.
[0109] FIG. 69C 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. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide.
[0110] FIG. 69D 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. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype.
[0111] FIG. 70 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), e.g. 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).
[0112] FIG. 71A and FIG. 71B depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., a gut barrier enhancer molecule, e.g., IL-22 or GLP-2, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpI. Optionally, 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. 71A) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter, FIG. 71B), 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. In certain embodiments, the one or more cassettes are under the control of constitutive promoters.
[0113] FIG. 72A, FIG. 72B, and FIG. 72C depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, e.g., anti-cancer/immune modulatory effectors described herein, e.g., a gut barrier enhancer molecule, e.g., IL-22 or GLP-2, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, is assembled behind a fliC-5’UTR, and is driven by the native fliC and/or fliD promoter (FIG. 72A and FIG. 72B) or a tet-inducible promoter (FIG. 72C). In alternate embodiments, 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. In certain embodiments, 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). Optionally, an N terminal part of FliC is included in the construct, as shown in FIG. 72B and FIG. 72C.
[0114] FIG. 73 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. In a
nonlimiting example a bacterial strain which has one or more diffusible outer membrane phenotype (“leaky membrane”) mutation, e.g., as described herein.
[0115] FIG. 74 depicts an exemplary L-homoserine and L-methionine biosynthesis pathway. Circles indicate genes repressed by MetJ, and deletion of metJ leads to constitutive expression of these genes and activation of the pathway.
[0116] FIG. 75 depicts an exemplary histidine biosynthesis pathway.
[0117] FIG. 76 depicts an exemplary lysine biosynthesis pathway.
[0118] FIG. 77 depicts an exemplary asparagine biosynthesis pathway.
[0119] FIG. 78 depicts an exemplary glutamine biosynthesis pathway.
[0120] FIG. 79 depicts an exemplary tryptophan biosynthesis pathway. [0121] FIG. 80 depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters and sequences described herein. Different FNR-responsive promoters were used to create a library of anaerobic/low oxygen conditions inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+O2) or anaerobic conditions (-O2). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays.
[0122] FIG. 81A depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (PfnrS). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. FIG. 81B depicts FNR promoter activity as a function of β-galactosidase activity in an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high- level gene expression within 1 hr. under anaerobic and/or low oxygen conditions. FIG. 81C depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen.
[0123] FIG. 82 depicts ATC (FIG. 82A) or nitric oxide-inducible (FIG. 82B) 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 PnsrR-GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units. FIG. 82C depicts a schematic of the constructs.
[0124] FIG. 83 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.
[0125] FIG. 84A 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).
[0126] FIG. 84B depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct (SYN133) or the pLogic046- nsrR-norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001).
[0127] 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. To improve acetate production, while still maintaining high levels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of competing routes (shown in in rounded boxes) 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. Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0128] FIG. 86A and FIG.86B depict line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) (FIG. 86A) and in 0.5% glucuronic acid MOPS (pH6.3) (FIG.86B). 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).
[0129] FIG. 86C and FIG. 86D depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) (FIG.86C) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) (FIG. 86D). Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter- pbt-buk butyrate cassettes. SYN2006 comprises a FNRS ter-tesB cassette integrated at the HA1/2 locus and a deletion in the endogenous adhE gene. SYN2007 comprises a FNRS ter-tesB cassette integrated at the HA1/2 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.
[0130] FIG. 86E 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 HA1/2 locus of the chromosome (SYN1004) was compared with a strain comprising the same integrated cassette and additionally a deletion in the endogenous frd gene (SYN2005).
[0131] FIG. 86F 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 HA1/2 locus and deletions in the endogenous adhE and pta genes. SYN2003 comprises FNRS- ter-pbt-buk butyrate cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene.
[0132] FIG. 86G and FIG. 86H depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFNγ secretion in primary human PBMC cells from donor 1 (D1) (FIG.86G ) and donor 2 (D2) (FIG. 86H).
[0133] FIG. 87A and FIG.87B depict schematics of indole metabolite mode of action (FIG.36A) and indole biosynthesis (FIG. 87B). FIG. 87A depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (I3A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function. I3A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production. 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. 87B depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598–605 (2016). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in FIG. 87A and 87B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 87A and 87B, including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde.
[0134] FIG. 88A and FIG. 88B depict diagrams of bacterial tryptophan metabolism pathways. FIG. 88A 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 (EC1.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2- monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines (–––––) indicate a spontaneous reaction. FIG. 88B 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: Indole-3-pyruvic acid; IAM: Indole-3-acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3- acetonitrile; N-formyl Kyn: N-formylkynurenine;; Kyn:Kynurenine; KynA: Kynurenic acid; I3C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3’-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole. Enzymes are numbered as follows: 1. EC 1.13.11.11 (Tdo2, Bna2), EC 1.13.11.11 (Ido1); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (lad1), EC 1.2.3.7 (Aao1); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclb1, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125
(CYP79B2, CYP79B3); 9. EC 1.4.3.2 (StaO), EC 2.6.1.27 (Aro9, aspC), EC 2.6.1.99 (Taa1), EC 1.4.1.19 (TrpDH); 10. EC 1.13.12.3 (laaM); 11. EC 4.1.1.74 (IpdC); 12. EC 1.14.13.168 (Yuc2); 13. EC 3.5.1.4 (IaaH); 14. EC 3.5.5.1. (Nit1); 15. EC 4.2.1.84 (Nit1); 16. EC 4.99.1.6 (CYP71A13); 17. EC 3.2.1.147 (Pen2). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in FIG. 88A and 88B. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in FIG. 88A and 88B. In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0135] FIG. 89 depicts a schematic of the E. coli tryptophan synthesis pathway. In Escherichia coli, tryptophan is biosynthesized from chorismate, the principal common precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and enterobactin (enterochelin), as shown in the superpathway of chorismate metabolism. Five genes encode five enzymes that catalyze tryptophan biosynthesis from chorismate. The five genes trpE trpD trpC trpB trpA form a single transcription unit, the trp operon. A weak internal promoter also exists within the trpD structural gene that provides low, constitutive levels of mRNA.
[0136] FIG. 90A, FIG. 90B, FIG. 90C, and FIG. 90D 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. 90A 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. 90B, and/or FIG.90C, and/or FIG. 90D. FIG. 90B 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. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 90A and/or described in the description of FIG. 90A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90C, and/or FIG. 90D. Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. FIG.90C depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene. Escherichia coli serA-encoded 3- phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major
phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD1 to NADH. E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 90A and/or described in the description of FIG. 90A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90B, and/or FIG.90D. Optionally, 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. 90D 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. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in FIG. 90A and/or described in the description of FIG. 90A. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in FIG. 90B, and/or FIG. 90C. Optionally, 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. Optionally, 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.
[0137] FIG. 91A, FIG. 91B, FIG. 91D, FIG. 91D, FIG.91E, FIG. 91F, FIG. 91G, and FIG. 91H depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included. FIG. 91A depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. In certain embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in FIG. 90A and/or and/or FIG. 90B, and/or FIG.90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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. 91B 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. 90A and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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 taa1 (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 ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 91C 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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. coli), which converts tryptophan to indole-3- acetaldehyde and FICZ, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 91D 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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. 91E 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. 90A and/or and/or FIG. 90B, and/or FIG. 90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (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) which together convert tryptophan to kynurenine, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG.91F 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. 90A and/or and/or FIG. 90B, and/or FIG.90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (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 (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens, which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter. FIG.91G 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. 90A and/or and/or FIG. 90B, and/or FIG.90C, and/or FIG. 90D for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from E. coli), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter. FIG. 91H 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. 91A, FIG. 91B, FIG. 91D, FIG. 91D, FIG. 91E, FIG. 91F, FIG.91G and FIG. 91H 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. [0138] FIG. 92A, FIG. 92B, FIG. 92C, FIG. 92D, and FIG. 92E depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In certain
embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. In FIG. 92A, the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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 taa1 (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 ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) and iad1 ( Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3- acetaldehyde oxidase, e.g., from Arabidopsis thaliana) which together produce indole-3- acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 92B the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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. coli) and or iad1 (Indole-3-acetaldehyde
dehydrogenase, e.g., from Ustilago maydis) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana), e.g., under the control of an inducible promoter e.g., an FNR promoter. In FIG. 92C the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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 taa1 (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. In FIG. 92D the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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. In FIG. 92E the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, 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 cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana) and nit1 (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. In FIG. 92F the optional circuits for tryptophan production are as depicted and described in FIG. 90A. The strain optionally comprises additional circuits as depicted and/or described in FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Alternatively, optionally, tryptophan can be imported through a transporter.
Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from
Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3- acetate.
[0139] The engineered bacterium shown in any of FIG. 92A, FIG. 92B, FIG. 92C, FIG. 92D, and FIG. 92E, FIG. 92F 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.
[0140] FIG. 93A, FIG. 93B, and FIG. 93C depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. 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. 93A 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. 90A and/or FIG. 90B and/or FIG.90C and/or FIG. 90D. Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine. FIG. 93B 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. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG. 90D. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3-acetate. FIG. 93C 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. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG.90D. Additionally, the strain comprises a circuit as described in FIG. 48, comprising 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 AcuI: (indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl- CoA). The circuits further comprise fldH1 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).
[0141] FIG. 94A and FIG.94B depict schematics showing exemplary engineering strategies which can be employed for tryptophan production. FIG.
94A 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). DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon.
Alternatively, chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the serine biosynthesis pathway, 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). There are three isozymes of DAHP synthase, each specifically feedback regulated by tyrosine (AroF), phenylalanine (AroG) or 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 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. There are two shikimate kinase enzymes, I (AroK) and II (AroL). AroA: 3- Phosphoshikimate-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. This reaction introduces a second double bond into the aromatic ring system. TrpEDCAB (E coli trp operon): TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L- glutamate. Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5'-phosphoribosyl)-anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional phosphoribosylanthranilate isomerase / indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway. The phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into
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 α) functions as the α subunit of the tetrameric (α2-β2) tryptophan synthase complex. The TrpB polypeptide functions as the β subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the β 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 biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional chorismate mutase / prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine
aminotransferase (TyrB), also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. TyrB catalyzes the transamination of 2-ketoisocaproate, p- hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and
phenylalanine, respectively. TyrB overlaps with the catalytic activities of branched- chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine. SerA: D- 3-phosphoglycerate dehydrogenase catalyzes the first committed step in the
biosynthesis of L-serine. SerC: The serC-encoded enzyme,
phosphoserine/phosphohydroxythreonine aminotransferase, functions in the
biosynthesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5'- phosphate is a cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated. FIG. 94B 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. In one embodiment of the disclosure, bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEfbr, and/or TrpD, and/or TrpC, and/or TrpA, and/or TrpB. In one embodiment, endogenous TnaA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise one or more additional copies of SerA. In one embodiment, bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter. In one embodiment, endogenous PheA is knocked out through mutation(s) and/or deletion(s). In one embodiment, two or more of the strategies depicted in the schematic of FIG. 94B are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed.
[0142] FIG.95A and FIG. 95B and FIG. 95C depict bar graphs showing tryptophan production by various engineered bacterial strains. FIG.95A depicts a bar graph showing tryptophan production by various tryptophan producing strains. The data show expressing a feedback resistant form of AroG (AroGfbr) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE fbr) has a positive effect on tryptophan production. FIG. 95B shows tryptophan production from a strain comprising a tet-trpEfbrDCBA, tet-aroGfbr construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes E. coli two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically). FIG. 95C depicts a bar graph showing improved tryptophan production by engineered strain comprising∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbr through the addition of serine.
[0143] FIG. 96 depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476.
SYN2126∆trpR∆tnaA.∆trpR∆tnaA, tet-aroGfbr. SYN2339 comprises∆trpR∆tnaA, tet-aroGfbr, tet-trpEfbrDCBA. SYN2473 comprises∆trpR∆tnaA, tet-aroGfbr-serA, tet- trpEfbrDCBA. SYN2476 comprises∆trpR∆tnaA, 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.
[0144] FIG. 97 depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit. IPA produced by the gut microbiota has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate
Gastrointestinal Bardrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296–310, August 21, 2014). In some embodiments, 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). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0145] FIG. 98 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 Nostoc punctiforme NIES-2108;
FldH1/FldH2: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC: indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI:
acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. lpdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; lad1: Indole-3-acetaldehyde
dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
[0146] 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 (FldA ) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (FldD ) and acrylyl-CoA reductase (AcuI) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC ) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate
decarboxylase (lpdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3- acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3- acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0147] FIG. 99 depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises∆trpR and∆tnaA (∆trpR∆tnaA). SYN2339 comprises circuitry for the production of tryptophan (∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iad1 incorporated at the end of the second construct (∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH- ipdC-iad1 (p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA.
[0148] FIG. 100 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 ∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A). SYN2340 comprises∆trpR∆tnaA,
Figure imgf000056_0001
(p15A). SYN2794 comprises∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr- tdcCs (p15A). Results indicate that TdcCs from Clostridium sporogenes is more efficient the TdcCr from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine.
[0149] FIG. 101A and FIG. 101B depict line graphs of ELISA results. FIG. 101A 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. 101B depicts a line graph, showing an phopho- STAT3 (Tyr705) ELISA showing an antibody completion assay. Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody. The data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821.
[0150] FIG. 101C depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation.
[0151] FIG. 101D depicts a line graph showing that anti IL-22 neutralizing antibody inhibits SYN3001-induced STAT3 activation (n=3).
[0152] FIG. 102 depicts the gene organization of exemplary construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction.
[0153] FIG. 103A depicts a“Oxygen bypass switch” useful for aerobic pre- induction of a strain comprising one or proteins of interest (POI), e.g., one or more anti- cancer molecules or immune modulatory effectors (POI1) and a second set of one or more proteins of interest (POI2), e.g., one or more transporter(s)/importer(s) and/or exporter(s), under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active effector molecules prior to administration. This can be done by pre-inducing the expression of these effectors as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more effectors or proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more effectors or proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites.
[0154] 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 O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, 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. Thus, 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. In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.
[0155] FIG. 103B depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of
FNRS24Y. By using a ribosome binding site optimization strategy, 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. Bioinformatics tools for optimization of RBS are known in the art.
[0156] FIG. 103C depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bioinformatics tools for optimization of RBS are known in the art. In one strategy, 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.
[0157] FIG. 104 depicts the gene organization of an exemplary construct, e.g., comprised in SYN-PKU401, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene.
[0158] FIG. 105 depicts the gene organization of an exemplary construct comprising LacI in reverse orientation, and a IPTG inducible promoter driving the expression of one or more POIs. In some embodiments, 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. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting.
[0159] In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some
embodiments, the construct is used in combination with construct expressing a second POI, e.g., a transporter, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be
constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is employed in a biosafety system, such as the system shown in FIG. 67A, FIG. 67B, FIG.67C, and FIG. 67D, FIG. 67E. In some embodiments, the construct is integrated into the genome at one or more locations described herein.
[0160] FIG. 106A, FIG. 106B, and FIG. 106C depict schematics of non- limiting examples of constructs for the expression of proteins of interest POI(s). FIG. 106A depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone. In some embodiments, 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. In some embodiments, the construct allows pre-induction and pre-loading of a POI1 and/or a POI2 prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct.
[0161] In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a strain comprising deltaThyA or deltaDapA, 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).
[0162] FIG. 106B 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. For the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi- copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose inducible construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, temperature sensitive, or IPTG inducible constructs. In some embodiments, the construct allows pre- induction and pre-loading of POI and/or POI2 and/or POI3 prior to in vivo
administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct.
[0163] FIG. 106C depicts a schematic of a non-limiting example of the organization of a construct for the expression of protein(s) of interest POI(s) 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 POI. 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 POI1 and/or POI2 and/or POI3 prior to in vivo
administration. In a non-limiting example, the construct is useful for pre-induction and is combined with low-oxygen inducible constructs. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct.
[0164] FIG. 107A 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.
[0165] FIG. 107B depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, 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. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch).
[0166] FIG. 108A depicts a schematic diagram of a wild-type clbA construct.
[0167] FIG. 108B depicts a schematic diagram of a clbA knockout construct.
[0168] FIG. 109 depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animal disease model; 9. Assimilate into the microbiome; 10.
Develop understanding of in vivo PK and dosing regimen. [0169] FIGs. 110A, 110B, 110C, 110D, and 110E depict a schematic of non- limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. FIG. 110A depicts the parameters for starter culture 1 (SC1): loop full– glycerol stock, duration overnight, temperature 37° C, shaking at 250 rpm. FIG. 110B depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C, shaking at 250 rpm. FIG. 110C 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. 110D depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1X 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. FIG. 110E depicts the parameters for vial fill/storage: 1-2 mL aliquots, -80° C.
[0170] FIG. 111 depicts graphs of breath versus blood in a subset of 10 subjects. Description of Embodiments
[0171] The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating or treating disorders associated with hyperammonemia, e.g., urea cycle disorders, hepatic encephalopathy and other disorders associated with excess ammonia or elevated ammonia levels. The genetically engineered bacteria are capable of reducing excess ammonia, particularly under certain environmental conditions, such as those in the mammalian gut. In some embodiments, the genetically engineered bacteria reduce excess ammonia by incorporating excess nitrogen in the body into non-toxic molecules, e.g., arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, or tryptophan. In some embodiments, the genetically engineered bacteria reduce excess ammonia and also reduce one or more other toxic substances, e.g., GABA and/or manganese. In some embodiments, the genetically engineered bacteria reduce excess ammonia and also reduce GABA levels, e.g., by importing GABA and/or by metabolizing GABA. In some embodiments, the genetically engineered bacteria reduce excess ammonia and also reduce manganese levels, e.g., by importing manganese. The genetically engineered bacteria may additionally produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia (e.g., UCDs, HE, etc). Thus, in any of the described embodiments, the genetically engineered bacteria may also produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia. In some embodiments, the genetically engineered bacteria produce a short chain fatty acid, e.g., butyrate, propionate, and/or acetate. In some embodiments, the engineered bacteria reduce excess ammonia and produce one or molecules that improve gut barrier function or otherwise alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate. In some embodiments, the engineered bacteria reduce excess ammonia, reduce one or more other toxic substances, e.g., GABA and/or manganese, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate. In some embodiments, the genetically engineered bacteria reduce excess ammonia, reduce GABA levels, e.g., by importing GABA and/or by metabolizing GABA, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate. In some embodiments, the genetically engineered bacteria reduce excess ammonia, reduce manganese levels, e.g., by importing manganese, and produce one or molecules that improve gut barrier function or alleviate a symptom of a disorder associated with elevated ammonia, e.g., produce a short chain fatty acid, such as butyrate, propionate, and/or acetate.
[0172] In any of the described embodiments, the engineered bacteria may further comprise one or more of more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. [0173] In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by a constitutive promoter. In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by a tissue-specific promoter. In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by an inducible promoter. In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be regulated by an inducible promoter that is responsive to environmental conditions, factors, or cues, e.g., environmental conditions, factors, or cues found in the mammalian gut. Exemplary inducible promoters include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by HE-specific 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.
[0174] In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be present on one or more low copy or high copy plasmids. In some embodiments, any one or more of the payload or therapeutic circuits (e.g., ammonia consuming, GABA reducing, manganese reducing, short chain fatty acid producing circuits) and/or any one or more of the additional circuits (e.g., auxotrophies, kill switch circuits, antibiotic resistance circuits, transporters, and secretion circuits) may be integrated into the bacterial chromosome. In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.
[0175]“Hyperammonemia,”“hyperammonemic,” or“excess ammonia” is used to refer to increased concentrations of ammonia in the body. Hyperammonemia is caused by decreased detoxification and/or increased production of ammonia. Decreased detoxification may result from urea cycle disorders (UCDs), such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; or from bypass of the liver, e.g., open ductus hepaticus; and/or deficiencies in glutamine synthetase (Hoffman et al., 2013; Häberle et al., 2013). Decreased detoxification may also result from liver disorders such as hepatic encephalopathy, acute liver failure, or chronic liver failure; and neurodegenerative disorders such as Huntington’s disease (Chen et al., 2015; Chiang et al., 2007). Increased production of ammonia may result from infections, drugs, neurogenic bladder, and intestinal bacterial overgrowth (Häberle et al., 2013). Other disorders and conditions associated with hyperammonemia include, but are not limited to, liver disorders such as hepatic encephalopathy, acute liver failure, or chronic liver failure; organic acid disorders; isovaleric aciduria; 3- methylcrotonylglycinuria; methylmalonic acidemia; propionic aciduria; propinic acidemia; fatty acid oxidation defects; carnitine cycle defects; carnitine deficiency; β- oxidation deficiency; lysinuric protein intolerance; pyrroline-5-carboxylate synthetase deficiency; pyruvate carboxylase deficiency; ornithine aminotransferase deficiency; carbonic anhydrase deficiency; hyperinsulinism-hyperammonemia syndrome;
mitochondrial disorders; valproate therapy; asparaginase therapy; total parenteral nutrition; cystoscopy with glycine-containing solutions; post-lung/bone marrow transplantation; portosystemic shunting; urinary tract infections; ureter dilation; multiple myeloma; and chemotherapy; gastrointestinal bleeding (Hoffman et al., 2013; Häberle et al., 2013; Pham et al., 2013; Lazier et al., 2014). Another disorder associated with hyperammonemia is Reye’s syndrome. Reye's syndrome is a rare condition primarily affecting the liver and brain. The disorder most commonly develops in children ages 5 to 14 after an otherwise unremarkable viral illness. An elevated blood ammonia level characteristically occurs in patients with Reye's syndrome, leading to brain swelling and ammonia toxicity.
[0176] In healthy subjects, plasma ammonia concentrations are typically less than about 50 µmol/L (Leonard, 2006). In some embodiments, a diagnostic signal of hyperammonemia is a plasma ammonia concentration of at least about 50 µmol/L, at least about 80 µmol/L, at least about 150 µmol/L, at least about 180 µmol/L, or at least about 200 µmol/L (Leonard, 2006; Hoffman et al., 2013; Häberle et al., 2013).
[0177]“Ammonia” is used to refer to gaseous ammonia (NH3), ionic ammonia (NH4+), or a mixture thereof. In bodily fluids, gaseous ammonia and ionic ammonium exist in equilibrium:
[0178] NH3 + H+↔ NH4+
[0179] Some clinical laboratory tests analyze total ammonia (NH3 + NH4+) (Walker, 2012). In any embodiment of the invention, unless otherwise indicated, “ammonia” may refer to gaseous ammonia, ionic ammonia, and/or total ammonia.
[0180]“Detoxification” of ammonia is used to refer to the process or processes, natural or synthetic, by which toxic ammonia is removed and/or converted into one or more non-toxic molecules, including but not limited to: arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, tryptophan, or urea. The urea cycle, for example, enzymatically converts ammonia into urea for removal from the body in the urine. Because ammonia is a source of nitrogen for many amino acids, which are synthesized via numerous biochemical pathways, enhancement of one or more of those amino acid biosynthesis pathways may be used to incorporate excess nitrogen into non- toxic molecules. For example, arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms, thereby incorporating excess nitrogen into non-toxic molecules. In humans, arginine is not reabsorbed from the large intestine, and as a result, excess arginine in the large intestine is not considered to be harmful. Likewise, citrulline is not reabsorbed from the large intestine, and as a result, excess citrulline in the large intestine is not considered to be harmful. Arginine biosynthesis may also be modified to produce citrulline as an end product; citrulline comprises three nitrogen atoms and thus the modified pathway is also capable of incorporating excess nitrogen into non-toxic molecules.
[0181]“Arginine regulon,”“arginine biosynthesis regulon,” and“arg regulon” are used interchangeably to refer to the collection of operons in a given bacterial species that comprise the genes encoding the enzymes responsible for converting glutamate to arginine and/or intermediate metabolites, e.g., citrulline, in the arginine biosynthesis pathway. The arginine regulon also comprises operators, promoters, ARG boxes, and/or regulatory regions associated with those operons. The arginine regulon includes, but is not limited to, the operons encoding the arginine biosynthesis enzymes N- acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine
transcarbamylase, argininosuccinate synthase, argininosuccinate lyase,
carbamoylphosphate synthase, operators thereof, promoters thereof, ARG boxes thereof, and/or regulatory regions thereof. In some embodiments, the arginine regulon comprises an operon encoding ornithine acetyltransferase and associated operators, promoters, ARG boxes, and/or regulatory regions, either in addition to or in lieu of N- acetylglutamate synthetase and/or N-acetylornithinase. In some embodiments, one or more operons or genes of the arginine regulon may be present on a plasmid in the bacterium. In some embodiments, a bacterium may comprise multiple copies of any gene or operon in the arginine regulon, wherein one or more copies may be mutated or otherwise altered as described herein.
[0182] One gene may encode one enzyme, e.g., N-acetylglutamate synthetase (argA). Two or more genes may encode distinct subunits of one enzyme, e.g., subunit A and subunit B of carbamoylphosphate synthase (carA and carB). In some bacteria, two or more genes may each independently encode the same enzyme, e.g., ornithine transcarbamylase (argF and argI). In some bacteria, the arginine regulon includes, but is not limited to, argA, encoding N-acetylglutamate synthetase; argB, encoding N- acetylglutamate kinase; argC, encoding N-acetylglutamylphosphate reductase; argD, encoding acetylornithine aminotransferase; argE, encoding N-acetylornithinase; argG, encoding argininosuccinate synthase; argH, encoding argininosuccinate lyase; one or both of argF and argI, each of which independently encodes ornithine transcarbamylase; carA, encoding the small subunit of carbamoylphosphate synthase; carB, encoding the large subunit of carbamoylphosphate synthase; operons thereof; operators thereof; promoters thereof; ARG boxes thereof; and/or regulatory regions thereof. In some embodiments, the arginine regulon comprises argJ, encoding ornithine acetyltransferase (either in addition to or in lieu of N-acetylglutamate synthetase and/or N- acetylornithinase), operons thereof, operators thereof, promoters thereof, ARG boxes thereof, and/or regulatory regions thereof.
[0183]“Arginine operon,”“arginine biosynthesis operon,” and“arg operon” are used interchangeably to refer to a cluster of one or more of the genes encoding arginine biosynthesis enzymes under the control of a shared regulatory region comprising at least one promoter and at least one ARG box. In some embodiments, the one or more genes are co-transcribed and/or co-translated. Any combination of the genes encoding the enzymes responsible for arginine biosynthesis may be organized, naturally or synthetically, into an operon. For example, in B. subtilis, the genes encoding N- acetylglutamylphosphate reductase, N-acetylglutamate kinase, N-acetylornithinase, N- acetylglutamate kinase, acetylornithine aminotransferase, carbamoylphosphate synthase, and ornithine transcarbamylase are organized in a single operon, argCAEBD-carAB- argF, under the control of a shared regulatory region comprising a promoter and ARG boxes. In E. coli K12 and Nissle, the genes encoding N-acetylornithinase, N- acetylglutamylphosphate reductase, N-acetylglutamate kinase, and argininosuccinate lyase are organized in two bipolar operons, argECBH. The operons encoding the enzymes responsible for arginine biosynthesis may be distributed at different loci across the chromosome. In unmodified bacteria, each operon may be repressed by arginine via ArgR. In some embodiments, arginine and/or intermediate byproduct production may be altered in the genetically engineered bacteria of the invention by modifying the expression of the enzymes encoded by the arginine biosynthesis operons as provided herein. Each arginine operon may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any arginine operon, or a gene or regulatory region within an arginine operon, may be present in the bacterium, wherein one or more copies of the operon or gene or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same product (e.g., operon or gene or regulatory region) to enhance copy number or to comprise multiple different components of an operon performing multiple different functions. [0184]“ARG box consensus sequence” refers to an ARG box nucleic acid sequence, the nucleic acids of which are known to occur with high frequency in one or more of the regulatory regions of argR, argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and/or carB. As described above, each arg operon comprises a regulatory region comprising at least one 18-nucleotide imperfect palindromic sequence, called an ARG box, that overlaps with the promoter and to which the repressor protein binds (Tian et al., 1992). The nucleotide sequences of the ARG boxes may vary for each operon, and the consensus ARG box sequence is A/T nTGAAT A/T A/T T/A T/A ATTCAn T/A (Maas, 1994). The arginine repressor binds to one or more ARG boxes to actively inhibit the transcription of the arginine biosynthesis enzyme(s) that are operably linked to that one or more ARG boxes.
[0185]“Mutant arginine regulon” or“mutated arginine regulon” is used to refer to an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of each of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct, e.g., citrulline, in the arginine biosynthesis pathway, such that the mutant arginine regulon produces more arginine and/or intermediate byproduct than an unmodified regulon from the same bacterial subtype under the same conditions. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr, and a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
argininosuccinate lyase, and carbamoylphosphate synthase, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr, a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr and a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr and a mutant or deleted arginine repressor. In some embodiments, the mutant arginine regulon comprises an operon encoding wild-type N-acetylglutamate synthetase and one or more nucleic acid mutations in at least one ARG box for said operon. In some embodiments, the mutant arginine regulon comprises an operon encoding wild-type N- acetylglutamate synthetase and mutant or deleted arginine repressor. In some embodiments, the mutant arginine regulon comprises an operon encoding ornithine acetyltransferase (either in addition to or in lieu of N-acetylglutamate synthetase and/or N-acetylornithinase) and one or more nucleic acid mutations in at least one ARG box for said operon.
[0186] The ARG boxes overlap with the promoter in the regulatory region of each arginine biosynthesis operon. In the mutant arginine regulon, the regulatory region of one or more arginine biosynthesis operons is sufficiently mutated to disrupt the palindromic ARG box sequence and reduce ArgR binding, but still comprises sufficiently high homology to the promoter of the non-mutant regulatory region to be recognized as the native operon-specific promoter. The operon comprises at least one nucleic acid mutation in at least one ARG box such that ArgR binding to the ARG box and to the regulatory region of the operon is reduced or eliminated. In some
embodiments, bases that are protected from DNA methylation and bases that are protected from hydroxyl radical attack during ArgR binding are the primary targets for mutations to disrupt ArgR binding (see, e.g., Table 3). The promoter of the mutated regulatory region retains sufficiently high homology to the promoter of the non-mutant regulatory region such that RNA polymerase binds to it with sufficient affinity to promote transcription of the operably linked arginine biosynthesis enzyme(s). In some embodiments, the G/C:A/T ratio of the promoter of the mutant differs by no more than 10% from the G/C:A/T ratio of the wild-type promoter. [0187] In some embodiments, more than one ARG box may be present in a single operon. In one aspect of these embodiments, at least one of the ARG boxes in an operon is altered to produce the requisite reduced ArgR binding to the regulatory region of the operon. In an alternate aspect of these embodiments, each of the ARG boxes in an operon is altered to produce the requisite reduced ArgR binding to the regulatory region of the operon.
[0188]“Reduced” ArgR binding is used to refer to a reduction in repressor binding to an ARG box in an operon or a reduction in the total repressor binding to the regulatory region of said operon, as compared to repressor binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions. In some embodiments, ArgR binding to a mutant ARG box and regulatory region of an operon is at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, at least about 90% lower, or at least about 95% lower than ArgR binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions. In some embodiments, reduced ArgR binding to a mutant ARG box and regulatory region results in at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold increased mRNA expression of the one or more genes in the operon.
[0189]“ArgR” or“arginine repressor” is used to refer to a protein that is capable of suppressing arginine biosynthesis by regulating the transcription of arginine biosynthesis genes in the arginine regulon. When expression of the gene that encodes for the arginine repressor protein (“argR”) is increased in a wild-type bacterium, arginine biosynthesis is decreased. When expression of argR is decreased in a wild-type bacterium, or if argR is deleted or mutated to inactivate arginine repressor function, arginine biosynthesis is increased.
[0190] Bacteria that“lack any functional ArgR” and“ArgR deletion bacteria” are used to refer to bacteria in which each arginine repressor has significantly reduced or eliminated activity as compared to unmodified arginine repressor from bacteria of the same subtype under the same conditions. Reduced or eliminated arginine repressor activity can result in, for example, increased transcription of the arginine biosynthesis genes and/or increased concentrations of arginine and/or intermediate byproducts, e.g., citrulline. Bacteria in which arginine repressor activity is reduced or eliminated can be generated by modifying the bacterial argR gene or by modifying the transcription of the argR gene. For example, the chromosomal argR gene can be deleted, can be mutated, or the argR gene can be replaced with an argR gene that does not exhibit wild-type repressor activity.
[0191]“Operably linked” refers a nucleic acid sequence, e.g., a gene encoding feedback resistant ArgA, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis.
A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
[0192] 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. In some embodiments, the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage. In some embodiments, the metabolites may be gut specific. In some embodiments, the metabolite may be associated with hepatic encephalopathy, e.g., bilirubin. Non-limiting examples of molecules or metabolites include, 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, and manganese in their blood and intestines. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein. In some embodiments, the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter. In some embodiments, the genetically engineered bacteria comprise a promoter 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.
[0193]“Exogenous environmental condition(s)” refer to setting(s) or circumstance(s) under which the promoter described herein is induced. The phrase “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. Thus,“exogenous” and“endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are
endogenous to a mammalian body, but external or exogenous to an intact
microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous
environmental conditions are specific to the small intestine of a mammal. In some embodiments, exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease state (e.g., HE). In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some
embodiments, the exogenous environmental condition is a tissue-specific or disease- specific metabolite or molecule(s). In some embodiments, the exogenous
environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the 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.
[0194] 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.
[0195] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1.
[0196] In a non-limiting example, a promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression.
However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS.
Table 1. Examples of transcription factors and responsive genes and regulatory regions
Figure imgf000075_0001
[0197]“Gut barrier function enhancer molecules” include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF-β1, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, and kynurenine. A gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene. Alternatively, a gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. These molecules may also be referred to as therapeutic molecules.
[0198] As used herein, a“gene cassette” or“operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce a gut barrier function enhancer molecule, e.g., butyrate, propionate. In addition to encoding a set of genes capable of producing said molecule, the gene cassette or operon may also comprise additional transcription and translation elements, e.g., a ribosome binding site.
[0199] A“butyrogenic gene cassette,”“butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to, Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio,
Eubacterium, and Treponema. The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3- hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, 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. Thus, a butyrogenic gene cassette may comprise thiA1, 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. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB.
[0200] Likewise, a“propionate gene cassette” or“propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway. Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to, Clostridium propionicum,
Megasphaera elsdenii, and Prevotella ruminicola. The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, which encode propionate CoA- transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering Escherichia coli with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyzes the reduction of lactate to propionate. Dehydration of (R)-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). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the acuI 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). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI 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 pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L- threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase,
dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.
[0201] In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from E. coli (sbm, ygfD, ygfG, ygfH).
Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering Escherichia coli for high^level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057–1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure. 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) converts succinyl CoA to L- methylmalonylCoA, YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG (methylmalonylCoA decarboxylase) converts L-methylmalonylCoA into
PropionylCoA, and ygfH (propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli; Froese 2009). 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). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA.
[0202] 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.
[0203] An“acetate gene cassette” or“acetate operon” refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria“synthesize acetate from a number of carbon and energy sources,” including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria. Escherichia coli are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and Thermoacetogenium, are acetogenic anaerobes that are capable of converting CO or CO2 + H2 into acetate, e.g., using the Wood- Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
[0204] Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, 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. In some embodiments, 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.
[0205]“GABA” and“γ-aminobutyric acid” are used to refer to the predominant inhibitory neurotransmitter (C4H9NO2) in the mammalian central nervous system. In humans, GABA is also directly responsible for regulating muscle tone. GABA is capable of activating the GABAA receptor, which is part of a ligand-gated ion channel complex, as well as the GABAB metabotropic G protein-coupled receptor. Neurons that produce GABA are known as“GABAergic” neurons, and activation of GABA receptors is described as GABAergic tone (i.e., increased activation of GABA receptors refers to increased GABAergic tone).
[0206]“GABA transporter” and“GabP” are used to refer to a membrane transport protein that is capable of transporting GABA into bacterial cells (see, e.g., Li et al., 2001). In Escherichia coli, the gabP gene encodes a high-affinity GABA permease responsible for GABA transport (Li et al., 2001). In some embodiments, the GABA transporter is encoded by a gabP gene derived from a bacterial species, including but not limited to, Bacillus subtilis and Escherichia coli. These endogenous GABA transporter genes may be a source of genes for the genetically engineered bacteria of the invention. Any suitable gene(s) encoding a GABA transporter may be used.
[0207]“Manganese” refers to a chemical element with the symbol“Mn” and atomic number 25. In biological systems, manganese is an essential trace metal and plays an important role in enzyme-mediated catalysis, but can also have deleterious effects. Cells maintain manganese under tight homeostatic control in order to avoid toxicity. Some disorders associated with hyperammonemia may also be characterized by elevated levels of manganese; manganese may contribute to disease pathogenesis (e.g., hepatic encephalopathy) (Rivera-Mancía et al., 2012).
[0208]“Manganese transporter” and“MntH” refer to a membrane transport protein that is capable of transporting manganese into bacterial cells (see, e.g., Jensen and Jensen, 2014). In Escherichia coli, the mntH gene encodes a proton-stimulated, divalent metal cation uptake system involved in manganese transport (Porcheron et al., 2013). In some embodiments, the manganese transporter is encoded by a mntH gene derived from a bacterial species, including but not limited to, Salmonella typhimurium, Shigella flexneri, Yersinia pestis, and Escherichia coli. These endogenous manganese transporter genes may be a source of genes for the genetically engineered bacteria of the invention. Any suitable gene(s) encoding a manganese transporter may be used.
[0209] As used herein, a“non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments,“non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non- native nucleic acid sequence, e.g., gene or gene cassette, may be present on a plasmid or bacterial chromosome. In some embodiments, the genetically engineered bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR- responsive promoter operably linked to a butyrogenic gene cassette, or an arginine production cassette. In addition, multiple copies of the gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same non-native nucleic acid sequence, e.g., 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.
[0210]“Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive
Escherichia coli σS 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 σ32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli σ70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter
(BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105),
M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter
(BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis σA promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive Bacillus subtilis σB promoter (e.g., promoter ctc (BBa_K143010), promoter gsiB
(BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella
(BBa_K112706), Pspv from Salmonella (BBa_K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997;
BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180;
BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof.
[0211] As used herein, genetically engineered bacteria that“overproduce” arginine or an intermediate byproduct, e.g., citrulline, refer to bacteria that comprise a mutant arginine regulon. For example, the engineered bacteria may comprise a feedback resistant form of ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or intermediate byproduct than unmodified bacteria of the same subtype under the same conditions. The genetically engineered bacteria may alternatively or further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes. The genetically engineered bacteria may alternatively or further comprise a mutant or deleted arginine repressor. In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more arginine than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300- fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more citrulline or other intermediate byproduct than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the mRNA transcript levels of one or more of the arginine biosynthesis genes in the genetically engineered bacteria are at least about 1.5-fold, at least about 2- fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold higher than the mRNA transcript levels in unmodified bacteria of the same subtype under the same conditions. In certain embodiments, the unmodified bacteria will not have detectable levels of arginine, intermediate byproduct, and/or transcription of the gene(s) in such operons. However, protein and/or transcription levels of arginine and/or intermediate byproduct will be detectable in the corresponding genetically engineered bacterium having the mutant arginine regulon. Transcription levels may be detected by directly measuring mRNA levels of the genes. Methods of measuring arginine and/or intermediate byproduct levels, as well as the levels of transcript expressed from the arginine biosynthesis genes, are known in the art. Arginine and citrulline, for example, may be measured by mass spectrometry.
[0212]“Gut” refers to the organs, glands, tracts, and systems that are
responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal 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.
[0213] As used herein, the term“low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., <21% O2; <160 torr O2)). Thus, the term“low oxygen condition or conditions” or“low oxygen environment” refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere. In some embodiments, the term“low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term “low oxygen” is meant to refer to a level, amount, or concentration of O2 that is 0-60 mmHg O2 (0-60 torr O2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O2), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O2, 0.75 mmHg O2, 1.25 mmHg O2, 2.175 mmHg O2, 3.45 mmHg O2, 3.75 mmHg O2, 4.5 mmHg O2, 6.8 mmHg O2, 11.35 mmHg O2, 46.3 mmHg O2, 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments,“low oxygen” refers to about 60 mmHg O2 or less (e.g., 0 to about 60 mmHg O2). The term“low oxygen” may also refer to a range of O2 levels, amounts, or concentrations between 0-60 mmHg O2 (inclusive), e.g., 0-5 mmHg O2, < 1.5 mmHg O2, 6-10 mmHg, < 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al., Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al., J Clin. Invest., 41(11): 1971- 1980 (1962); Crompton et al., J Exp. Biol., 43: 473-478 (1965); He et al., PNAS (USA), 96: 4586-4591 (1999);
McKeown, Br. J. Radiol., 87:20130676 (2014) (doi: 10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorporated by reference herewith in their entireties. In some embodiments, the term“low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments,“low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table 2 summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (O2) is expressed as the amount of dissolved oxygen (“DO”) which refers to the level of free, non-compound oxygen (O2) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; 1mg/L = 1 ppm), or in micromoles (umole) (1 umole O2 = 0.022391 mg/L O2). Fondriest Environmental, Inc.,“Dissolved Oxygen”, Fundamentals of Environmental Measurements, 19 Nov 2013,
www.fondriest.com/environmental-measurements/parameters/water-quality/dissolved- oxygen/>. In some embodiments, the term“low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (O2) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term“low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g., 0-5%, 0.05– 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5– 2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term“low oxygen” is meant to refer to 9% O2 saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O2 saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of O2 saturation levels between 0-9%, inclusive (e.g., 0-5%, 0.05– 0.1%, 0.1-0.2%, 0.1- 0.5%, 0.5– 2.0%, 0-8%, 5-7%, 0.3-4.2% O2, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
Table 2.
Figure imgf000086_0001
[0214] As used herein, the term“gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence.
[0215]“Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell.
Examples of microorganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules. In certain aspects, the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.
[0216]“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, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent No.6,835,376; U.S. Patent No.6,203,797; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
[0217] As used herein,“payload” refers to one or more polynucleotides and/or polypeptides of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus. In some embodiments, the payload is encoded by a gene or multiple genes or an operon. In some embodiments, the one or more genes and/or operon(s) comprising the payload are endogenous to the microorganism. In some embodiments, the one or more elements of the payload is derived from a different microorganism and/or organism. In some embodiments, the payload is a therapeutic payload. In some embodiments, the payload is encoded by genes for the biosynthesis of a molecule. In some embodiments, the payload is encoded by genes for the metabolism, catabolism, or degradation of a molecule. In some embodiments, the payload is encoded by genes for the importation of a molecule. In some embodiments, the payload is encoded by genes for the exportation of a molecule. In some embodiments, the payload is a regulatory molecule(s), e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads. Non-limiting examples of payload(s) include one or more of the following: (1) ArgAfbr, (2) mutated Arg Boxes, (3) mutated ArgR, (4) mutated ArgG, (5) butyrate biosynthetic cassette, (6) proprionate biosynthetic cassette, (7) acetate biosynthetic cassette; (8) GABA-metabolizing cassette, (9) GABA-transporter, (10) Mn-transporter (11) tryptophan or any of its metabolites, e.g., kynurenine, kynurenic acid, and indole metabolites described herein (11) secreted or surface displayed polypeptides, e.g., GLP-2 or IL-22. Other exemplary payloads include IL-10, IL-27, TGF-β1, TGF-β2, elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2,. Payloads also include mutated endogenous genes, which allow the production of certain metabolites or polypeptides or improve the production of certain metabolites or polypeptides. Other exemplary payloads include mutated sequence(s) that result in an auxotrophy, e.g., thyA auxotrophy, kill switch circuit, antibiotic resistance circuits, transporter sequence for importing biological molecules or substrates, secretion circuit.
[0218]“Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an
appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and
Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No.6,203,797; U.S. Patent 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
[0219] As used herein,“stably maintained” or“stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a feedback resistant argA gene, mutant arginine repressor, and/or other mutant arginine regulon that is incorporated into the host genome or propagated on a self-replicating extra- chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically engineered bacterium comprising an argAfbr gene, in which the plasmid or chromosome carrying the argAfbr gene is stably maintained in the bacterium, such that argAfbr can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo.
[0220] As used herein, the terms“modulate” and“treat” and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment,“modulate” and“treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment,“modulate” and“treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment,“modulate” and“treat” refer to slowing the
progression or reversing the progression of a disease, disorder, and/or condition. As used herein,“prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.
[0221] Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Primary hyperammonemia is caused by UCDs, which are autosomal recessive or X-linked inborn errors of metabolism for which there are no known cures.
Hyperammonemia can also be secondary to other disruptions of the urea cycle, e.g., toxic metabolites, infections, and/or substrate deficiencies. Hyperammonemia can also contribute to other pathologies. For example, Huntington’s disease is an autosomal dominant disorder for which there are no known cures. Urea cycle abnormalities characterized by hyperammonemia, high blood citrulline, and suppression of urea cycle enzymes may contribute to the pathology of Huntington’s disease, an autosomal dominant disorder for which there are no known cures. Treating hyperammonemia may encompass reducing or eliminating excess ammonia and/or associated symptoms, and does not necessarily encompass the elimination of the underlying hyperammonemia- associated disorder.
[0222] As used herein a "pharmaceutical composition" refers to a preparation of genetically engineered bacteria of the invention with other components such as a physiologically suitable carrier and/or excipient.
[0223] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.
[0224] The term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.
[0225] The terms“therapeutically effective dose” and“therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g.,
hyperammonemia. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder associated with elevated ammonia concentrations. 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.
[0226] As used herein, the term“polypeptide” includes“polypeptide” as well as “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term“polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus,“peptides,”“dipeptides,”“tripeptides,“oligopeptides,”“protein,”“amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of“polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any of these terms. The term “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. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term“peptide” or“polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.
[0227] An“isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms “fragment,”“variant,”“derivative” and“analog” include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non- naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions.
[0228] Polypeptides also include fusion proteins. As used herein, the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term“fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins.“Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.“Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution.
Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation,
Washington, D.C. (1978), and in Argos, EMBO J.8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.
[0229] As used herein, the term“sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and 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.
[0230] As used herein the term“linker”,“linker peptide” or“peptide linkers” or “linker” refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains. As used herein the term“synthetic” refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety.
[0231] As used herein the term“codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism.
[0232] 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.
[0233] As used herein, the terms“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. In some embodiments, the polypeptide to be secreted include a“secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polypeptide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the“passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antinflammatory or barrier enhancer molecule(s) into the extracellular milieu. In some embodiments, 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, tolB, 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 nlpI, are deactivated. Thus, in some embodiments, 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. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.
[0234] As used herein, the term“transporter” is meant to refer to a mechanism, e.g., protein or proteins, for importing a molecule, e.g., amino acid, toxin, metabolite, substrate, etc. into the microorganism from the extracellular milieu.
[0235] The articles“a” and“an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.
[0236] The phrase“and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example,“A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase“and/or” may be used interchangeably with“at least one of” or“one or more of” the elements in a list.
Bacteria
[0237] The genetically engineered bacteria disclosed herein are capable of reducing excess ammonia and converting ammonia and/or nitrogen into alternate byproducts. In some embodiments, the genetically engineered bacteria are naturally non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli,
Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.
[0238] In some embodiments, the genetically engineered bacteria are
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 al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that E. coli Nissle lacks prominent virulence factors (e.g., E. coli α-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E. coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn’s disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle’s therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007).
[0239] One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be modified and adapted for other species, strains, and subtypes of bacteria. It is known, for example, that arginine-mediated regulation is remarkably well conserved in very divergent bacteria, i.e., Gram-negative bacteria, such as E. coli, Salmonella enterica serovar Typhimurium, Thermotoga, and Moritella profunda, and Gram-positive bacteria, such as B. subtilis, Geobacillus
stearothermophilus, and Streptomyces clavuligerus, as well as other bacteria (Nicoloff et al., 2004). Furthermore, the arginine repressor is universally conserved in bacterial genomes and that its recognition signal (the ARG box), a weak palindrome, is also conserved between genomes (Makarova et al., 2001).
[0240] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum
(Sonnenborn et al., 2009). The residence time of bacteria in vivo can be determined using the methods described herein. In some embodiments, the residence time is calculated for a human subject. A non-limiting example using a streptomycin-resistant E. coli Nissle comprising a wild-type ArgR and a wild-type arginine regulon is provided herein. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention.
Reduction of Excess Ammonia
Arginine Biosynthesis Pathway
[0241] In bacteria such as Escherichia coli (E. coli), the arginine biosynthesis pathway is capable of converting glutamate to arginine in an eight-step enzymatic process involving the enzymes N-acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase (Cunin et al., 1986). The first five steps involve N-acetylation to generate an ornithine precursor. In the sixth step, ornithine transcarbamylase (also known as ornithine carbamoyltransferase) catalyzes the formation of citrulline. The final two steps involve carbamoylphosphate utilization to generate arginine from citrulline.
[0242] In some bacteria, e.g., Bacillus stearothermophilus and Neisseria gonorrhoeae, the first and fifth steps in arginine biosynthesis may be catalyzed by the bifunctional enzyme ornithine acetyltransferase. This bifunctionality was initially identified when ornithine acetyltransferase (argJ) was shown to complement both N- acetylglutamate synthetase (argA) and N-acetylornithinase (argE) auxotrophic gene mutations in E. coli (Mountain et al., 1984; Crabeel et al., 1997).
[0243] ArgA encodes N-acetylglutamate synthetase, argB encodes N- acetylglutamate kinase, argC encodes N-acetylglutamylphosphate reductase, argD encodes acetylornithine aminotransferase, argE encodes N-acetylornithinase, argF encodes ornithine transcarbamylase, argI also encodes ornithine transcarbamylase, argG encodes argininosuccinate synthase, argH encodes argininosuccinate lyase, and argJ encodes ornithine acetyltransferase. CarA encodes the small A subunit of carbamoylphosphate synthase having glutaminase activity, and carB encodes the large B subunit of carbamoylphosphate synthase that catalyzes carbamoylphosphate synthesis from ammonia. Different combinations of one or more of these arginine biosynthesis genes (i.e., argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB) may be organized, naturally or synthetically, into one or more operons, and such organization may vary between bacterial species, strains, and subtypes (see, e.g., Table 3). The regulatory region of each operon contains at least one ARG box, and the number of ARG boxes per regulatory region may vary between operons and bacteria.
[0244] All of the genes encoding these enzymes are subject to repression by arginine via its interaction with ArgR to form a complex that binds to the regulatory region of each gene and inhibits transcription. N-acetylglutamate synthetase is also subject to allosteric feedback inhibition at the protein level by arginine alone (Tuchman et al., 1997; Caldara et al., 2006; Caldara et al., 2008; Caldovic et al., 2010).
[0245] The genes that regulate arginine biosynthesis in bacteria are scattered across the chromosome and organized into multiple operons that are controlled by a single repressor, which Maas and Clark (1964) termed a“regulon.” Each operon is regulated by a regulatory region comprising at least one 18-nucleotide imperfect palindromic sequence, called an ARG box, that overlaps with the promoter and to which the repressor protein binds (Tian et al., 1992; Tian et al., 1994). The argR gene encodes the repressor protein, which binds to one or more ARG boxes (Lim et al., 1987).
Arginine functions as a corepressor that activates the arginine repressor. The ARG boxes that regulate each operon may be non-identical, and the consensus ARG box sequence is (Maas, 1994). In addition, the
Figure imgf000098_0001
regulatory region of argR contains two promoters, one of which overlaps with two ARG boxes and is autoregulated.
[0246] In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon and produce more arginine and/or an intermediate byproduct, e.g., citrulline, than unmodified bacteria of the same subtype under the same conditions. The mutant arginine regulon comprises one or more nucleic acid mutations that reduce or prevent arginine-mediated repression– via ArgR binding to ARG boxes and/or arginine binding to N-acetylglutamate synthetase– of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine in the arginine biosynthesis pathway, thereby enhancing arginine and/or intermediate byproduct biosynthesis.
[0247] In alternate embodiments, the bacteria are genetically engineered to consume excess ammonia via another metabolic pathway, e.g., a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, and a tryptophan biosynthesis pathway. As used herein,“an ammonia conversion circuit” refers to a metabolic pathway by which excess ammonia may be consumed and/or reduced.
[0248] Histidine Biosynthesis Pathway
[0249] Histidine biosynthesis, for example, is carried out by eight genes located within a single operon in E. coli. Three of the eight genes of the operon (hisD, hisB, and hisI) encode bifunctional enzymes, and two (hisH and hisF) encode polypeptide chains which together form one enzyme to catalyze a single step, for a total of 10 enzymatic reactions (Alifano et al., 1996). The product of the hisG gene, ATP phosphoribosyltransferase, is inhibited at the protein level by histidine. In some embodiments, the genetically engineered bacteria of the invention comprise a feedback- resistant hisG. Bacteria may be mutagenized and/or screened for feedback-resistant hisG mutants using techniques known in the art. Bacteria engineered to comprise a feedback-resistant hisG would have elevated levels of histidine production, thus increasing ammonia consumption and reducing hyperammonemia. Alternatively, one or more genes required for histidine biosynthesis could be placed under the control of an inducible promoter, such as a FNR-inducible promoter, and allow for increased production of rate-limiting enzymes. Any other suitable modification(s) to the histidine biosynthesis pathway may be used to increase ammonia consumption.
Methionine Biosynthesis Pathway
[0250] The bacterial methionine regulon controls the three-step synthesis of methionine from homoserine (i.e., acylation, sulfurylation, and methylation). The metJ gene encodes a regulatory protein that, when combined with methionine or a derivative thereof, causes repression of genes within the methionine regulon at the transcriptional level (Saint-Girons et al., 1984; Shoeman et al., 1985). In some embodiments, the genetically engineered bacteria of the invention comprise deleted, disrupted, or mutated metJ. Bacteria engineered to delete, disrupt, or mutate metJ would have elevated levels of methionine production, thus increasing ammonia consumption and reducing hyperammonemia. Any other suitable modification(s) to the methionine biosynthesis pathway may be used to increase ammonia consumption. Lysine Biosynthesis Pathway
[0251] Microorganisms synthesize lysine by one of two pathways. The diaminopimelate (DAP) pathway is used to synthesize lysine from aspartate and pyruvate (Dogovski et al., 2012), and the aminoadipic acid pathway is used to synthesize lysine from alpha-ketoglutarate and acetyl coenzyme A. The
dihydrodipicolinate synthase (DHDPS) enzyme catalyzes the first step of the DAP pathway, and is subject to feedback inhibition by lysine (Liu et al., 2010; Reboul et al., 2012). In some embodiments, the genetically engineered bacteria of the invention comprise a feedback-resistant DHDPS. Bacteria engineered to comprise a feedback- resistant DHDPS would have elevated levels of histidine production, thus increasing ammonia consumption and reducing hyperammonemia. Alternatively, lysine production could be optimized by placing one or more genes required for lysine biosynthesis under the control of an inducible promoter, such as a FNR-inducible promoter. Any other suitable modification(s) to the lysine biosynthesis pathway may be used to increase ammonia consumption.
Asparagine Biosynthesis Pathway
[0252] Asparagine is synthesized directly from oxaloacetate and aspartic acid via the oxaloacetate transaminase and asparagine synthetase enzymes, respectively. In the second step of this pathway, either L-glutamine or ammonia serves as the amino group donor. In some embodiments, the genetically engineered bacteria of the invention overproduce asparagine as compared to unmodified bacteria of the same subtype under the same conditions, thereby consuming excess ammonia and reducing hyperammonemia. Alternatively, asparagine synthesis may be optimized by placing one or both of these genes under the control of an inducible promoter, such as a FNR- inducible promoter. Any other suitable modification(s) to the asparagine biosynthesis pathway may be used to increase ammonia consumption.
Glutamine Biosynthesis Pathway
[0253] The synthesis of glutamine and glutamate from ammonia and
oxoglutarate is tightly regulated by three enzymes. Glutamate dehydrogenase catalyzes the reductive amination of oxoglutarate to yield glutamate in a single step. Glutamine synthetase catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine (Lodeiro et al., 2008). Glutamine synthetase also acts with glutamine– oxoglutarate amino transferase (also known as glutamate synthase) in a cyclic reaction to produce glutamate from glutamine and oxoglutarate. In some embodiments, the genetically engineered bacteria of the invention express glutamine synthetase at elevated levels as compared to unmodified bacteria of the same subtype under the same conditions. Bacteria engineered to have increased expression of glutamine synthetase would have elevated levels of glutamine production, thus increasing ammonia consumption and reducing hyperammonemia. Alternatively, expression of glutamate dehydrogenase and/or glutamine–oxoglutarate amino transferase could be modified to favor the consumption of ammonia. Since the production of glutamine synthetase is regulated at the transcriptional level by nitrogen (Feng et al., 1992; van Heeswijk et al., 2013), placing the glutamine synthetase gene under the control of different inducible promoter, such as a FNR-inducible promoter, may also be used to improve glutamine production. Any other suitable modification(s) to the glutamine and glutamate biosynthesis pathway may be used to increase ammonia consumption.
Tryptophan Biosynthesis Pathway
[0254] In most bacteria, the genes required for the synthesis of tryptophan from a chorismate precursor are organized as a single transcriptional unit, the trp operon. The trp operon is under the control of a single promoter that is inhibited by the tryptophan repressor (TrpR) when high levels of tryptophan are present. Transcription of the trp operon may also be terminated in the presence of high levels of charged tryptophan tRNA. In some embodiments, the genetically engineered bacteria of the invention comprise a deleted, disrupted, or mutated trpR gene. The deletion, disruption, or mutation of the trpR gene, and consequent inactivation of TrpR function, would result in elevated levels of both tryptophan production and ammonia consumption. Alternatively, one or more enzymes required for tryptophan biosynthesis could be placed under the control of an inducible promoter, such as a FNR-inducible promoter. Any other suitable modification(s) to the tryptophan biosynthesis pathway may be used to increase ammonia consumption.
Engineered Bacteria Comprising a Mutant Arginine Regulon
[0255] In some embodiments, the genetically engineered bacteria comprise an arginine biosynthesis pathway and are capable of reducing excess ammonia. In a more specific aspect, the genetically engineered bacteria comprise a mutant arginine regulon in which one or more operons encoding arginine biosynthesis enzyme(s) is derepressed to produce more arginine or an intermediate byproduct, e.g., citrulline, than unmodified bacteria of the same subtype under the same conditions. In some embodiments, the genetically engineered bacteria overproduce arginine. In some embodiments, the genetically engineered bacteria overproduce citrulline; this may be additionally beneficial, because citrulline is currently used as a therapeutic for particular urea cycle disorders (National Urea Cycle Disorders Foundation). In some embodiments, the genetically engineered bacteria overproduce an alternate intermediate byproduct in the arginine biosynthesis pathway, such as any of the intermediates described herein. In some embodiments, the genetically engineered bacterium consumes excess ammonia by producing more arginine, citrulline, and/or other intermediate byproduct than an unmodified bacterium of the same bacterial subtype under the same conditions.
Enhancement of arginine and/or intermediate byproduct biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules in order to treat conditions associated with hyperammonemia, including urea cycle disorders and hepatic encephalopathy.
[0256] One of skill in the art would appreciate that the organization of arginine biosynthesis genes within an operon varies across species, strains, and subtypes of bacteria, e.g., bipolar argECBH in E. coli K12, argCAEBD-carAB-argF in B. subtilis, and bipolar carAB-argCJBDF in L. plantarum. Non-limiting examples of operon organization from different bacteria are shown in Table 3 (in some instances, the genes are putative and/or identified by sequence homology to known sequences in Escherichia coli; in some instances, not all of the genes in the arginine regulon are known and/or shown below). In certain instances, the arginine biosynthesis enzymes vary across species, strains, and subtypes of bacteria.
Table 3: Examples of arg operon organization
Figure imgf000102_0001
Figure imgf000103_0001
[0257] Each operon is regulated by a regulatory region comprising at least one promoter and at least one ARG box, which control repression and expression of the arginine biosynthesis genes in said operon.
[0258] In some embodiments, the genetically engineered bacteria of the invention comprise an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct in the arginine biosynthesis pathway. Reducing or eliminating arginine-mediated repression may be achieved by reducing or eliminating ArgR repressor binding (e.g., by mutating or deleting the arginine repressor or by mutating at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes) and/or arginine binding to N-acetylglutamate synthetase (e.g., by mutating the N-acetylglutamate synthetase to produce an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr).
ARG Box
[0259] In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, and carbamoylphosphate synthase, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis. In either of these embodiments, the genetically engineered bacteria may further comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr. Thus, in some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr. In some embodiments, the genetically engineered bacteria comprise a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr. In some
embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr, a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes, and/or a mutant or deleted arginine repressor.
[0260] In some embodiments, the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for one or more of the operons that encode N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
argininosuccinate lyase, carbamoylphosphate synthase, and wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis.
[0261] In some embodiments, the ARG boxes for the operon encoding argininosuccinate synthase (argG) maintain the ability to bind to ArgR, thereby driving citrulline biosynthesis. For example, the regulatory region of the operon encoding argininosuccinate synthase (argG) may be a constitutive, thereby driving arginine biosynthesis. In alternate embodiments, the regulatory region of one or more alternate operons may be constitutive. In certain bacteria, however, genes encoding multiple enzymes may be organized in bipolar operons or under the control of a shared regulatory region; in these instances, the regulatory regions may need to be
deconvoluted in order to engineer constitutively active regulatory regions. For example, in E. coli K12 and Nissle, argE and argCBH are organized in two bipolar operons, argECBH, and those regulatory regions may be deconvoluted in order to generate constitutive versions of argE and/or argCBH.
[0262] In some embodiments, all ARG boxes in one or more operons that comprise an arginine biosynthesis gene are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in one or more operons that encode an arginine biosynthesis enzyme are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in each operon that comprises an arginine biosynthesis gene are mutated to reduce or eliminate ArgR binding. In some embodiments, all ARG boxes in each operon that encodes an arginine biosynthesis enzyme are mutated to reduce or eliminate ArgR binding.
[0263] In some embodiments, the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase, argininosuccinate synthase driven by a ArgR-repressible regulatory region, and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for each of the operons that encode N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, carbamoylphosphate synthase, and optionally, wild-type N-acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and enhancing citrulline biosynthesis. In some embodiments, the genetically engineered bacteria capable of producing citrulline is particularly advantageous, because citrulline further serves as a
therapeutically effective supplement for the treatment of certain urea cycle disorders (National Urea Cycle Disorders Foundation).
[0264] In some embodiments, the genetically engineered bacteria encode an arginine feedback resistant N-acetylglutamate synthase, argininosuccinate synthase driven by a constitutive promoter, and further comprise a mutant arginine regulon comprising one or more nucleic acid mutations in each ARG box for each of the operons that encode N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate lyase, carbamoylphosphate synthase, and optionally, wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby derepressing the regulon and enhancing arginine biosynthesis. [0265] In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon and a feedback resistant ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or an intermediate byproduct than unmodified bacteria of the same subtype under the same conditions. In any of these embodiments, the mutant arginine regulon and/or a feedback resistant ArgA may be integrated into the bacterial chromosome at one or more integration sites or may be present on one or more plasmids.
Arginine Repressor Binding Sites (ARG Boxes)
[0266] In some embodiments, the genetically engineered bacteria additionally comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase,
acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, and carbamoylphosphate synthase, such that the arginine regulon is derepressed and biosynthesis of arginine and/or an intermediate byproduct, e.g., citrulline, is enhanced.
[0267] In some embodiments, the mutant arginine regulon comprises an operon encoding ornithine acetyltransferase and one or more nucleic acid mutations in at least one ARG box for said operon. The one or more nucleic acid mutations results in the disruption of the palindromic ARG box sequence, such that ArgR binding to that ARG box and to the regulatory region of the operon is reduced or eliminated, as compared to ArgR binding to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions. In some embodiments, nucleic acids that are protected from DNA methylation and hydroxyl radical attack during ArgR binding are the primary targets for mutations to disrupt ArgR binding. In some embodiments, the mutant arginine regulon comprises at least three nucleic acid mutations in one or more ARG boxes for each of the operons that encode the arginine biosynthesis enzymes described above. The ARG box overlaps with the promoter, and in the mutant arginine regulon, the G/C:A/T ratio of the mutant promoter region differs by no more than 10% from the G/C:A/T ratio of the wild-type promoter region (Table 4). The promoter retains sufficiently high homology to the non-mutant promoter such that RNA polymerase binds with sufficient affinity to promote transcription. [0268] The wild-type genomic sequences comprising ARG boxes and mutants thereof for each arginine biosynthesis operon in E. coli Nissle are shown in Table 4. For exemplary wild-type sequences, the ARG boxes are indicated in italics, and the start codon of each gene is boxed. The RNA polymerase binding sites are underlined (Cunin, 1983; Maas, 1994). In some embodiments, the underlined sequences are not altered. Bases that are protected from DNA methylation during ArgR binding are highlighted, and bases that are protected from hydroxyl radical attack during ArgR binding are bolded (Charlier et al., 1992). The highlighted and bolded bases are the primary targets for mutations to disrupt ArgR binding.
Figure imgf000107_0001
Figure imgf000108_0001
[0269] In some embodiments, more than one ARG box may be present in a single operon. In one aspect of these embodiments, at least one of the ARG boxes in an operon is mutated to produce the requisite reduced ArgR binding to the regulatory region of the operon. In an alternate aspect of these embodiments, each of the ARG boxes in an operon is mutated to produce the requisite reduced ArgR binding to the regulatory region of the operon. One of skill in the art would appreciate that the number of ARG boxes per regulatory region may vary across bacteria, and the nucleotide sequences of the ARG boxes may vary for each operon. For example, the carAB operon in E. coli Nissle comprises two ARG boxes, and one or both ARG box sequences may be mutated. The argG operon in E. coli Nissle comprises three ARG boxes, and one, two, or three ARG box sequences may be mutated, disrupted, or deleted. In some embodiments, all three ARG box sequences are mutated, disrupted, or deleted, and a constitutive promoter, e.g., BBa_J23100, is inserted in the regulatory region of the argG operon. One of skill in the art would appreciate that the number of ARG boxes per regulatory region may vary across bacteria, and the nucleotide sequences of the ARG boxes may vary for each operon. [0270] An exemplary embodiment of a constitutively expressed argG construct in E. coli Nissle is depicted in Table 5. Table 5 depicts the wild-type genomic sequence of the regulatory region and 5’ portion of the argG gene in E. coli Nissle, and a constitutive mutant thereof. The promoter region of each sequence is underlined, and a 5’ portion of the argG gene is boxed. In the wild-type sequence, ArgR binding sites are in uppercase and underlined. In the mutant sequence, the 5’ untranslated region is in uppercase and underlined. Bacteria expressing argG under the control of the constitutive promoter are capable of producing arginine. Bacteria expressing argG under the control of the wild-type, ArgR-repressible promoter are capable of producing citrulline. A map of the wild-type argG operon E. coli Nissle and a constitutively expressing mutant thereof is shown in FIG. 12.
Figure imgf000109_0001
[0271] In some embodiments, the ArgR binding affinity to a mutant ARG box or regulatory region of an operon is at least about 50% lower, at least about 60% lower, at least about 70% lower, at least about 80% lower, at least about 90% lower, or at least about 95% lower than the ArgR binding affinity to an unmodified ARG box and regulatory region in bacteria of the same subtype under the same conditions. In some embodiments, the reduced ArgR binding to a mutant ARG box and regulatory region increases mRNA expression of the gene(s) in the associated operon by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold.
[0272] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the arginine biosynthesis genes. Primers specific for arginine biosynthesis genes, e.g., argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB, may be designed and used to detect mRNA in a sample according to methods known in the art (Fraga et al., 2008). In some embodiments, a fluorophore is added to a sample reaction mixture that may contain arg mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the arginine biosynthesis genes.
[0273] In some embodiments, the genetically engineered bacteria comprising one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
argininosuccinate lyase, and carbamoylphosphate synthase additionally comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr.
[0274] In some embodiments, the genetically engineered bacteria comprise a feedback resistant form of ArgA, as well as one or more nucleic acid mutations in each ARG box of one or more of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, ornithine acetyltransferase, and carbamoylphosphate synthase.
[0275] In some embodiments, the genetically engineered bacteria comprise a feedback resistant form of ArgA, argininosuccinate synthase driven by a ArgR- repressible regulatory region, as well as one or more nucleic acid mutations in each ARG box of each of the operons that encode the arginine biosynthesis enzymes N- acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate lyase, ornithine acetyltransferase, and carbamoylphosphate synthase. In these embodiments, the bacteria are capable of producing citrulline.
[0276] In some embodiments, the genetically engineered bacteria comprise a feedback resistant form of ArgA, argininosuccinate synthase expressed from a constitutive promoter, as well as one or more nucleic acid mutations in each ARG box of each of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
argininosuccinate lyase, ornithine acetyltransferase, and carbamoylphosphate synthase. In these embodiments, the bacteria are capable of producing arginine.
[0277] Table 6 shows examples of mutant constructs in which one or more nucleic acid mutations reduce or eliminate arginine-mediated repression of each of the arginine operons. The mutant constructs comprise feedback resistant form of ArgA driven by an oxygen level-dependent promoter, e.g., a FNR promoter. Each mutant arginine regulon comprises one or more nucleic acid mutations in at least one ARG box for one or more of the operons that encode N-acetylglutamate kinase, N- acetylglutamylphosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase,
argininosuccinate lyase, carbamoylphosphate synthase, and wild-type N- acetylglutamate synthetase, such that ArgR binding is reduced or eliminated, thereby enhancing arginine and/or intermediate byproduct biosynthesis. Non-limiting examples of mutant arginine regulon constructs are shown in Table 6.
Table 6: Examples of ARG Box mutant constructs
Figure imgf000112_0001
[0278] The mutations may be present on a plasmid or chromosome. In some embodiments, the arginine regulon is regulated by a single repressor protein. In particular species, strains, and/or subtypes of bacteria, it has been proposed that the arginine regulon may be regulated by two putative repressors (Nicoloff et al., 2004). Thus, in certain embodiments, the arginine regulon of the invention is regulated by more than one repressor protein. [0279] In certain embodiments, the mutant arginine regulon is expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the mutant arginine regulon is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria.
Arginine Repressor (ArgR)
[0280] The genetically engineered bacteria of the invention comprise an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of one or more of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct in the arginine biosynthesis pathway. In some embodiments, the reduction or elimination of arginine-mediated repression may be achieved by reducing or eliminating ArgR repressor binding, e.g., by mutating at least one ARG box for one or more of the operons that encode the arginine biosynthesis enzymes (as discussed above) or by mutating or deleting the arginine repressor (discussed here) and/or by reducing or eliminating arginine binding to N-acetylglutamate synthetase (e.g., by mutating the N- acetylglutamate synthetase to produce an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr).
[0281] Thus, in some embodiments, the genetically engineered bacteria lack a functional ArgR repressor and therefore ArgR repressor-mediated transcriptional repression of each of the arginine biosynthesis operons is reduced or eliminated. In some embodiments, the engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive. In some embodiments, the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis. In some embodiments, each copy of a functional argR gene normally present in a corresponding wild-type bacterium is independently deleted or rendered inactive by one or more nucleotide deletions, insertions, or substitutions. In some embodiments, each copy of the functional argR gene normally present in a corresponding wild-type bacterium is deleted.
[0282] In some embodiments, the arginine regulon is regulated by a single repressor protein. In particular species, strains, and/or subtypes of bacteria, it has been proposed that the arginine regulon may be regulated by two distinct putative repressors (Nicoloff et al., 2004). Thus, in certain embodiments, two distinct ArgR proteins each comprising a different amino acid sequence are mutated or deleted in the genetically engineered bacteria.
[0283] In some embodiments, the genetically modified bacteria comprising a mutant or deleted arginine repressor additionally comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr. In some embodiments, the genetically engineered bacteria comprise a feedback resistant form of ArgA, lack any functional arginine repressor, and are capable of producing arginine. In certain embodiments, the genetically engineered bacteria further lack functional ArgG and are capable of producing citrulline. In some embodiments, the argR gene is deleted in the genetically engineered bacteria. In some embodiments, the argR gene is mutated to inactivate ArgR function. In some embodiments, the argG gene is deleted in the genetically engineered bacteria. In some embodiments, the argG gene is mutated to inactivate ArgR function. In some embodiments, the genetically engineered bacteria comprise argAfbr and deleted ArgR. In some embodiments, the genetically engineered bacteria comprise argAfbr, deleted ArgR, and deleted argG. In some embodiments, the deleted ArgR and/or the deleted argG is deleted from the bacterial genome and the argAfbris present in a plasmid. In some embodiments, the deleted ArgR and/or the deleted argG is deleted from the bacterial genome and the argAfbris chromosomally integrated. In one specific embodiment, the genetically modified bacteria comprise chromosomally integrated argAfbr, deleted genomic ArgR, and deleted genomic argG. In another specific embodiment, the genetically modified bacteria comprise
argAfbrpresent on a plasmid, deleted genomic ArgR, and deleted genomic argG. In any of the embodiments in which argG is deleted, citrulline rather than arginine is produced.
[0284] In some embodiments, under conditions where a feedback resistant form of ArgA is expressed, the genetically engineered bacteria of the invention produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more arginine, citrulline, other intermediate byproduct, and/or transcript of the gene(s) in the operon as compared to unmodified bacteria of the same subtype under the same conditions. [0285] In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the arginine biosynthesis genes. Primers specific for arginine biosynthesis genes, e.g., argA, argB, argC, argD, argE, argF, argG, argH, argI, argJ, carA, and carB, may be designed and used to detect mRNA in a sample according to methods known in the art (Fraga et al., 2008). In some embodiments, a fluorophore is added to a sample reaction mixture that may contain arg mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the arginine biosynthesis genes.
[0286] In any of these embodiments in which the ArgR is mutated, the mutant ArgR and/or a feedback resistant ArgA may be integrated into the bacterial
chromosome at one or more integration sites or may be present on one or more plasmids.
Feedback Resistant N-acetylglutamate Synthetase
[0287] In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N-acetylglutamate synthase mutant, e.g., argAfbr. In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising an arginine feedback resistant ArgA, and when the arginine feedback resistant ArgA is expressed, are capable of producing more arginine and/or an intermediate byproduct than unmodified bacteria of the same subtype under the same conditions. The arginine feedback resistant N-acetylglutamate synthetase protein (argAfbr) is significantly less sensitive to L-arginine than the enzyme from the feedback sensitive parent strain (see, e.g., Eckhardt et al., 1975; Rajagopal et al., 1998). The feedback resistant argA gene can be present on a plasmid or chromosome. In some embodiments, expression from the plasmid may be useful for increasing argAfbr expression. In some embodiments, expression from the chromosome may be useful for increasing stability of argAfbr expression.
[0288] In some embodiments, any of the described mutant sequences involved in the arginine biosynthetic pathway (e.g., ArgR, argAfbr , Arg Box sequence) are integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of the sequence encoding the arginine feedback resistant N- acetylglutamate synthase may be integrated into the bacterial chromosome. Having multiple copies of the arginine feedback resistant N-acetylglutamate synthase integrated into the chromosome allows for greater production of the N-acetylglutamate synthase and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the transporter or kill-switch circuits, in addition to the arginine feedback resistant N-acetylglutamate synthase could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions. Multiple distinct feedback resistant N-acetylglutamate synthetase proteins are known in the art and may be combined in the genetically engineered bacteria. In some embodiments, the argAfbr gene is expressed under the control of a constitutive promoter. In some embodiments, the argAfbr gene is expressed under the control of a promoter that is induced by exogenous environmental conditions. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut in a healthy or disease state, e.g., propionate. In some embodiments, 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. In some embodiments, the exogenous environmental conditions are low- oxygen or anaerobic conditions, such as the environment of the mammalian gut.
[0289] The nucleic acid sequence of an exemplary
Figure imgf000116_0001
sequence is shown in Table 7. The polypeptide sequence of an exemplary sequence is shown in Table
Figure imgf000116_0002
8.
Table 7
Figure imgf000117_0001
Bold underline: mutated amino acid resulting feedback resistance. (mutation is Y19C) [0290] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 30 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 SEQ ID NO: 30 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 30 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 30 or a functional fragment thereof.
[0291] In some embodiments, the genetically engineered bacteria encode a polypeptide sequence of SEQ ID NO: 31 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria encode a polypeptide sequence encodes a polypeptide, which contains one or more conservative amino acid
substitutions relative to SEQ ID NO: 31 or a functional fragment thereof. In some embodiments, genetically engineered bacteria encode 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 DNA sequence of SEQ ID NO: 31 or a functional fragment thereof.
[0292] In some embodiments, arginine feedback inhibition of N-acetylglutamate synthetase is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in the genetically engineered bacteria when the arginine feedback resistant N-acetylglutamate synthetase is active, as compared to a wild-type N-acetylglutamate synthetase from bacteria of the same subtype under the same conditions.
[0293] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the argAfbr gene, such that argAfbr can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the feedback resistant argA gene. In some embodiments, the feedback resistant argA gene is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the feedback resistant argA gene is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing argAfbr expression. In some
embodiments, the feedback resistant argA gene is expressed on a chromosome. [0294] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 18. For example, the genetically engineered bacteria may include four copies of argAfbr inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of argAfbr inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three mutant arginine regulons, e.g., two producing citrulline and one producing arginine, inserted at three different insertion sites dapA, cea, and araC/BAD.
[0295] In some embodiments, the plasmid or chromosome also comprises wild- type ArgR binding sites, e.g., ARG boxes. In some instances, the presence and/or build- up of functional ArgR may result in off-target binding at sites other than the ARG boxes, which may cause off-target changes in gene expression. A plasmid or chromosome that further comprises functional ARG boxes may be used to reduce or eliminate off-target ArgR binding, i.e., by acting as an ArgR sink. In some
embodiments, the plasmid or chromosome does not comprise functional ArgR binding sites, e.g., the plasmid or chromosome comprises modified ARG boxes or does not comprise ARG boxes.
[0296] In some embodiments, the feedback resistant argA gene is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the feedback resistant argA gene is present in the chromosome and operably linked to a promoter that is induced under low- oxygen or anaerobic conditions. In some embodiments, the feedback resistant argA gene is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the feedback resistant argA gene is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the feedback resistant argA gene is present on a
chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the feedback resistant argA gene is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. [0297] In some embodiments, the genetically engineered bacteria comprise a variant or mutated oxygen level-dependent transcriptional regulator, e.g., FNR, ANR, or DNR, in addition to the corresponding oxygen level-dependent promoter. The variant or mutated oxygen level-dependent transcriptional regulator increases the transcription of operably linked genes in a low-oxygen or anaerobic environment. In some embodiments, the corresponding wild-type transcriptional regulator retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity. In certain embodiments, the mutant oxygen level-dependent transcriptional regulator is a FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., 2006).
[0298] In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent transcriptional regulator from a different bacterial species that reduces and/or consumes ammonia in low-oxygen or anaerobic environments. In certain embodiments, the mutant oxygen level-dependent transcriptional regulator is a FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011). In some
embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.
[0299] In some embodiments, the genetically engineered bacteria comprise argAfbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter, as well as wild-type argA expressed under the control of a mutant regulatory region comprising one or more ARG box mutations as discussed above. In certain embodiments, the genetically engineered bacteria comprise argAfbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter and do not comprise wild-type argA. In still other embodiments, the mutant arginine regulon comprises argAfbr expressed under the control of an oxygen level-dependent promoter, e.g., a FNR promoter, and further comprises wild-type argA without any ARG box mutations.
[0300] In some embodiments, the genetically engineered bacteria express argAfbr from a plasmid and/or chromosome. In some embodiments, the argAfbr gene is expressed under the control of a constitutive promoter. In some embodiments, the argAfbr gene is expressed under the control of an inducible promoter. In one embodiment, argAfbr is expressed under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, e.g., a FNR fbr promoter. The nucleic acid sequence of an exemplary FNR promoter-driven argA
fbr sequence is shown in Table 9. The FNR promoter sequence is bolded and the argA sequence is boxed. The nucleic acid sequence of a FNR promoter-driven argAfbr plasmid is shown in Table 10, with the FNR promoter sequence bolded and argAfbr sequence boxed. Table 11 shows the nucleic acid sequence of an exemplary pSC101 plasmid. Any suitable FNR promoter(s) may be combined with any suitable feedback- resistant ArgA. Non-limiting FNR promoter sequences are provided in Table 6. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 16, SEQ ID NO: 17, nirB1 promoter (SEQ ID NO: 18), nirB2 promoter (SEQ ID NO: 19), nirB3 promoter (SEQ ID NO: 20), ydfZ promoter (SEQ ID NO: 21), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 22), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 23), fnrS, an
anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 24 or fnrS2 promoter SEQ ID NO: 25), nirB promoter fused to a crp binding site (SEQ ID NO: 26), and fnrS fused to a crp binding site (SEQ ID NO: 27). Table 12 depicts the nucleic acid fbr
sequence of an exemplary fnrS promoter-driven argA sequence. The FNR promoter fbr
sequence is bolded, the ribosome binding site is highlighted, and the argA sequence is
Figure imgf000121_0001
[0301] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 32 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 SEQ ID NO: 32. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 32, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 32.
[0302] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 33 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 SEQ ID NO: 33. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 33, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 33.
[0303] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 35 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 SEQ ID NO: 35. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 35, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 35.
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
[0304] In some embodiments, the genetically engineered bacteria comprise argAfbr integrated into the chromosome. In some embodiments, the integrated fbrArgA is under the control of the fnrS promoter. In some embodiments, an antibiotic resistance cassette is also present at the same site. In some embodiments, no antibiotic resistance cassette is present. In some embodiments, the antibiotic resistance is chloramphenicol. In some embodiments, the antibiotic resistance is kanamycin. In some embodiments, the genetically engineered bacteria comprising argAfbr integrated into the chromosome is a thyA auxotroph. In some embodiments, the genetically engineered bacteria comprise argAfbr integrated into the chromosome and also comprise an ArgR mutation or have ArgR deleted. In one specific embodiment, the genetically engineered bacteria comprise argAfbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, and comprise a thyA auxotrophy. In another specific embodiment, the genetically engineered bacteria comprise argAfbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, comprise a thyA auxotrophy, and comprise an antibiotic resistance cassette. In another specific embodiment, the genetically engineered bacteria comprise argAfbr under the control of the fnrS promoter and integrated into the chromosome, comprise an ArgR mutation or have ArgR deleted, comprise a thyA auxotrophy, and comprise a kanamycin resistance cassette. In one specific embodiment, the genetically engineered bacteria is SYN- UCD305. In another specific embodiment, the genetically engineered bacteria is SYN_UCD303.
[0305] Table 13 shows non-limiting examples of FNRS-fbrArgA constructs which are integrated into the chromosome.
[0306] SEQ ID NO: 36 comprises FNRS-fbrArgA and chloramphenicol resistance, e.g., as comprised in SYN-UCD301, SYN-UCD302. SEQ ID NO: 37 comprises FNRS-fbrArgA and kanamycin resistance, e.g., as comprised inSYN- UCD303, SYN-UCD306, SYN-UCD307, and SYN-UCD309. SEQ ID NO: 38 FNRS- fbrArgA and no antibiotic resistance, e.g., as comprised in SYN-UCD305 SYN- UCD304, SYN-UCD308, SYNUCD310.
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
[0307] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 36 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 SEQ ID NO: 36 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 36 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 37 or a functional fragment thereof.
[0308] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 37 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 SEQ ID NO: 37 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 37 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 37 or a functional fragment thereof.
[0309] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 38 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 SEQ ID NO: 38 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 38 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 38 or a functional fragment thereof.
[0310] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0311] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0312] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX and X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or
Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, is modified and/or mutated, e.g., to enhance stability, or increase ammonia catalysis.
[0313] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, 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.
[0314] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences described herein for the consumption of ammonia.
[0315] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, 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.
[0316] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0317] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate. [0318] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the reduction of ammonia levels, e.g., ArgAfbr, and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0319] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”). [0320] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance. Arginine Catabolism
[0321] An important consideration in practicing the invention is to ensure that ammonia is not overproduced as a byproduct of arginine and/or citrulline catabolism. In the final enzymatic step of the urea cycle, arginase catalyzes the hydrolytic cleavage of arginine into ornithine and urea (Cunin et al., 1986). Urease, which may be produced by gut bacteria, catalyzes the cleavage of urea into carbon dioxide and ammonia (Summerskill, 1966; Aoyagi et al., 1966; Cunin et al., 1986). Thus, urease activity may generate ammonia that can be toxic for human tissue (Konieczna et al., 2012). In some bacteria, including E. coli Nissle, the gene arcD encodes an arginine/ornithine antiporter, which may also liberate ammonia (Vander Wauven et al., 1984; Gamper et al., 1991; Meng et al., 1992).
[0322] AstA is an enzyme involved in the conversion of arginine to succinate, which liberates ammonia. SpeA is an enzyme involved in the conversion of arginine to agmatine, which can be further catabolized to produce ammonia. Thus, in some instances, it may be advantageous to prevent the breakdown of arginine. In some embodiments, the genetically engineered bacteria comprising a mutant arginine regulon additionally includes mutations that reduce or eliminate arginine catabolism, thereby reducing or eliminating further ammonia production. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate ArcD activity. In certain embodiments, ArcD is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate AstA activity. In certain embodiments, AstA is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate SpeA activity. In certain embodiments, SpeA is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate arginase activity. In certain embodiments, arginase is deleted. In some embodiments, the genetically engineered bacteria also comprise mutations that reduce or eliminate urease activity. In certain embodiments, urease is deleted. In some embodiments, one or more other genes involved in arginine catabolism are mutated or deleted.
Other Hyperammonemia Disorder Circuits
[0323] Hepatic encephalopathy (HE) is characterized by neurocognitive changes in patients and biochemical derangements have been implicated in pathogenesis.
Specifically, elevated ammonia levels are suspected to partly contribute to disease pathophysiology. In addition to hyperammonemia, elevated levels of cerebral GABA and manganese levels have been noted and suspected to contribute to clinical presentation.
[0324] In some embodiments, the disclosure provides genetically engineered microorganisms, e.g., bacteria and virus, pharmaceutical compositions thereof, and methods of modulating or treating diseases or disorders associated with
hyperammonemia, e.g., hepatic encephalopathy and Huntington’s disease. The genetically engineered bacteria are capable of reducing excess ammonia in a mammal. In some embodiments, the genetically engineered bacteria reduce excess ammonia by incorporating excess nitrogen in the body into non-toxic molecules, e.g., arginine, citrulline, methionine, histidine, lysine, asparagine, glutamine, or tryptophan. In some embodiments, the genetically engineered bacteria further comprise one or more circuits (genetic sequence) to reduce the levels of other toxic or deleterious molecule(s), e.g., GABA, manganese. In some embodiments, the genetically engineered bacteria further comprise one or more circuits to produce a gut barrier enhancer molecule, e.g., a short chain fatty acid such as butyrate, propionate, and acetate. This disclosure also provides compositions and therapeutic methods for reducing excess ammonia and other deleterious molecules, e.g., GABA and manganese. In certain aspects, the disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules. In certain embodiments, the disclosure provides genetically engineered bacteria that are capable of reducing excess ammonia and other deleterious molecules and further producing one or more therapeutic molecules, such as a gut barrier function enhancer molecule, e.g., butyrate. In some embodiments, the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia in which the circuits are under the control of an inducible promoter. In some embodiments, the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia and one or more circuits for reducing other deleterious molecules in which one or more of the circuits are under the control of an inducible promoter. In some embodiments, the disclosure provides genetically engineered bacteria comprising one or more circuits for reducing excess ammonia and one or more circuits for reducing other deleterious molecules and further producing one or more therapeutic molecules, such as a gut barrier function enhancer molecule, e.g., butyrate in which one or more of the circuits and/or therapeutic molecule(s) are under the control of an inducible promoter. In certain aspects, the compositions and methods disclosed herein may be used for treating a disease or disorder associated with excess ammonia, for example, hepatic encephalopathy or Huntington’s disease, and/or one or more symptoms associated with disease or disorder associated with excess ammonia, such as hepatic encephalopathy or Huntington’s disease.
GABA Transport and Metabolism
[0325] γ-Aminobutyric acid (GABA) is the predominant inhibitory
neurotransmitter in the mammalian central nervous system. In humans, GABA activates the post-synaptic GABAA receptor, which is part of a ligand-gated chloride- specific ion channel complex. Activation of this complex on a post-synaptic neuron allows chloride ions to enter the neuron and exert an inhibitory effect. Alterations of such GABAergic neurotransmission have been implicated in the pathophysiology of several neurological disorders, including epilepsy (Jones-Davis and MacDonald, 2003), Huntington’s disease (Krogsgaard-Larsen, 1992), and hepatic encephalopathy (Jones and Basile, 1997).
[0326] Neurons in the brain that are modulated by GABA are said to be under inhibitory GABAergic tone. This inhibitory tone prevents neuronal firing until a sufficiently potent stimulatory stimulus is received, or until the inhibitory tone is otherwise released. Increased GABAergic tone in hepatic encephalopathy was initially described in the early 1980s, based on a report of similar visual response patterns in rabbits with galactosamine-induced liver failure and rabbits treated with allosteric modulators of the GABAA receptor (e.g., pentobarbital, diazepam) (Jones and Basile, 1997). Clinical improvements in HE patients treated with a highly selective
benzodiazapene antagonist at the GABAA receptor, flumazenil, further confirmed these observations (Banksy et al., 1985; Scollo-Lavizzari and Steinmann, 1985). Increased GABAergic tone in HE has since been proposed as a consequence of one or more of the following: (1) increased GABA concentrations in the brain, (2) altered integrity of the GABAA receptor, and/or (3) increased concentrations of endogenous modulators of the GABAA receptor (Ahboucha and Butterworth, 2004).
[0327] GABA uptake in E. coli is driven by membrane potential and facilitated by the membrane transport protein, GabP (Li et al., 2001). GabP is a member of the amino acid/polymaine/organocation (APC) transporter superfamily, one of the two largest families of secondary active transporters (Jack et al., 2000). GabP protein, encoded by the gabP gene, consists of 466 amino acids and 12 transmembrane alpha- helices, wherein both N- and C- termini face the cytosol (Hu and King, 1998a). The GabP residue sequence also includes a consensus amphipathic region (CAR), which is conserved between members of the APC family from bacteria to mammals (Hu and King, 1998b). Upon entry into the cell, GABA is converted to succinyl semialdehyde (SSA) by GABA α-ketoglutarate transaminase (GSST). Succinate-semialdehyde dehydrogenase (SSDH) then catalyzes the second and only other specific step in GABA catabolism, the oxidation of succinyl semialdehyde to succinate (Dover and Halpern, 1972). Ultimately, succinate becomes a substrate for the citric acid (TCA) cycle.
[0328] In some embodiments, the bacteria are genetically engineered to consume excess ammonia via a metabolic pathway, e.g., an arginine biosynthesis pathway, a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, or a tryptophan biosynthesis pathway as described herein (an“ammonia conversion circuit”). In some embodiments, the genetically engineered bacteria comprise an arginine biosynthesis pathway and are capable of reducing excess ammonia. In some embodiments, the ammonia conversion circuit is under the control of an inducible promoter. In some embodiments, the ammonia conversion circuit is under the control of an oxygen level-dependent promoter, e.g., an FNR-inducible promoter. In some embodiments, the ammonia conversion circuit is under the control of a promoter induced by a molecule or metabolite associated with hepatic
encephalopathy, e.g., bilirubin, aspartate aminotransferase, alanine aminotransferase, ,transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, or manganese
[0329] In some embodiments, the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA membrane transport protein(s), e.g., GabP, and are capable of transporting GABA into the cell (a“GABA transport circuit”) (FIG. 41).
[0330] In some embodiments, the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA catabolism enzyme(s), e.g., GSST, SSDH, and/or COT (a“GABA metabolic circuit”) (FIG. 41). In some embodiments, the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing one or more GABA membrane transport protein(s), e.g., GabP, and one or more circuits for producing one or more GABA catabolism enzyme(s), e.g., GSST, SSDH, and/or COT (a“GABA metabolic circuit”) (FIG. 41).
[0331] In a more specific aspect, the genetically engineered bacteria comprise an ammonia conversion circuit, a GABA transport circuit, and a GABA metabolic circuit. In some embodiments, the ammonia conversion circuit, GABA transport circuit, and GABA metabolic circuit are under the control of the same promoter. In alternate embodiments, the ammonia conversion circuit, GABA transport circuit, and GABA metabolic circuit are under the control of different promoters. Exemplary promoters include any of the promoters disclosed herein. For example, in some embodiments, the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage. Non-limiting examples of molecules or metabolites include, 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, and manganese in their blood and intestines. In some embodiments, the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter. In some embodiments, the genetically engineered bacteria comprise a promoter 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.
[0332] The amino acid sequence of an exemplary GabP transporter is shown in Table 48. In some embodiments, the genetically engineered bacteria comprise the amino acid sequence of SEQ ID NO: 105 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 SEQ ID NO: 105 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise an amino 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 amino acid sequence of SEQ ID NO: 105 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 105 or a functional fragment thereof.
[0333] A non-limiting example of a polynucleotide sequence is shown in Table 49 (SEQ ID NO: 106).
[0334] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. [0335] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0336] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in
Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA is modified and/or mutated, e.g., to enhance stability, or increase GABA catalysis.
[0337] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA 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.
[0338] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences described herein for the consumption of ammonia.
[0339] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA 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.
[0340] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0341] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0342] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of tryptophan and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0343] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake and/or catabolism of GABA further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”)
[0344] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Manganese Transport
[0345] Manganese is a biologically important trace metal and is required for the survival of most living organisms. In mammals, manganese is excreted in the bile, but its disposal is affected by the impaired flow of bile from the liver to the duodenum (i.e., cholestasis) that accompanies liver failure. Similar to ammonia, elevated concentrations of manganese play a role in the development of hepatic encephalopathy (Rivera-Mancía et al., 2012). Astrocytes in the brain which detoxify ammonia in a reaction catalyzed by glutamine synthetase, require manganese as a cofactor and thus have a tendency to accumulate this metal (Aschner et al., 1999). In vitro studies have demonstrated that manganese can result in the inhibition of glutamate transport (Hazell and Norenberg, 1996), abnormalities in astrocyte morphology (Hazell et al., 2006), and increased cell volume (Rama Rao et al., 2007). Manganese and ammonia have also been shown to act synergistically in the pathogenesis of hepatic encephalopathy (Jayakumar et al., 2004).
[0346] Metal ion homeostasis in prokaryotic cells, which lack internal compartmentalization, is maintained by the tight regulation of metal ion flux across in cytoplasmic membrane (Jensen and Jensen, 2014). Manganese uptake in bacteria predominantly involves two major types of transporters: proton-dependent Nramp- related transporters, and/or ATP-dependent ABC transporters. The Nramp (Natural resistance-associated macrophage protein) transporter family was first described in plants, animals, and yeasts (Cellier et al., 1996), but MntH has since been characterized in several bacterial species (Porcheron et al., 2013). Selectivity of the Nramp1 transporter for manganese has been shown in metal accumulation studies, wherein overexpression of Staphylococcus aureus mntH resulted in increased levels of cell- associated manganese, but no accumulation of calcium, copper, iron, magnesium, or zinc (Horsburgh et al., 2002). Additionally, Bacillus subtilis strains comprising a mutation in the mntH gene exhibited impaired growth in metal-free medium that was rescued by the addition of manganese (Que and Helmann, 2000). The amino acid sequence of an exemplary MntH transporter is shown in Table 50. In some
embodiments, the genetically engineered bacteria comprise the amino acid sequence of SEQ ID NO: 107 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 SEQ ID NO: 107 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise an amino 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 amino acid sequence of SEQ ID NO: 107 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 107 or a functional fragment thereof. A non-limiting example of a polynucleotide sequence is shown in Table 51 (SEQ ID NO: 108).
[0347] High-affinity manganese uptake may also be mediated by ABC (ATP- binding cassette) transporters. Members of this transporter superfamily utilize the hydrolysis of ATP to fuel the import or export of diverse substrates, ranging from ions to macromolecules, and are well characterized for their role in multi-drug resistance in both prokaryotic and eukaryotic cells. Non-limiting examples of bacterial ABC transporters involved in manganese import include MntABCD (Bacilis subtilis, Staphylococcus aureus), SitABCD (Salmonella typhimurium, Shigella flexneri), PsaABCD (Streptococcus pneumoniae), and YfeABCD (Yersinia pestis) (Bearden and Perry, 1999; Kehres et al., 2002; McAllister et al., 2004; Zhou et al., 1999). The MntABCD transporter complex consists of three subunits, wherein MntC and MntD are integral membrane proteins that comprise the permease subunit mediate cation transport, MntB is the ATPase, and MntA binds and delivers manganese to the permease submit. Other ABC transporter operons, such as sitABCD, psaABCD, and yfeABCD, exhibit similar subunit organization and function (Higgins, 1992; Rees et al., 2009).
[0348] In some embodiments, the genetically engineered bacteria comprising an ammonia conversion circuit further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”) (FIG. 42).
[0349] In some embodiments, the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, and a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, and a GABA transport circuit. In some embodiments, the genetically engineered bacteria comprise an ammonia conversion circuit, a manganese transport circuit, a GABA transport circuit, and a GABA metabolic circuit. In some embodiments, the circuits are under the control of the same promoter. In alternate embodiments, the circuits are under the control of different promoters. In some embodiments, the genetically engineered bacteria of the invention comprise an oxygen level-dependent promoter induced by low-oxygen, microaerobic, or anaerobic conditions. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite, for example, a tissue-specific molecule or metabolite or a molecule or metabolite indicative of liver damage. Non-limiting examples of molecules or metabolites include, 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, and manganese in their blood and intestines. In some embodiments, the genetically engineered bacteria comprise a promoter induced by inflammation or an inflammatory response, e.g., RNS or ROS promoter. In some embodiments, the genetically engineered bacteria comprise a promoter 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.
[0350] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0351] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein. [0352] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table XI or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese is modified and/or mutated, e.g., to enhance stability, or increase manganese uptake or catalysis.
[0353] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the uptake of manganese 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.
[0354] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences described herein for the consumption of ammonia.
[0355] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese 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.
[0356] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0357] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0358] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese 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. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1. [0359] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the uptake of manganese further comprise a GABA transport circuit and/or a GABA metabolic circuit.
[0360] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance. Short chain Fatty Acids and Tryptophan Metabolites
[0361] One strategy in the treatment, prevention, and/or management of certain diseases, disorders, or conditions associated with hyperammonemia, including HE, 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.
[0362] For example, 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, e.g., in the context of hyperammonemia in HE, may be to provide a treatment which contains one or more of such metabolites.
[0363] For example, butyrate and other SCFA, e.g., derived from the
microbiota, are known to promote maintaining 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) anti-inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, Il-6, and IFN-gamma), and inhibition of NF-κB. Many of these actions of SCFAs in gut homeostatis can be ascribed to GPR43 and GPR109A, which 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.
[0364] In addition, a number of 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 Th17 cell activity, and the maintenance of intraepithelial lymphocytes and RORγt+ innate lymphoid cells.
[0365] 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.
[0366] In addition, some indole metabolites, e.g., indole 3-propionic acid (IPA), may exert their effect as an activating ligand of Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function, through downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial
Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296–310, August 21, 2014). As a result, indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health.
[0367] Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryprophan metabolites.
Acetate
[0368] In some embodiments, 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. In other embodiments, 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. In some embodiments, 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 embodiments, 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. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, 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. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid.
[0369] In E. coli Nissle, acetate is generated as an end product of fermentation. In E coli, 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. Without wishing to be bound by theory, prevention or reduction of flux through one or more metabolic arm(s) generating metabolites other than acetate, e.g. through mutation, deletion and/or inhibition of one or more gene(s) encoding key enzymes in these metabolic arms, results in an increase in production of acetate for NAD recycling. As disclosed herein, e.g., in Example 20, 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).
[0370] 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. E. coli carrying ldhA mutations show no observable growth defect and can still ferment sugars to a variety of products other than lactate.
[0371] In some embodiments, 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.
[0372] 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 ethanol. Deletion of adhE has been employed to enhance production of certain metabolites industrially, including succinate, D-lactate, and polyhydroxyalkanoates (Singh et al, Manipulating redox and ATP balancing for improved production of succinate in E. coli.; Metab Eng. 2011 Jan;13(1):76-81; Zhou et al., Evaluation of genetic manipulation strategies on D-lactate production by
Escherichia coli, Curr Microbiol. 2011 Mar;62(3):981-9; Jian et al., Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways, Appl Microbiol Biotechnol. 2010 Aug;87(6):2247-56).
[0373] In some embodiments, 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.
[0374] 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. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA gene, thereby reducing or eliminating the activity of FrdA.
[0375] In some embodiments, 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.
[0376] In some embodiments, 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. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0377] In some embodiments, the genetically engineered bacteria comprising one or more of these mutations also comprise a butyrate cassette. [0378] In some embodiments, 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 acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0379] In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g., E. coli Nissle.
Without wishing to be bound by theory, 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.
[0380] Phosphate acetyltransferase (Pta) 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. The acetate formation from 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(1):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). [0381] In some embodiments, the genetically engineered bacteria produce lower amounts of acetate than the amounts produced by the wild type bacterium under the same conditions.
[0382] In some embodiments, 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. In some embodiments, 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.
[0383] In some embodiments, the genetically engineered bacteria further comprise one or more gene cassettes for the production of butyrate.
[0384] In some embodiments, 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% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. [0385] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of acetate is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0386] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0387] In any of the embodiments described above and elsewhere 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 constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of acetate is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, 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.
[0388] In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, 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.
[0389] In any of the embodiments described above and elsewhere 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 described herein for the consumption of ammonia.
[0390] In any of the embodiments described above and elsewhere 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 production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules known in the art or described herein.
[0391] In any of the embodiments described above and elsewhere 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. [0392] In any of the embodiments described above and elsewhere 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 propionate. In any of the embodiments described above and elsewhere 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 or catabolism of tryptophan and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere 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. In any of the embodiments described above and elsewhere 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 IL-22. In any of the embodiments described above and elsewhere 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 GLP2. In any of the embodiments described above and elsewhere 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 a satiety effector, e.g., GLP1.
[0393] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising one or more gene sequences for the production of acetate further comprise a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprising one or more gene sequences for the production of acetate further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0394] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Butyrate
[0395] In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 2 and Table 3). Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to, Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium, and Treponema. In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, 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, thiA1, 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, thiA1, hbd, crt2, pbt, and buk. In some embodiments, 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. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etfB3, etfA3, and thiA1 from
Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296. Alternatively, 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. Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from Peptoclostridium difficile and ter from Treponema denticola. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from E coli). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
[0396] In some embodiments, 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). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0397] Table 14 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes.
Table 14. Exemplary Butyrate Cassette Sequences
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0002
[0398] Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 15.
Table 15. Exemplary Polypeptide Sequences for Butyrate Production
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
[0399] 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. In some embodiments, because 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-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from Treponema denticola, which can functionally replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from Peptoclostridium difficile. In this embodiment, the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and ter, e.g., from Treponema denticola, and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites , in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose..
[0400] In some embodiments, the genetically engineered bacteria of the invention comprise thiA1, 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 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. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0401] The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd2, etfB3, etfA3, thiA1, hbd, and crt2, e.g., from
Peptoclostridium difficile, and tesB from E. Coli and produce butyrate in low-oxygen conditions, in the presence of 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. In one embodiment, the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from Treponema denticola, thiA1, hbd, crt2, pbt, and buk, e.g., from Peptoclostridium difficile, and tesB from E. Coli , and produce butyrate in low-oxygen conditions, in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0402] In some embodiments, 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. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis. In some embodiments, local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders.
[0403] In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 39. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 39. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 39. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 39. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. Accordingly, in one embodiment, the bcd2 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: 39. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 39. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 39.
[0404] In one embodiment, the etfB3 gene has at least about 80% identity with SEQ ID NO: 40. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 40. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 40. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 40. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. Accordingly, in one embodiment, 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: 40. In another embodiment, the etfB3 gene comprises the sequence of SEQ ID NO: 40. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 40.
[0405] In one embodiment, the etfA3 gene has at least about 80% identity with SEQ ID NO: 41. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 41. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 41. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 41. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. Accordingly, in one embodiment, 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: 41. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 41. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 41.
[0406] In one embodiment, the thiA1 gene has at least about 80% identity with SEQ ID NO: 42. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 42. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 42. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 42. In another embodiment, the thiA1 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42. Accordingly, in one embodiment, the thiA1 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: 42. In another embodiment, the thiA1 gene comprises the sequence of SEQ ID NO: 42. In yet another embodiment the thiA1 gene consists of the sequence of SEQ ID NO: 42.
[0407] In one embodiment, the hbd gene has at least about 80% identity with SEQ ID NO: 43. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 43. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 43. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 43. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43. 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: 43. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 43. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 43.
[0408] In one embodiment, the crt2 gene has at least about 80% identity with SEQ ID NO: 44. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 44. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 44. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 44. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. 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: 44. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 44. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 44.
[0409] In one embodiment, the pbt gene has at least about 80% identity with SEQ ID NO: 45. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 45. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 45. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 45. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. 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: 45. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 45. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 45.
[0410] In one embodiment, the buk gene has at least about 80% identity with SEQ ID NO: 46. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 46. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 46. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 46. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46. 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: 46. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 46. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 46.
[0411] In one embodiment, the ter gene has at least about 80% identity with SEQ ID NO: 47. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 47. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 47. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 47. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. 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: 47. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 47. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 47.
[0412] In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. Accordingly, in one embodiment, 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: 48. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 48. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 48.
[0413] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Bcd2. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 164. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 164. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 164. Accordingly, in some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 164. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 164. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 164.
[0414] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding etfB3. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 165. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 165. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 165. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 165. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 165. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 165.
[0415] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) etfA3. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 166. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 166. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 166.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 166. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 166. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 166.
[0416] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Ter. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 167. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 167. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 167. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 167. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 167.
[0417] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding ThiA. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 168. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 168. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 168. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 168. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 168. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 168.
[0418] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) Hbd. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 169. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 169. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 169.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 169. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 169.
[0419] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Crt2: Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 170. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 170. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 170.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 170. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 170. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 170.
[0420] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Pbt. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 171. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 171. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 171. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 171. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 171. [0421] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Buk. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 172. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 172. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 172. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 172. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 172. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 172.
[0422] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TesB. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 173. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 173. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 173. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 173. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 173. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 173.
[0423] In some embodiments, 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. [0424] To improve or maintain acetate production, while maintaining or improving levels of butyrate production, one or more targeted deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more butyrate- producing cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE genes.
[0425] In some embodiments, the genetically engineered bacteria comprise one or more butyrate producing cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene.
[0426] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous adhE gene. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA gene. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
[0427] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk 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 ter-thiA1-hbd-crt2-pbt- buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk 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 ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) 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) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0428] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB 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 ter- thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB 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 ter-thiA1-hbd- crt2-tesB gene cassette(s) 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) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes.
[0429] In some embodiments, 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 acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0430] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0431] In certain situations, the need may arise to prevent and/or reduce acetate production of an engineered or naturally occurring strain, e.g., E. coli Nissle, while maintaining high levels of butyrate production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for butyrate production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for butyrate synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes.
[0432] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes.
[0433] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and ldhA genes.
[0434] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk 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) comprising one or more ter- thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk 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) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) 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) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk 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) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) 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) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt- buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0435] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and ldhA genes.
[0436] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB 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) comprising one or more ter-thiA1-hbd- crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB 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) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) 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) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB 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) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) 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) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter- thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes.
[0437] In some embodiments, 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% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0438] In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%,6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%,25% to 30%, 30% to 35%, 35% to 40%,40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0439] In some embodiments, 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, alone or in combination with various mutations in genes of the mixed acid fermentation pathway, as described herein. In some embodiments, 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 some embodiments, the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation. In some embodiments, 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. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose.
[0440] In one embodiment, 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.
[0441] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0442] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0443] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate is modified and/or mutated, e.g., to enhance stability, or increase butyrate production.
[0444] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of butyrate 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.
[0445] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences described herein for the consumption of ammonia.
[0446] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate 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.
[0447] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate.
[0448] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate 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. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of butyrate further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0449] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising one or more gene sequences for the production of butyrate further comprise a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprising one or more gene sequences for the production of butyrate further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0450] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Propionate
[0451] In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing an anti-inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes.
[0452] In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 16A, Table 16B, and Table 16C). 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. In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from R. sphaeroides, which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI in E coli, yhdH is used. This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, and optionally further comprise tesB. In another embodiment, 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, and ygfH converts propionylCoA into propionate and succinate into succinylCoA.
[0453] 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). There are three genes which encode methylmalonyl-CoA
carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl- CoA to propionyl-CoA.
[0454] The genes may be codon-optimized, and translational and transcriptional elements may be added. Table 16A, B, and C lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 16D lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes. Table 16A. Propionate Cassette Sequences (Acrylate Pathway)
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
ATTATGATCTTCGAAGTTGAAGGCGCAGCGCCTGCGGCAGCTCC
Figure imgf000203_0001
GGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTA
Figure imgf000204_0001
ATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGAT
Figure imgf000205_0001
AACCTGCTGGCTGCCGCTGTTAATGCCGCTCGCGTTCGCGCCA
Figure imgf000206_0001
GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA
Figure imgf000207_0001
Figure imgf000208_0001
GATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGAC
Figure imgf000209_0001
Figure imgf000210_0001
TCAGACCCGCCCGGCGAAGAAGCATGGAAACTTCCCCTGCTG
Figure imgf000211_0001
[0455] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 16A (SEQ ID NO: 49-62 SEQ ID NO: 174, and SEQ ID NO: 48) 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 16A (SEQ ID NO: 49-62 SEQ ID NO: 174, and SEQ ID NO: 48) or a functional fragment thereof. In some embodiments, 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 16A (SEQ ID NO: 49-62 SEQ ID NO: 174, and SEQ ID NO: 48) 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 16A (SEQ ID NO: 49-62 SEQ ID NO: 174, and SEQ ID NO: 48) or a functional fragment thereof.
[0456] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 16B (SEQ ID NO: 175- SEQ ID NO: 178) 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 16B (SEQ ID NO: 175- SEQ ID NO: 178) or a functional fragment thereof. In some embodiments, 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 16B (SEQ ID NO: 175- SEQ ID NO: 178) 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 16B (SEQ ID NO: 175- SEQ ID NO: 178) or a functional fragment thereof.
[0457] In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 16C (SEQ ID NO: 179- SEQ ID NO: 184) 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 16C (SEQ ID NO: 179- SEQ ID NO: 184) or a functional fragment thereof. In some embodiments, 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 16C (SEQ ID NO: 179- SEQ ID NO: 184) 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 16C (SEQ ID NO: 179- SEQ ID NO: 184) or a functional fragment thereof.
[0458] Table 16D lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cassette(s) of the genetically engineered bacteria.
Table 16D. Polypeptide Sequences for Propionate Synthesis
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
[0459] In one embodiment, the bacterial cell comprises a non-native or heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a non-native or heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, 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. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette.
[0460] Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, 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, Megasphaera elsdenii, or Prevotella ruminicola.
[0461] In one embodiment, 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. 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). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat autoimmune disease, such as IBD.
[0462] The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, 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. For example, 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. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N-terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment.
[0463] As used herein, the term“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. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).
[0464] 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. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing 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, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).
[0465] In some embodiments, 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.
[0466] In one embodiment, 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.
[0467] In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from Clostridium propionicum. In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate
embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, and optionally further comprise tesB. The genes may be codon-optimized, and translational and transcriptional elements may be added.
[0468] In one embodiment, the pct gene has at least about 80% identity with SEQ ID NO: 49. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 49. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 49. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 49. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. Accordingly, in one embodiment, the pct gene has at least about 80%, 821%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 921%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. In another embodiment, the pct gene comprises the sequence of SEQ ID NO: 49. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 49. [0469] In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 50. In another embodiment, the lcdA gene has at least about 85% identity with SEQ ID NO: 50. In one embodiment, the lcdA gene has at least about 90% identity with SEQ ID NO: 50. In one embodiment, the lcdA gene has at least about 95% identity with SEQ ID NO: 50. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 822%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 922%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 50. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO: 50. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO: 50.
[0470] In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 51. In another embodiment, the lcdB gene has at least about 85% identity with SEQ ID NO: 51. In one embodiment, the lcdB gene has at least about 90% identity with SEQ ID NO: 51. In one embodiment, the lcdB gene has at least about 95% identity with SEQ ID NO: 51. In another embodiment, the lcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. Accordingly, in one embodiment, the lcdB gene has at least about 80%, 81%, 82%, 823%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 923%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 51. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO: 51. In yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO: 51.
[0471] In one embodiment, the lcdC gene has at least about 80% identity with SEQ ID NO: 52. In another embodiment, the lcdC gene has at least about 85% identity with SEQ ID NO: 52. In one embodiment, the lcdC gene has at least about 90% identity with SEQ ID NO: 52. In one embodiment, the lcdC gene has at least about 95% identity with SEQ ID NO: 52. In another embodiment, the lcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 52. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 824%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 924%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 52. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO: 52. In yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO: 52. [0472] In one embodiment, the etfA gene has at least about 80% identity with SEQ ID NO: 53. In another embodiment, the etfA gene has at least about 825% identity with SEQ ID NO: 53. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the etfA gene has at least about 925% identity with SEQ ID NO: 53. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. Accordingly, in one embodiment, the etfA gene has at least about 80%, 81%, 82%, 83%, 84%, 825%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 925%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. In another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 53. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 53.
[0473] In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 54. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the acrB gene has at least about 926%, 97%, 98%, or 99% identity with SEQ ID NO: 54. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 826%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 926%, 97%, 98%, or 99% identity with SEQ ID NO: 54. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 54. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 54.
[0474] In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the acrC gene has at least about 96%, 927%, 98%, or 99% identity with SEQ ID NO: 55. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 827%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 927%, 98%, or 99% identity with SEQ ID NO: 55. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 55. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 55. [0475] In one embodiment, the thrAfbr gene has at least about 280% identity with SEQ ID NO: 56. In another embodiment, the thrAfbr gene has at least about 285% identity with SEQ ID NO: 56. In one embodiment, the thrAfbr gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the thrAfbr gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the thrAfbr gene has at least about 96%, 97%, 928%, or 99% identity with SEQ ID NO: 56. Accordingly, in one embodiment, the thrAfbr gene has at least about 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 2828%, 289%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 928%, or 99% identity with SEQ ID NO: 56. In another embodiment, the thrAfbr gene comprises the sequence of SEQ ID NO: 56. In yet another embodiment the thrAfbr gene consists of the sequence of SEQ ID NO: 56.
[0476] In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the thrB gene has at least about 290% identity with SEQ ID NO: 57. In one embodiment, the thrB gene has at least about 295% identity with SEQ ID NO: 57. In another embodiment, the thrB gene has at least about 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 57. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 829%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 57. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 57.
[0477] In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. Accordingly, in one embodiment, 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: 58. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 58. [0478] In one embodiment, the ilvAfbr gene has at least about 80% identity with SEQ ID NO: 59. In another embodiment, the ilvAfbr gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the ilvAfbr gene has at least about 90% identity with SEQ ID NO: 59. In one embodiment, the ilvAfbr gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the ilvAfbr gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. Accordingly, in one embodiment, the ilvAfbr 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 ilvAfbr gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the ilvAfbr gene consists of the sequence of SEQ ID NO: 59.
[0479] In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 60. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 60. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. Accordingly, in one embodiment, 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: 60. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 60.
[0480] In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 61. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 61. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. Accordingly, in one embodiment, 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: 61. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 61. [0481] In one embodiment, the lpd gene has at least about 80% identity with SEQ ID NO: 62. In another embodiment, the lpd gene has at least about 85% identity with SEQ ID NO: 62. In one embodiment, the lpd gene has at least about 90% identity with SEQ ID NO: 62. In one embodiment, the lpd gene has at least about 95% identity with SEQ ID NO: 62. In another embodiment, the lpd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 62. Accordingly, in one embodiment, the lpd 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: 62. In another embodiment, the lpd gene comprises the sequence of SEQ ID NO: 62. In yet another embodiment the lpd gene consists of the sequence of SEQ ID NO: 62.
[0482] In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 48. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 48. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. Accordingly, in one embodiment, 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: 48. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 48. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 48.
[0483] In one embodiment, the acuI gene has at least about 80% identity with SEQ ID NO: 174. In another embodiment, the acuI gene has at least about 85% identity with SEQ ID NO: 174. In one embodiment, the acuI gene has at least about 90% identity with SEQ ID NO: 174. In one embodiment, the acuI gene has at least about 95% identity with SEQ ID NO: 174. In another embodiment, the acuI gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 174. Accordingly, in one embodiment, the acuI 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: 174. In another embodiment, the acuI gene comprises the sequence of SEQ ID NO: 174. In yet another embodiment the acuI gene consists of the sequence of SEQ ID NO: 174. [0484] In one embodiment, the sbm gene has at least about 80% identity with SEQ ID NO: 175. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 175. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 175. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 175. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 175.0. Accordingly, in one embodiment, 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: 175. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 175. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 175.
[0485] In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 176. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 176. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 176. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 176. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 176. Accordingly, in one embodiment, 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: 176. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 176. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 176.
[0486] In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 177. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 177. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 177. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 177. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 177.. Accordingly, in one embodiment, 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: 177. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 177. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 177. [0487] In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 178. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 178. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 178. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 178. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 178. Accordingly, in one embodiment, 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: 178. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 178. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 178.
[0488] In one embodiment, the mutA gene has at least about 80% identity with SEQ ID NO: 179. In another embodiment, the mutA gene has at least about 85% identity with SEQ ID NO: 179. In one embodiment, the mutA gene has at least about 90% identity with SEQ ID NO: 179. In one embodiment, the mutA gene has at least about 95% identity with SEQ ID NO: 179. In another embodiment, the mutA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 179. Accordingly, in one embodiment, the mutA 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: 179. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO: 179. In yet another embodiment the mutA gene consists of the sequence of SEQ ID NO: 179.
[0489] In one embodiment, the mutB gene has at least about 80% identity with SEQ ID NO: 180. In another embodiment, the mutB gene has at least about 85% identity with SEQ ID NO: 180. In one embodiment, the mutB gene has at least about 90% identity with SEQ ID NO: 180. In one embodiment, the mutB gene has at least about 95% identity with SEQ ID NO: 180. In another embodiment, the mutB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 180. Accordingly, in one embodiment, the mutB 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: 180. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO: 180. In yet another embodiment the mutB gene consists of the sequence of SEQ ID NO: 180. [0490] In one embodiment, the GI 18042134 gene has at least about 80% identity with SEQ ID NO: 181. In another embodiment, the GI 18042134 gene has at least about 85% identity with SEQ ID NO: 181. In one embodiment, the GI 18042134 gene has at least about 90% identity with SEQ ID NO: 181. In one embodiment, the GI 18042134 gene has at least about 95% identity with SEQ ID NO: 181. In another embodiment, the GI 18042134 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 181. Accordingly, in one embodiment, the GI 18042134 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: 181. In another embodiment, the GI 18042134 gene comprises the sequence of SEQ ID NO: 181. In yet another embodiment the GI 18042134 gene consists of the sequence of SEQ ID NO: 181.
[0491] In one embodiment, the mmdA gene has at least about 80% identity with SEQ ID NO: 182. In another embodiment, the mmdA gene has at least about 85% identity with SEQ ID NO: 182. In one embodiment, the mmdA gene has at least about 90% identity with SEQ ID NO: 182. In one embodiment, the mmdA gene has at least about 95% identity with SEQ ID NO: 182. In another embodiment, the mmdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 182.
Accordingly, in one embodiment, the mmdA 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: 182. In another embodiment, the mmdA gene comprises the sequence of SEQ ID NO: 182. In yet another embodiment the mmdA gene consists of the sequence of SEQ ID NO: 182.
[0492] In one embodiment, the PFREUD_188870 gene has at least about 80% identity with SEQ ID NO: 183. In another embodiment, the PFREUD_188870 gene has at least about 85% identity with SEQ ID NO: 183. In one embodiment, the PFREUD_188870 gene has at least about 90% identity with SEQ ID NO: 183. In one embodiment, the PFREUD_188870 gene has at least about 95% identity with SEQ ID NO: 183. In another embodiment, the PFREUD_188870 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 183. Accordingly, in one embodiment, the PFREUD_188870 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: 183. In another embodiment, the PFREUD_188870 gene comprises the sequence of SEQ ID NO: 183. In yet another embodiment the PFREUD_188870 gene consists of the sequence of SEQ ID NO: 183.
[0493] In one embodiment, the Bccp gene has at least about 80% identity with SEQ ID NO: 184. In another embodiment, the Bccp gene has at least about 85% identity with SEQ ID NO: 184. In one embodiment, the Bccp gene has at least about 90% identity with SEQ ID NO: 184. In one embodiment, the Bccp gene has at least about 95% identity with SEQ ID NO: 184. In another embodiment, the Bccp gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 184. Accordingly, in one embodiment, the Bccp 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: 184. In another embodiment, the Bccp gene comprises the sequence of SEQ ID NO: 184. In yet another embodiment the Bccp gene consists of the sequence of SEQ ID NO: 184.
[0494] In one embodiment, 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: 185 through SEQ ID NO: 209. 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: 185 through SEQ ID NO: 209. 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: 185 through SEQ ID NO: 209. In one embodiment, 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: 185 through SEQ ID NO: 209. 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: 185 through SEQ ID NO: 209. Accordingly, in one embodiment, 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: 185 through SEQ ID NO: 209. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria 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: 185 through SEQ ID NO: 209. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or more of SEQ ID NO: 185 through SEQ ID NO: 209.
[0495] In some embodiments, 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.
[0496] To improve acetate production, while maintaining high levels of propionate production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non- limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more propionate cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE.
[0497] In some embodiments, 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 ldhA gene, the frdA gene and the adhE gene.
[0498] 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 one or more endogenous genes selected from in the ldhA 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 ldhA 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. 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 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 ldhA 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 ldhA genes 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 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 ldhA, the 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 one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes.
[0499] In some embodiments, 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 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 ldhA gene. In some
embodiments, 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. In some embodiments, 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. In some embodiments, 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 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 ldhA and frdA genes. In some embodiments, 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 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 ldhA genes and adhE genes. In some embodiments, 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. In some embodiments, 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. 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 ldhA, the frdA, and adhE genes.
[0500] In some embodiments, 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 acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0501] In some embodiments, 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. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0502] In certain situations, 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. Without wishing to be bound by theory, 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. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid
fermentation, 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.
[0503] 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 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 pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE 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 in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of 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. 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, 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, ldhA, frdA, and adhE genes.
[0504] In some embodiments, 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. 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 and/or deletion in the endogenous pta gene. 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 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. 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 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. 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 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 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 ldhA genes.
[0505] 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 and ldhA 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 frdA 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 and frdA 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 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) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) 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) selected from sbm, ygfD, ygfG, and/or ygfHand further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some
embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) 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. 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, ldhA, 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, ldhA, frdA, and adhE genes.
[0506] In some embodiments, 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% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0507] In some embodiments, 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. In yet another embodiment, 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. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions.
[0508] In some embodiments, 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. In some embodiments, 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. In some embodiments, the local production of propionate reduces food intake and improves gut barrier function and reduces inflammation In some embodiments, 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. In some embodiments, 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. [0509] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0510] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0511] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of propionate is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, 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.
[0512] In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, 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.
[0513] In any of the embodiments described above and elsewhere 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 described herein for the consumption of ammonia.
[0514] In any of the embodiments described above and elsewhere 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 production of one or more gut barrier enhancer molecules and/or anti- inflammatory molecules known in the art or described herein.
[0515] In any of the embodiments described above and elsewhere 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. [0516] In any of the embodiments described above and elsewhere 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 acetate. In any of the embodiments described above and elsewhere 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 or catabolism of tryptophan and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere 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. In any of the embodiments described above and elsewhere 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 IL-22. In any of the embodiments described above and elsewhere 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 GLP2. In any of the embodiments described above and elsewhere 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 a satiety effector, e.g., GLP1.
[0517] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising one or more gene sequences for the production of propionate further comprise a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprising one or more gene sequences for the production of propionate further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0518] In any of the embodiments described above and herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Tryptophan and Tryptophan Metabolism
Kynurenine [0519] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. Kynurenine is a metabolite produced in the first, rate-limiting step of tryptophan catabolism. This step involves the conversion of tryptophan to kynurenine, and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-dioxygenase (IDO-1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily localized to the liver (Alvarado et al., 2015). Biopsies from human patients with IBD show elevated levels of IDO-1 expression compared to biopsies from healthy individuals, particularly near sites of ulceration (Ferdinande et al., 2008; Wolf et al., 2004). IDO-1 enzyme expression is similarly upregulated in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced mouse models of IBD; inhibition of IDO-1 significantly augments the inflammatory response caused by each inducer (Ciorba et al., 2010; Gurtner et al., 2003; Matteoli et al., 2010). Kynurenine has also been shown to directly induce apoptosis in neutrophils (El-Zaatari et al., 2014). Together, these observations suggest that IDO-1 and kynurenine play a role in limiting inflammation. The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some
embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions.
[0520] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible
transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine- oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene, genes, or gene cassettes for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some
embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions
Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites Tryptophan and the Kynurenine Pathway
[0521] Tryptophan (TRP) is an essential amino acid that, after consumption, is either incorporated into proteins via new protein synthesis, or converted a number of biologically active metabolites with a number of differing roles in health and disease (Perez-De La Cruz et al., 2007 Kynurenine Pathway and Disease: An Overview;
CNS&Neurological Disorders -Drug Targets 2007, 6,398-410). Along one arm of tryptophan catabolism, trytophan is converted to the neurotransmitter serotonin (5- hydroxytryptamine, 5-HT) by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. A large share of tryptophan, however, is metabolized to a number of bioactive metabolites, collectively called kynurenines, along a second arm called the kynurenine pathway (KP). In the first step of catabolism, TRP is converted to Kynurenine, (KYN), which has well-documented immune suppressive functions in several types of immune cells, and has recently been shown to be an activating ligand for the arylcarbon receptor (AhR; also known as dioxin receptor). KYN was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival. In the gut, kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism.
[0522] More recently, additional tryptophan metabolites, collectively termed “indoles”, herein, including for example, indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc. which are generated by the microbiota, some by the human host, some from the diet, which are also able to function as AhR agonists, see e.g., Table 17 and elsewhere herein, and Lama et al., Nat Med. 2016 Jun;22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands.
[0523] Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in additiona to kynurenine, tryptophan metabolites L-kynurenine, 6-formylindolcarbazole (FICZ, a photoproduct of TRP), and KYNA are have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-κB subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT associated transcription of IL-6, which induced autocrine activation of IDO1 via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated immunosuppression enables the immune escape of tumor cells.
[0524] In the gut, tryptophan may also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell response and promotion of Treg cells.
[0525] The rate-limiting conversion of TRP to KYN may be mediated by either of two forms of indoleamine 2, 3-dioxygenase (IDO) or by tryptophan 2,3-dioxygenase (TDO). One characteristic of TRP metabolism is that the rate-limiting step of the catalysis from TRP to KYN is generated by both the hepatic enzyme tryptophan 2,3- dioxygenase (TDO) and the ubiquitous expressed enzyme IDO1. TDO is essential for homeostasis of TRP concentrations in organisms and has a lower affinity to TRP than IDO1. Its expression is activated mainly by increased plasma TRP concentrations but can also be activated by glucocorticoids and glucagon. The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (as shown in the figures and the examples, and Sci Transl Med. 2013 July 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of TRP:KYN. Along one pathway, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors and can also bind AHR and also GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMDA)-receptors, and others. Along a third pathway of the KP, KYN can be converted to anthranilic acid (AA) and further downstream quinolinic acid (QUIN), which is a glutamate receptor agonist and has a neurotoxic role.
[0526] Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of a number of diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning trypophan and tryptophan metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites. and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases.
Other Indole Tryptophan Metabolites [0527] In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles; Nature Scientific Reports 5:12689).
[0528] In the gastrointestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372–385, August 22, 2013). AHR is essential for IL-22-production in the intestinal lamina propria (Lee et al., Nature Immunology 13, 144–151 (2012); AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch).
[0529] Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of Il-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function.
[0530] Table 17 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure. Thus, in some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes for the production of one or more metabolites listed in Table 17 or shown in FIG. 87B, FIG. 88A, and/or FIG.88B. Table 17. Indole Tryptophan Metabolites
Figure imgf000247_0001
[0531] In addition, some indole metabolites may exert their effect through Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nr1i2-/-) mice showed a distinctly ‘‘leaky’’ gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296–310, August 21, 2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo.
[0532] As a result of PXR agonism, indole levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. I.e., low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinal barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health.
[0533] In other embodiments, IPA producing circuits comprise enzymes depicted and described in the figures and elsewhere herein. Thus, in some
embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes selected from TrpDH: tryptophan dehydrogenase (e.g., from Nostoc punctiforme NIES-2108); FldH1/FldH2: indole-3-lactate dehydrogenase (e.g., from Clostridium sporogenes); FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase (e.g., from Clostridium sporogenes); FldBC: indole-3-lactate dehydratase, (e.g., from Clostridium sporogenes); FldD: indole-3-acrylyl-CoA reductase (e.g., from Clostridium sporogenes); AcuI: acrylyl-CoA reductase (e.g., from Rhodobacter sphaeroides); lpdC: Indole-3-pyruvate decarboxylase (e.g., from Enterobacter cloacae); lad1: Indole-3-acetaldehyde dehydrogenase (e.g., from Ustilago maydis); and Tdc: Tryptophan decarboxylase (e.g., from Catharanthus roseus or from Clostridium sporogenes). In some embodiments, the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the production of one or more of the following: indole-3- propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis(TrA).
[0534] 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 (FldA ) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (FldD ) and acrylyl-CoA reductase (AcuI) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC ) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate
decarboxylase (lpdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3- acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3- acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA).
[0535] Although microbial degradation of tryptophan to indole-3-propionate has been shown in a number of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr 1;107(3):283-8), to date, the bacterial entire biosynthetic pathway from tryptophan to IPA is unknown. In Clostridium sporogenes, tryptophan is catabolized via indole-3- pyruvate, indole-3-lactate, and indole-3-acrylate to indole-3-propionate (O’Neill and DeMoss, Tryptophan transaminase from Clostridium sporogenes, Arch Biochem Biophys. 1968 Sep 20;127(1):361-9). Two enzymes that have been purified from C. sporogenes are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from Clostridium sporogenes, Can J Microbiol. 1968 Apr;14(4):429-35). Lactococcus lactis, catabolizes tryptophan by an
aminotransferase to indole-3-pyruvate. In Lactobacillus casei and Lactobacillus helveticus tryptophan is also catabolized to indole-3-lactate through successive transamination and dehydrogenation (see, e.g., Tryptophan catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein).
[0536] L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g., Clostridium sporogenes or Lactobacillus casei) converts L-tryptophan and 2-oxoglutarate to (indol- 3yl)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g., Clostridium sporogenes orLactobacillus casei) converts (indol-3yl) pyruvate and NADH and H+ to indole-3 lactate and NAD+.
[0537] In some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes selected from tryptophan transaminase (e.g., from C. sporogenes) and/or indole-3-lactate dehydrogenase (e.g., from C.
sporogenes), and/or indole-3-pyruvate aminotransferase (e.g., from Lactococcus lactis). In other embodiments, such enzymes encoded by the bacteria are from Lactobacillus casei and/or Lactobacillus helveticus. [0538] In other embodiments, IPA producing circuits comprise enzymes depicted and described in FIG. 97 and FIG. 98 and elsewhere herein.
[0539] In some embodiments, the bacteria comprise gene sequence for producing one or more tryptophan metabolites, e.g.,“indoles”. In some embodiments, the bacteria comprise gene sequence for producing and indole selected from indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ. In some embodiments, the bacteria comprise gene sequence for producing an indole that functions as an AhR agonist, see e.g., Table 17, FIG. 87B, FIG. 88A, and/or FIG. 88B..
[0540] In some embodiments, the bacteria comprise any one or more of the circuits described and depicted in the figures and examples. Methoxyindole pathway, Serotonin and Melatonin
[0541] The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tph1 or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5-HTP). Subsequently, 5-HTP undergoes decarboxylation to serotonin.
[0542] The majority (95%–98%) of total body serotonin is found in the gut (Berger et al., 2009). Peripheral serotonin acts autonomously on many cells, tissues, and organs, including the cardiovascular, gastrointestinal, hematopoietic, and immune systems as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin functions as a ligand for any of 15 membrane-bound mostly G protein-coupled serotonin receptors (5-HTRs) that are involved in various signal transduction pathways in both CNS and periphery. Intestinal serotonin is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. Gut microbiota are interconnected with serotonin signaling and are for example capable of increasing serotonin levels through host serotonin production (Jano et al., Cell. 2015 Apr 9;161(2):264-76. doi:
10.1016/j.cell.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis). [0543] Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders, including IBD.
[0544] In some embodiments, the engineered bacteria comprise gene sequence encoding one or more tryptophan hydroxylase genes (Tph1 or Tph2). In some embodiments, the engineered bacteria further comprise gene sequence for
decarboxylating 5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for the production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered bacteria comprise gene sequence for the production of seratonin.
[0545] In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g., decrease or increase serotonin levels, e.g, in the gut and in the circulation. In certain embodiments, the genetically engineered bacteria influence serotonin synthesis, release, and/or degradation. In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to improve gut barrier function, modulate the inflammatory status, otherwise ameliorate symptoms of A gastrointestinal disorder or inflammatory disorder. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In some embodiments, the genetically engineered bacteria release serotonin into the environment, e.g., the gut. In some embodiments, the genetically engineered modulate or influence serotonin levels produced by the host. In some embodiments, the genetically engineered bacteria counteract microbiota which are responsible for altered serotonin function in many metabolic diseases.
[0546] In some embodiments, the genetically engineered bacteria comprise gene sequence encoding tryptophan hydroxylase (TpH (1and/or2)) and/or l-amino acid decarboxylase, e.g. for the treatment of constipation-associated metabolic disorders. In some embodiments, the genetically engineered bacteria comprise genetic cassettes which allow trptophan uptake and catalysis, reducing trptophan availability for serotonin synthesis (serotonin depletion). In some embodiments, the genetically engineered bacteria comprise cassettes which promote serotonin uptake from the environment, e.g., the gut, and serotonin catalysis.
[0547] Additionally, serotonin also functions a substrate for melatonin biosynthesis. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. [0548] In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d-erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As a non-limiting example, the cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). "Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources". Journal of Chemical Biology 5 (1): 5–17. doi:10.1007/s12154-011-0064-8.
[0549] In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase (TPH), hydroxyl-O-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic– amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents.
Exemplary Tryptophan and Tryptophan Metabolite Circuits Decreasing Exogenous Tryptophan
[0550] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria.
[0551] The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in E. coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole, et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93).
[0552] In some embodiments, the engineered bacteria comprise gene
sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. In one
embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the
Escherichia coli aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the Escherichia coli tnaB gene.
[0553] In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to, Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or Corynebacterium glutamicum. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is Escherichia coli strain Nissle.
[0554] Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein.
[0555] In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another
embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty- fold, or fifty-fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
[0556] In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine metabolites, or indole tryptophan metabolites as shown in Table 17, FIG. 87B, FIG. 88A, and/or FIG. 88B.
[0557] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52;
producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.
[0558] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein. In some embodiments, expression of the gene sequences(s) is driven by an inducible promoter, described in more detail herein. In some embodiments, the expression of the gene sequences(s) is driven by a constitutive promoter. Increasing Kynurenine
[0559] In some embodiments, the genetically engineered bacteria are capable of producing kynurenine.
[0560] In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013.
[0561] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non-limiting example of such gene sequence(s) are shown the figures and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S.
cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine-- oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.
[0562] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.
[0563] In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in the figures and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0564] The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some
embodiments, the genetically engineered bacteria are capable of producing kynurenine 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 and others described herein. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0565] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some
embodiments the enzymes are derived from the species listed in table S7 of Vujkovic- Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety.
[0566] In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine 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 and others described herein.
[0567] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Increasing Tryptophan
[0568] In some embodiments, the genetically engineered microorganisms of the present disclosure are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in the figures.
[0569] In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of E. coli. (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of B. subtilis. (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC- F, trypB, and trpA genes from E. Coli. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis.
[0570] Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from E. Coli and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from B. subtilis and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes.
[0571] In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 10). Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+ to NADH. As part of Tryptophan biosynthesis, E. coli uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved.
[0572] In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 10
[0573] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function.
[0574] In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 10.
[0575] The inner membrane protein YddG of Escherichia coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over- express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.
[0576] In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, liver damage, and/or metabolic disease, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[04] Table 18A and 18B lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure.
Table 18A. Tr to han S nthesis Cassette Se uences
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
[0577] In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 24A. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 18A. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 18A. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 18A. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of
Table 18A. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which 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 one or more sequences of Table 18A. In another embodiment, the genetically engineered bacteria comprise the sequence of Table 18A. In one embodiment, the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 18A.
[0578] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a Tryptophan operon. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 210. In another embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which has at least about 85% identity with SEQ ID NO: 210. In one embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which has at least about 90% identity with SEQ ID NO: 210. In one embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which has at least about 95% identity with SEQ ID NO: 210. In another embodiment, the Tryptophan operon gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 210.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a Tryptophan operon gene sequence which 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: 210. In another embodiment, the genetically engineered bacteria comprise the Tryptophan operon gene sequence of SEQ ID NO: 210. In yet another embodiment the genetically engineered bacteria comprise a Tryptophan operon gene sequence which consists of the sequence of SEQ ID NO: 210.
[0579] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpE. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 211. In another embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 85% identity with SEQ ID NO: 211. In one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 90% identity with SEQ ID NO: 211. In one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which has at least about 95% identity with SEQ ID NO: 211. In another embodiment, the TrpE gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 211.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpE gene sequence which 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: 211. In another embodiment, the genetically engineered bacteria comprise the TrpE gene sequence of SEQ ID NO: 211. In yet another embodiment the genetically engineered bacteria comprise a TrpE gene sequence which consists of the sequence of SEQ ID NO: 211.
[0580] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpD. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 212. In another embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 85% identity with SEQ ID NO: 212. In one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 90% identity with SEQ ID NO: 212. In one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which has at least about 95% identity with SEQ ID NO: 212. In another embodiment, the TrpD gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 212.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpD gene sequence which 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: 212. In another embodiment, the genetically engineered bacteria comprise the TrpD gene sequence of SEQ ID NO: 212. In yet another embodiment the genetically engineered bacteria comprise a TrpD gene sequence which consists of the sequence of SEQ ID NO: 212.
[0581] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpC. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 213. In another embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 85% identity with SEQ ID NO: 213. In one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 90% identity with SEQ ID NO: 213. In one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which has at least about 95% identity with SEQ ID NO: 213. In another embodiment, the TrpC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 213.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpC gene sequence which 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: 213. In another embodiment, the genetically engineered bacteria comprise the TrpC gene sequence of SEQ ID NO: 213. In yet another embodiment the genetically engineered bacteria comprise a TrpC gene sequence which consists of the sequence of SEQ ID NO: 213.
[0582] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpB. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 214. In another embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 85% identity with SEQ ID NO: 214. In one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 90% identity with SEQ ID NO: 214. In one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which has at least about 95% identity with SEQ ID NO: 214. In another embodiment, the TrpB gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 214.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpB gene sequence which 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: 214. In another embodiment, the genetically engineered bacteria comprise the TrpB gene sequence of SEQ ID NO: 214. In yet another embodiment the genetically engineered bacteria comprise a TrpB gene sequence which consists of the sequence of SEQ ID NO: 214.
[0583] In one embodiment, the genetically engineered bacteria comprise a gene sequence encoding a TrpA. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 215. In another embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 85% identity with SEQ ID NO: 215. In one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 90% identity with SEQ ID NO: 215. In one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which has at least about 95% identity with SEQ ID NO: 215. In another embodiment, the TrpA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 215.
Accordingly, in one embodiment, the genetically engineered bacteria comprise a TrpA gene sequence which 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: 215. In another embodiment, the genetically engineered bacteria comprise the TrpA gene sequence of SEQ ID NO: 215. In yet another embodiment the genetically engineered bacteria comprise a TrpA gene sequence which consists of the sequence of SEQ ID NO: 215. Table 18B. Polypeptide Sequences
Figure imgf000269_0001
Figure imgf000270_0001
[0584] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpE. In some embodiments, TrpE has at least about 80% identity with SEQ ID NO: 216. In some embodiments, TrpE has at least about 85% identity with one or more of SEQ ID NO: 216. In some embodiments, TrpE has at least about 90% identity with SEQ ID NO: 216. In some embodiments, TrpE has at least about 95% identity with SEQ ID NO: 216. In some embodiments, TrpE has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:75. Accordingly, In some embodiments, TrpE 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: 216. In another embodiment, TrpE comprises the sequence of SEQ ID NO: 216. In another embodiment, TrpE consists of the sequence of one or more of SEQ ID NO: 216.
[0585] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpA. In some embodiments, TrpA has at least about 80% identity with SEQ ID NO: 217. In some embodiments, TrpA has at least about 85% identity with one or more of SEQ ID NO: 217. In some embodiments, TrpA has at least about 90% identity with SEQ ID NO: 217. In some embodiments, TrpA has at least about 95% identity with SEQ ID NO: 217. In some embodiments, TrpA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:83. Accordingly, In some embodiments, TrpA 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: 217. In another embodiment, TrpA comprises the sequence of SEQ ID NO: 217. In another embodiment, TrpA consists of the sequence of one or more of SEQ ID NO: 217.
[0586] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpB. In some embodiments, TrpB has at least about 80% identity with SEQ ID NO: 218. In some embodiments, TrpB has at least about 85% identity with one or more of SEQ ID NO: 218. In some embodiments, TrpB has at least about 90% identity with SEQ ID NO: 218. In some embodiments, TrpB has at least about 95% identity with SEQ ID NO: 218. In some embodiments, TrpB has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:81. Accordingly, In some embodiments, TrpB 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: 218. In another embodiment, TrpB comprises the sequence of SEQ ID NO: 218. In another embodiment, TrpB consists of the sequence of one or more of SEQ ID NO: 218.
[0587] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpD. In some embodiments, TrpD has at least about 80% identity with SEQ ID NO: 219. In some embodiments, TrpD has at least about 85% identity with one or more of SEQ ID NO: 219. In some embodiments, TrpD has at least about 90% identity with SEQ ID NO: 219. In some embodiments, TrpD has at least about 95% identity with SEQ ID NO: 219. In some embodiments, TrpD has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:77. Accordingly, In some embodiments, TrpD 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: 219. In another embodiment, TrpD comprises the sequence of SEQ ID NO: 219. In another embodiment, TrpD consists of the sequence of one or more of SEQ ID NO: 219.
[0588] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpC. In some embodiments, TrpC has at least about 80% identity with SEQ ID NO: 220. In some embodiments, TrpC has at least about 85% identity with one or more of SEQ ID NO: 220. In some embodiments, TrpC has at least about 90% identity with SEQ ID NO: 220. In some embodiments, TrpC has at least about 95% identity with SEQ ID NO: 220. In some embodiments, TrpC has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:79. Accordingly, In some embodiments, TrpC 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: 220. In another embodiment, TrpC comprises the sequence of SEQ ID NO: 220. In another embodiment, TrpC consists of the sequence of one or more of SEQ ID NO: 220.
[0589] Table 19A and B depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 19A and B also depicts an exemplary TnaA
(tryptophanase from E. coli) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodiments, the sequence is deleted from the E coli chromosome to increase levels of tryptophan.
Table 19A. Feedback resistant AroG and TrpE and tryptophanase sequences
Figure imgf000272_0001
Figure imgf000273_0001
[0590] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AroGfbr. In some embodiments, AroGfbr has at least about 80% identity with SEQ ID NO: 221. In some embodiments, AroGfbr has at least about 85% identity with one or more of SEQ ID NO: 221. In some embodiments, AroGfbr has at least about 90% identity with SEQ ID NO: 221. In some embodiments, AroGfbr has at least about 95% identity with SEQ ID NO: 221. In some embodiments, AroGfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:84.
Accordingly, In some embodiments, AroGfbr 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: 221. In some embodiments, AroGfbr comprises the sequence of SEQ ID NO: 221. In some embodiments, AroGfbr consists of the sequence of one or more of SEQ ID NO: 221.
[0591] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpEfbr. In some embodiments, TrpEfbr has at least about 80% identity with SEQ ID NO: 222. In some embodiments, TrpEfbr has at least about 85% identity with one or more of SEQ ID NO: 222. In some embodiments, TrpEfbr has at least about 90% identity with SEQ ID NO: 222. In some embodiments, TrpEfbr has at least about 95% identity with SEQ ID NO: 222. In some embodiments, TrpEfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:85.
Accordingly, In some embodiments, TrpEfbr 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: 222. In some embodiments, TrpEfbr comprises the sequence of SEQ ID NO: 222. In some embodiments, TrpEfbr consists of the sequence of one or more of SEQ ID NO: 222.
[0592] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding SerA. In some embodiments, SerA has at least about 80% identity with SEQ ID NO: 223. In some embodiments, SerA has at least about 85% identity with one or more of SEQ ID NO: 223. In some embodiments, SerA has at least about 90% identity with SEQ ID NO: 223. In some embodiments, SerA has at least about 95% identity with SEQ ID NO: 223. In some embodiments, SerA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:86. Accordingly, In some embodiments, SerA 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: 223. In some embodiments, SerA comprises the sequence of SEQ ID NO: 223. In some embodiments, SerA consists of the sequence of one or more of SEQ ID NO: 223.
[0593] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding SerAfbr. In some embodiments, SerAfbr has at least about 80% identity with SEQ ID NO: 224. In some embodiments, SerAfbr has at least about 85% identity with one or more of SEQ ID NO: 224. In some embodiments, SerAfbr has at least about 90% identity with SEQ ID NO: 224. In some embodiments, SerAfbr has at least about 95% identity with SEQ ID NO: 224. In some embodiments, SerAfbr has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO:87.
Accordingly, In some embodiments, SerAfbr has at least about 80%, 81%, 82%, 83%, 87%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 224. In some embodiments, SerAfbr comprises the sequence of SEQ ID NO: 224. In some embodiments, SerAfbr consists of the sequence of one or more of SEQ ID NO: 224.
[0594] In one embodiment, TnaA is mutated or deleted. [0595] Table 19B lists exemplary polynucleotide sequences useful for tryptophan production.
Table 19B. Sequences Useful for Tryptophan Production
Figure imgf000275_0001
Figure imgf000276_0001
[0596] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 25B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 19B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 19B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 19B. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 19B. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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 one or more sequences of Table 19B. In another
embodiment, the genetically engineered bacteria comprise the sequence of Table 19B embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 19B.
[0597] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding TrpEfbr. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 225. In another embodiment, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 85% identity with SEQ ID NO: 225. In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 90% identity with SEQ ID NO: 225. In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which has at least about 95% identity with SEQ ID NO: 225. In another embodiment, the TrpEfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 225. Accordingly, In some embodiments, the genetically engineered bacteria comprise a TrpEfbr gene sequence which 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: 225. In another embodiment, the genetically engineered bacteria comprise the TrpEfbr gene sequence of SEQ ID NO: 225. In yet another embodiment the genetically engineered bacteria comprise a TrpEfbr gene sequence which consists of the sequence of SEQ ID NO: 225.
[0598] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding feedback resistant AroG. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 80% identity with SEQ ID NO: 226. In another embodiment, the genetically engineered bacteria comprise a AroGfbr sequence which has at least about 85% identity with SEQ ID NO: 226. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 90% identity with SEQ ID NO: 226. In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which has at least about 95% identity with SEQ ID NO: 226. In another embodiment, the a AroGfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 226. Accordingly, In some embodiments, the genetically engineered bacteria comprise a AroGfbr gene sequence which 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: 226. In another embodiment, the genetically engineered bacteria comprise the a AroGfbr gene sequence of SEQ ID NO: 226. In yet another embodiment the genetically engineered bacteria comprise a AroGfbr gene sequence which consists of the sequence of SEQ ID NO: 226.
[0599] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SerA. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 80% identity with SEQ ID NO: 227. In another embodiment, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 85% identity with SEQ ID NO: 227. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 90% identity with SEQ ID NO: 227. In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which has at least about 95% identity with SEQ ID NO: 227. In another embodiment, the SerA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 227. Accordingly, In some embodiments, the genetically engineered bacteria comprise a SerA gene sequence which 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: 227. In another embodiment, the genetically engineered bacteria comprise the SerA gene sequence of SEQ ID NO: 227. In yet another embodiment the genetically engineered bacteria comprise a SerA gene sequence which consists of the sequence of SEQ ID NO: 227.
[0600] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding feedback resistant SerA. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 80% identity with SEQ ID NO: 228. In another embodiment, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 85% identity with SEQ ID NO: 228. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 90% identity with SEQ ID NO: 228. In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which has at least about 95% identity with SEQ ID NO: 228. In another embodiment, the SerAfbr gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 228. Accordingly, In some embodiments, the genetically engineered bacteria comprise a SerAfbr gene sequence which 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: 228. In another embodiment, the genetically engineered bacteria comprise the SerAfbr gene sequence of SEQ ID NO: 228. In yet another embodiment the genetically engineered bacteria comprise a SerAfbr gene sequence which consists of the sequence of SEQ ID NO: 228.
[0601] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and∆trpR, ∆tnaA. In some embodiments, the genetically engineered bacteria comprise and of the trypophan production combinations described herein.
[0602] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with
hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0603] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0604] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan is modified and/or mutated, e.g., to enhance stability, or increase tryptophan production.
[0605] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptophan 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.
[0606] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences described herein for the consumption of ammonia. [0607] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan 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.
[0608] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0609] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0610] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan 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. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0611] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptophan further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0612] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Producing Kynurenic Acid
[0613] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible
transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine- oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (also referred to as kynurenine
aminotransferases (e.g., KAT I, II, III)).
[0614] In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid 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. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0615] In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the circulation. In some embodiments the TRP:KYNA ratio is increased. In some embodiments, TRP:KYNA ratio is decreased.
[0616] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella, and Bacillus, and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some
embodiments the enzymes are derived from the species listed in table S7 of Vujkovic- Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV diseaseprogression and tryptophan catabolism Sci Transl Med. 2013 July 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety. [0617] In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine
aminotransferases.
[0618] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenic acid from tryptophan. Non-limiting example of such gene sequence(s) are shown in the figures and described elsewhere herein. In one
embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1(indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3- dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from S. cerevisiae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with IDO1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TDO2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine--oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from S. cerevisae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2.
[0619] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate
aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from homo sapiens. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 (Kynurenine--oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 from homo sapiens). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine--oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from homo sapiens.In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2.In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of . cclb1 and/or cclb2 and/or aadat and/or got2.
[0620] In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in the figures and the examples and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0621] In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell.
[0622] In some embodiments, 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 kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions.
[0623] In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid 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. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0624] In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0625] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. Producing Indole Tryptophan Metabolites and Tryptamine
[0626] In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of indole metabolites and/or tryptamine. Exemplary circuits for the production of indole metabolites/derivatives are shown in the figures.
[0627] In some embodiments, the genetically engineered bacteria comprise genetic circuitry for converting tryptophan to tryptamine. In some embodiments, the engineered bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from Catharanthus roseus. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli, taa1 (L- tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L- tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), and tynA (Monoamine oxidase, e.g., from E. coli). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2, (tryptophan N- monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N- monooxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynurenine from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynureninic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1(indoleamine 2,3-dioxygenase, e.g., from homo sapiens or TDO2 (tryptophan 2,3-dioxygenase, e.g., from homo sapiens), BNA2 (indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens), or CCLB1 (Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens) or CCLB2 (kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tnaA (tryptophanase, e.g., from E. coli). In some embodiments, the engineered bacteria comprise genetic circuitry for producing 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 gene sequence encoding pne2 (myrosinase, e.g., from Arabidopsis thaliana). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC (aspartate aminotransferase, e.g., from E. coli, taa1 (L- tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana), staO (L- tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae), iad1 ( Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc
(Tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), tynA (Monoamine oxidase, e.g., from E. coli), iad1 (Indole-3- acetaldehyde dehydrogenase, e.g., from Ustilago maydis), AAO1 (Indole-3- acetaldehyde oxidase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 ( L-tryptophan aminotransferase, e.g., from S. cerevisae), aspC
(aspartate aminotransferase, e.g., from E. coli, taa1 (L-tryptophan-pyruvate
aminotransferase, e.g., from Arabidopsis thaliana), staO (L-tryptophan oxidase, e.g., from streptomyces sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and yuc2 ( indole-3-pyruvate monoxygenase, e.g., from Arabidopsis thaliana). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IaaM (Tryptophan 2- monooxygenase e.g., from Pseudomonas savastanoi), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana), cyp79B3 (tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana, cyp71a13 (indoleacetaldoxime dehydratase, e.g., from Arabidopis thaliana), nit1 (Nitrilase, e.g., from Arabidopsis thaliana), iaaH (Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi). In some embodiments, the genetically engineered bacteria comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108), 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 iad1 (Indole-3- acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3- acetaldehyde into indole-3-acetate. [0628] In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. In some embodiments, the engineered bacteria produces tryptamine. Tryptophan is optionally produced from chorismate precursor, and the bacteria optionally comprises circuits as depicted and/or described in FIG. 90A and/or FIG. 90B and/or FIG.90C and/or FIG. 90D. Additionally, the bacteria comprises tdc (tryptophan decarboxylase, e.g., from Catharanthus roseus and/or Clostridium sporogenes), which converts tryptophan into tryptamine.
[0629] In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-acetate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from
Enterobacter cloacae) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis), which converts indole-3-acetaldehyde into indole-3- acetate.
[0630] In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-propionate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in FIG. 90A and/or FIG. 90B and/or FIG. 90C and/or FIG. 90D.
Additionally, the strain comprises a circuit as described in FIG. 98, comprising 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 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 AcuI:
(indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 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).
[0631] In some embodiments, the engineered bacteria comprises genetic circuitry for the production of indole-3-propionic acid (IPA). In some embodiments, the engineered bacteria comprises gene sequence encoding 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). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0632] In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or tryptamine synthesis(TrA) circuits. In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: TrpDH: tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108; FldH1/FldH2: indole-3- lactate dehydrogenase, e.g., from Clostridium sporogenes; FldA: indole-3-propionyl- CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes; FldBC:
indole-3-lactate dehydratase, e.g., from Clostridium sporogenes; FldD: indole-3- acrylyl-CoA reductase, e.g., from Clostridium sporogenes; AcuI: acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides. lpdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae; lad1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis; Tdc: Tryptophan decarboxylase, e.g., from Catharanthus roseus or from Clostridium sporogenes.
[0633] In some embodiments, the engineered bacteria comprise genetic circuitry for producing (indol-3-yl)pyruvate (IPyA). In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: tryptophan dehydrogenase (EC 1.4.1.19) (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 (FldA ) (converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (FldD ) and acrylyl-CoA reductase (AcuI) (convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA); Indole-3-lactate dehydratase (FldBC ) (converts indole-3- lactate-CoA to indole-3-acrylyl-CoA); Indole-3-pyruvate decarboxylase (lpdC:) (converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld)); lad1: Indole-3-acetaldehyde dehydrogenase (coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA)); Tdc: Tryptophan decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0634] In any of the described embodiments, 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).
[0635] In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in FIG. 90 (A-D) and FIG. 94 and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.
[0636] In some embodiments, 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 tryptophan metabolites bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 tryptophan metabolites than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan metabolites than unmodified bacteria of the same bacterial subtype under the same conditions.
[0637] In in any of these embodiments the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, indole, indole acetic acid FICZ, indole-3-propionic acid, is under the control of an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some
embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0638] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0639] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the
embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis. [0640] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites 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.
[0641] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences described herein for the consumption of ammonia.
[0642] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites 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.
[0643] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0644] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0645] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of indoles and other tryptophan metabolites and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0646] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of indoles and other tryptophan metabolites further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a “manganese transport circuit”).
[0647] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Tryptamine
[0648] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole-3-acetic acid (IAA) via the enzymes tryptophan monooxygenase (IaaM) and indole-3- acetamide hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, as described in the figures and examples.
[0649] A non-limiting example of such as strain is shown in FIG. 91A. Another non-limiting example of such as strain is shown in FIG. 93A. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from
Clostridium sporgenenes. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus.
[0650] Table 18-19 and Table A-C lists exemplary sequences for tryptamine production in genetically engineered bacteria.
[0651] In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0652] In some embodiments, 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 tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions.
[0653] In some embodiments, the genetically engineered bacteria are capable of producing Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine 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 and others described herein.
[0654] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0655] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0656] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[0657] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of tryptamine 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.
[0658] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences described herein for the consumption of ammonia.
[0659] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine 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.
[0660] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0661] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0662] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of tryptamine and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0663] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of tryptamine further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0664] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Indole-3-acetaldehyde and FICZ
[0665] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in FIG. 91B.
[0666] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 ( L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from
Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L- tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L- tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH and ipdC.
[0667] Further exemplary gene cassettes for the production of produce indole-3- acetaldehyde and FICZ from tryptophan are shown in FIG. 91C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA.
[0668] In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0669] In some embodiments, 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 Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four- fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty- fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions.
[0670] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetaldehyde and/or FICZ is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0671] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0672] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetaldehyde and/or FICZ is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the
embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ is modified and/or mutated, e.g., to enhance stability, or increase
tryptophan/tryptophan metabolite production or catalysis.
[0673] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetaldehyde and/or FICZ may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetaldehyde and/or FICZ 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.
[0674] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences described herein for the consumption of ammonia.
[0675] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ 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.
[0676] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0677] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0678] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Indole-3-acetaldehyde and/or FICZ and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences for the secretion of an anti- inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0679] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetaldehyde and/or FICZ further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0680] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Indole-3-acetic acid
[0681] In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in the tables. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole-3-aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter.
[0682] The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite , e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine 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.
[0683] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetic acid.
[0684] Non-limiting example of such genes encoding tryptophan catabolism enzymes are shown in FIG. 92A, FIG. 92B, FIG.92C, FIG. 92D, and FIG. 92E, and FIG. 93B and FIG. 93E.
[0685] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from S. cerevisae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP- A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from
Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1 (see, e.g., FIG. 92A).
[0686] Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 92B. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc
(Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from Catharanthus roseus). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from E. coli). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde
dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from Ustilago maydis). In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from Arabidopsis thaliana). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iad1 and/or aao1.
[0687] Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 92C. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole- 3-pyruvate monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from Enterobacter cloacae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from S. cerevisiae. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode aspC from E. coli. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode taa1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from streptomyces sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one
embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode trpDH and yuc2.. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH and yuc2.
[0688] Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 92D. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2- monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH.
[0689] Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in FIG. 92E. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13
(indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from
Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from Pseudomonas savastanoi).In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13 and nit1 and iaaH.
[0690] Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 92F. Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in FIG. 93E. In one embodiment, the genetically engineered bacteria comprise one or more gene
sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from Nostoc punctiforme NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole- 3-pyruvate decarboxylase, e.g., from Enterobacter cloacae). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from Ustilago maydis. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iad1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iad1.
[0691] In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0692] In some embodiments, 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 indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions.
[0693] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and∆trpR,∆tnaA.
[0694] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0695] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0696] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[0697] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid 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.
[0698] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences described herein for the consumption of ammonia.
[0699] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid 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.
[0700] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0701] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0702] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Indole-3-acetic acid and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0703] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetic acid further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0704] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance. Indole-3-acetonitrile
[0705] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3-acetonitrile from tryptophan is depicted in the figures and examples.
[0706] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from Arabidopis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13.
[0707] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from Arabidopsis thaliana. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13.
[0708] In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. [0709] In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0710] In some embodiments, 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 Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions.
[0711] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetonitrile is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0712] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0713] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetonitrile is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis. [0714] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetonitrile may be codon optimized, e.g., to improve expression in the host
microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- acetonitrile 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.
[0715] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences described herein for the consumption of ammonia.
[0716] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile 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.
[0717] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0718] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0719] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Indole-3-acetonitrile and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0720] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-acetonitrile further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0721] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance. Indole-3-propionic acid (IPA)
[0722] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan. FIG. 97 and FIG 98, and FIG. 93C depict schematics of exemplary circuits for the production of indole-3- propionic acid.
[0723] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from Rubrivivax benzoatilyticus. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole- 3-acrylate reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase from Clostridum botulinum. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3-acrylate reductase. In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein.
[0724] The genetically engineered bacteria comprise a circuit, comprising 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 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 AcuI:
(indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes and/or acrylyl-CoA reductase, e.g., from Rhodobacter sphaeroides, which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 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) (see, e.g., FIG. 98).
[0725] Another embodiment of the IPA producing strain is shown in FIG. 97. [0726] In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from Nostoc punctiforme NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from
Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI from Rhodobacter sphaeroides. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 (3-lactate dehydrogenase 1). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 from Clostridium sporogenes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole-3-lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from Clostridium sporogenes). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acul and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acul and fldH2.
[0727] In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0728] In some embodiments, 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 Indole-3-propionic acidthan unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 Indole-3-propionic acidthan unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven- fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-propionic acidthan unmodified bacteria of the same bacterial subtype under the same conditions.
[0729] In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan
metabolites selected from tryptamine and/or indole-3 acetaldehyde, indole-3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid.
[0730] In some embodiments, the genetically engineered bacteria are capable of producing such tryptophan metabolites under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing such tryptophan metabolites 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.
[0731] In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein.
[0732] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- propionic acid is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0733] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0734] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- propionic acid is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[0735] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- propionic acid may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole-3- propionic acid 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.
[0736] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences described herein for the consumption of ammonia.
[0737] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid 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.
[0738] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0739] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0740] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Indole-3-propionic acid and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0741] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole-3-propionic acid further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0742] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance. Indole
[0743] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non-limiting example of such gene sequence(s) are shown FIG. 91G and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA (tryptophanase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from E. coli.
[0744] In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0745] In some embodiments, 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 Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole than unmodified bacteria of the same bacterial subtype under the same conditions.
[0746] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0747] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0748] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[0749] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Indole 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.
[0750] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences described herein for the consumption of ammonia.
[0751] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole 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.
[0752] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0753] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0754] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Indole and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0755] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Indole further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0756] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Other indole metabolites
[0757] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which 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. Non- limiting example of such gene sequence(s) are shown FIG. 91H and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 from Arabidopsis thaliana.
[0758] In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in FIG. 90, FIG. 94A and/or FIG. 94B and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites.
[0759] In some embodiments, 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 Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions.
[0760] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0761] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0762] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[0763] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the production of Other indole metabolites 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.
[0764] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences described herein for the consumption of ammonia.
[0765] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites 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.
[0766] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0767] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0768] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of Other indole metabolites and/or one or more of its metabolites described herein. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences for the secretion of IL-22. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0769] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the production of Other indole metabolites further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0770] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Tryptophan Catabolic Pathway Enzymes
[0771] Table A and B comprises sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure. Table A. Tryptophan Pathway Enzymes
Figure imgf000340_0001
Figure imgf000341_0001
Figure imgf000342_0001
Figure imgf000343_0001
Figure imgf000344_0001
Figure imgf000345_0001
Figure imgf000346_0001
Figure imgf000347_0001
Figure imgf000348_0001
[0772] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more Trp aminotransferase. Accordingly, In some embodiments, the nucleic acid sequence comprising the Trp
aminotransferase gene has at least about 80% identity with SEQ ID NO: 229. In some embodiments, the gene sequence comprising the Trp aminotransferase gene has at least about 90% identity with SEQ ID NO: 229. In another embodiment, the gene sequence comprising the Trp aminotransferase gene has at least about 95% identity with SEQ ID NO: 229. Accordingly, In some embodiments, the gene sequence comprising the Trp aminotransferase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 229. In another embodiment, the gene sequence comprising the Trp
aminotransferase gene comprises SEQ ID NO: 229. In yet another embodiment the gene sequence comprising the Trp aminotransferase gene consists of SEQ ID NO: 229.
[0773] In some embodiments, the disclosure provides novel nucleic acids for producing one or more tryptophan metabolites. In some embodiments, the nucleic acid comprises gene sequence encoding one or more Tryptophan Decarboxylase
Accordingly, In some embodiments, the nucleic acid sequence comprising the
Tryptophan Decarboxylase gene has at least about 80% identity with SEQ ID NO: 230. In some embodiments, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 90% identity with SEQ ID NO: 230. In another embodiment, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 95% identity with SEQ ID NO: 230. Accordingly, In some embodiments, the gene sequence comprising the Tryptophan Decarboxylase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 230. In another embodiment, the gene sequence comprising the Tryptophan Decarboxylase gene comprises SEQ ID NO: 230. In yet another embodiment the gene sequence comprising the Tryptophan Decarboxylase gene consists of SEQ ID NO: 230.
[0774] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table A. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table A. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table A. In some
embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table A. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table A. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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 one or more sequences of Table A. In another embodiment, the genetically engineered bacteria comprise the sequence of Table A. In some embodiments, the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table A. [0775] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan amino transferase. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. roseus. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 231. In another embodiment, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 85% identity with SEQ ID NO: 231. In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 90% identity with SEQ ID NO: 231. In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which has at least about 95% identity with SEQ ID NO: 231. In another embodiment, the tryptophan amino transferase gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 231. Accordingly, In some embodiments, the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which 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: 231. In another embodiment, the genetically engineered bacteria comprise the tryptophan amino transferase gene sequence of SEQ ID NO: 231. In yet another embodiment the genetically engineered bacteria comprise a tryptophan amino transferase gene sequence which consists of the sequence of SEQ ID NO: 231.
[0776] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan decarboxylase. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. roseus. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 96. In another embodiment, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 85% identity with SEQ ID NO: 96. In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 90% identity with SEQ ID NO: 96. In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which has at least about 95% identity with SEQ ID NO: 96. In another embodiment, the tryptophan decarboxylase gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96. Accordingly, In some embodiments, the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which 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: 96. In another embodiment, the genetically engineered bacteria comprise the tryptophan decarboxylase gene sequence of SEQ ID NO: 96. In yet another embodiment the genetically engineered bacteria comprise a tryptophan decarboxylase gene sequence which consists of the sequence of SEQ ID NO: 96.
[0777] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding Tdc. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tdc from C. sporogenes. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 232. In another embodiment, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 85% identity with SEQ ID NO: 232. In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 90% identity with SEQ ID NO: 232. In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which has at least about 95% identity with SEQ ID NO: 232. In another embodiment, the Tdc gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 232. Accordingly, In some embodiments, the genetically engineered bacteria comprise a Tdc gene sequence which 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: 232. In another embodiment, the genetically engineered bacteria comprise the Tdc gene sequence of SEQ ID NO: 232. In yet another embodiment the genetically engineered bacteria comprise a Tdc gene sequence which consists of the sequence of SEQ ID NO: 232.
[0778] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding tryptophan dehydrogenase (trpDH). In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 80% identity with SEQ ID NO: 233. In another embodiment, the genetically engineered bacteria comprise a trpDH sequence which has at least about 85% identity with SEQ ID NO: 233. In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 90% identity with SEQ ID NO: 233. In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which has at least about 95% identity with SEQ ID NO: 233. In another embodiment, the a trpDH gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 233. Accordingly, In some embodiments, the genetically engineered bacteria comprise a trpDH gene sequence which 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: 233. In another embodiment, the genetically engineered bacteria comprise the a trpDH gene sequence of SEQ ID NO: 233. In yet another embodiment the genetically engineered bacteria comprise a trpDH gene sequence which consists of the sequence of SEQ ID NO: 233.
[0779] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding ipdC. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 80% identity with SEQ ID NO: 234. In another embodiment, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 85% identity with SEQ ID NO: 234. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 90% identity with SEQ ID NO: 234. In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which has at least about 95% identity with SEQ ID NO: 234. In another embodiment, the ipdC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 234. Accordingly, In some embodiments, the genetically engineered bacteria comprise a ipdC gene sequence which 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: 234. In another embodiment, the genetically engineered bacteria comprise the ipdC gene sequence of SEQ ID NO: 234. In yet another embodiment the genetically engineered bacteria comprise a ipdC gene sequence which consists of the sequence of SEQ ID NO: 234.
[0780] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding Iad1. In some embodiments, the genetically engineered bacteria comprise a Iad1 gene sequence which has at least about 80% identity with SEQ ID NO: 235. In another embodiment, the genetically engineered bacteria comprise a Iad1 gene sequence which has at least about 85% identity with SEQ ID NO: 235. In some embodiments, the genetically engineered bacteria comprise a Iad1 gene sequence which has at least about 90% identity with SEQ ID NO: 235. In some embodiments, the genetically engineered bacteria comprise a Iad1 gene sequence which has at least about 95% identity with SEQ ID NO: 235. In another embodiment, the Iad1 gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 235. Accordingly, In some embodiments, the genetically engineered bacteria comprise a Iad1 gene sequence which 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: 235. In another embodiment, the genetically engineered bacteria comprise the Iad1 gene sequence of SEQ ID NO: 235. In yet another embodiment the genetically engineered bacteria comprise a Iad1 gene sequence which consists of the sequence of SEQ ID NO: 235.
[0781] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldA. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 80% identity with SEQ ID NO: 236. In another embodiment, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 85% identity with SEQ ID NO: 236. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 90% identity with SEQ ID NO: 236. In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which has at least about 95% identity with SEQ ID NO: 236. In another embodiment, the fldA gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 236. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldA gene sequence which 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: 236. In another embodiment, the genetically engineered bacteria comprise the fldA gene sequence of SEQ ID NO: 236. In yet another embodiment the genetically engineered bacteria comprise a fldA gene sequence which consists of the sequence of SEQ ID NO: 236.
[0782] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldB. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 80% identity with SEQ ID NO: 237. In another embodiment, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 85% identity with SEQ ID NO: 237. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 90% identity with SEQ ID NO: 237. In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which has at least about 95% identity with SEQ ID NO: 237. In another embodiment, the fldB gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 237. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldB gene sequence which 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: 237. In another embodiment, the genetically engineered bacteria comprise the fldB gene sequence of SEQ ID NO: 237. In yet another embodiment the genetically engineered bacteria comprise a fldB gene sequence which consists of the sequence of SEQ ID NO: 237.
[0783] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldC. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 80% identity with SEQ ID NO: 238. In another embodiment, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 85% identity with SEQ ID NO: 238. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 90% identity with SEQ ID NO: 238. In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which has at least about 95% identity with SEQ ID NO: 238. In another embodiment, the fldC gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 238. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldC gene sequence which 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: 238. In another embodiment, the genetically engineered bacteria comprise the fldC gene sequence of SEQ ID NO: 238. In yet another embodiment the genetically engineered bacteria comprise a fldC gene sequence which consists of the sequence of SEQ ID NO: 238.
[0784] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding Acul. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 80% identity with SEQ ID NO: 239. In another embodiment, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 85% identity with SEQ ID NO: 239. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 90% identity with SEQ ID NO: 239. In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which has at least about 95% identity with SEQ ID NO: 239. In another embodiment, the Acul gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 239. Accordingly, In some embodiments, the genetically engineered bacteria comprise a Acul gene sequence which 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: 239. In another embodiment, the genetically engineered bacteria comprise the Acul gene sequence of SEQ ID NO: 239. In yet another embodiment the genetically engineered bacteria comprise a Acul gene sequence which consists of the sequence of SEQ ID NO: 239.
[0785] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldH1. In some embodiments, the genetically engineered bacteria comprise a fldH1 gene sequence which has at least about 80% identity with SEQ ID NO: 240. In another embodiment, the genetically engineered bacteria comprise a fldH1 gene sequence which has at least about 85% identity with SEQ ID NO: 240. In some embodiments, the genetically engineered bacteria comprise a fldH1 gene sequence which has at least about 90% identity with SEQ ID NO: 240. In some embodiments, the genetically engineered bacteria comprise a fldH1 gene sequence which has at least about 95% identity with SEQ ID NO: 240. In another embodiment, the fldH1 gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 240. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldH1 gene sequence which 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: 240. In another embodiment, the genetically engineered bacteria comprise the fldH1 gene sequence of SEQ ID NO: 240. In yet another embodiment the genetically engineered bacteria comprise a fldH1 gene sequence which consists of the sequence of SEQ ID NO: 240.
[0786] In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding fldD. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 80% identity with SEQ ID NO: 241. In another embodiment, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 85% identity with SEQ ID NO: 241. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 90% identity with SEQ ID NO: 241. In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which has at least about 95% identity with SEQ ID NO: 241. In another embodiment, the fldD gene sequence has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 241. Accordingly, In some embodiments, the genetically engineered bacteria comprise a fldD gene sequence which 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: 241. In another embodiment, the genetically engineered bacteria comprise the fldD gene sequence of SEQ ID NO: 241. In yet another embodiment the genetically engineered bacteria comprise a fldD gene sequence which consists of the sequence of SEQ ID NO: 241.
Table B. Tryptophan Pathway Catabolic Enzymes
Figure imgf000356_0001
Figure imgf000357_0001
Figure imgf000358_0001
Figure imgf000359_0001
Figure imgf000360_0001
Figure imgf000361_0001
Figure imgf000362_0001
Figure imgf000363_0001
Figure imgf000364_0001
[0787] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence or nucleic acid sequence encoding a polypeptide of Table B 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 sequence of Table B or a functional fragment thereof. In some embodiments, 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 of Table B 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 of Table B or a functional fragment thereof.
[0788] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding tryptophan decarboxylase. In some embodiments, the Tryptophan Decarboxylase encoded by bacterium has at least about 80% identity with the entire sequence selected from SEQ ID NO: 242, 243, 244. In some embodiments, the Tryptophan Decarboxylase gene has at least about 85% identity with the entire sequence selected from SEQ ID NO: 242, 243, 244. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 90% identity with the entire sequence selected from SEQ ID NO: 242, 243, 244. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 95% identity with the entire sequence selected from SEQ ID NO: 242, 243, 244. In another embodiment, the Tryptophan Decarboxylase polypeptide encoded by the bacteria has at least about 96%, 97%, 98%, or 99% identity with the entire sequence selected from SEQ ID NO: 242, 243, 244. Accordingly, In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria 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 the entire sequence selected from SEQ ID NO: 242, 243, 244. In some embodiments, the Tryptophan Decarboxylase polypeptide encoded by the bacteria comprises the sequence selected from SEQ ID NO: 242, 243, 244. In some embodiments, the Tryptophan
Decarboxylase polypeptide encoded by the bacteria consists of the sequence of selected from SEQ ID NO: 242, 243, 244..
[0789] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding tryptophan aminotransferase. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 80% identity with the entire sequence of selected form SEQ ID NO: 245 and 246. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 85% identity with the entire sequence of SEQ ID NO: 245 and 246. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 90% identity with the entire sequence of SEQ ID NO: 245 and 246. In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 95% identity with the entire sequence of SEQ ID NO: 245 and 246. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 245 and 246. Accordingly, In some embodiments, the Trp aminotransferase polypeptide encoded by the gene sequence 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 the entire sequence of SEQ ID NO: 245 and 246. In another embodiment, the Trp aminotransferase polypeptide encoded by the gene sequence comprises the sequence of SEQ ID NO: 245 and 246. In yet another embodiment the Trp aminotransferase polypeptide encoded by the gene sequence consists of the sequence of SEQ ID NO: 245 and 246.
[0790] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Monoamine oxidase (TYNA), e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 247. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 247. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 247. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 247. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 247. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 247.
[0791] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AAO1: Indole-3-acetaldehyde oxidase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 248. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 248. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 248. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 248. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 248. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 248.
[0792] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding aspC: aspartate aminotransferase, e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 249. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 249. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 249. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 249. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 249. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 249.
[0793] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TAA1: L-tryptophan-pyruvate aminotransferase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 250. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 250. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 250.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 250. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 250. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 250.
[0794] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding STAO: L-tryptophan oxidase, e.g., from
streptomyces sp. TP-A0274. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 251. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 251. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 251.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 251. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 251. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 251.
[0795] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding ipdC: Indole-3-pyruvate decarboxylase, e.g., from Enterobacter cloacae. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 252. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 252. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 252. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 252. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 252. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 252.
[0796] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IAD1: Indole-3-acetaldehyde dehydrogenase, e.g., from Ustilago maydis. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 253. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 253. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 253.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 253. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 253. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 253.
[0797] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding YUC2: indole-3-pyruvate monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 254. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 254. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 254.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 254. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 254. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 254.
[0798] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IaaM: Tryptophan 2-monooxygenase, e.g., from Pseudomonas savastanoi. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 255. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 255. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 255.
Accordingly, in some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 255. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 255. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 255.
[0799] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding iaaH: Indoleacetamide hydrolase, e.g., from Pseudomonas savastanoi. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 256. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 256. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 256.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 256. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 256.
[0800] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TrpDH: Tryptophan dehydrogenase, e.g., from Nostoc punctiforme NIES-2108. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 257. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 257. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 257.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 257. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 257.
[0801] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP79B2: tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 258. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 258. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 258.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 258. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 258. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 258.
[0802] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP79B3: tryptophan N-monooxygenase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 259. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 259. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 259.
Accordingly, in some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 259. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 259. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 259.
[0803] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CYP71A13: indoleacetaldoxime dehydratase, eg., from Arabidopis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 260. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 260. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 260.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 260. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 260. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 260.
[0804] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding PEN2: myrosinase, e.g, from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 261. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 261. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 261. Accordingly, In some
embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 261. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 261.
[0805] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Nit1: Nitrilase, e.g., from Arabidopsis thaliana. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 262. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 262. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 262. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 262. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 262. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 262.
[0806] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding IDO1: indoleamine 2,3-dioxygenase, e.g.,, from homo sapiens In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 263. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 263. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 263. Accordingly, In some
embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 263. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 263.
[0807] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TDO2: tryptophan 2,3-dioxygenase, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 264. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 264. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 264. Accordingly, In some
embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 264. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 264. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 264.
[0808] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding BNA2: indoleamine 2,3-dioxygenase, e.g., from S. cerevisiae. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 265. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 265. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 265. Accordingly, In some
embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 265. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 265. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 265.
[0809] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding Afmid: Kynurenine formamidase, e.g., from mouse. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 266. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 266. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 266. Accordingly, In some
embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 266. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 266. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 266.
[0810] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding BNA3: kynurenine--oxoglutarate transaminase, e.g., from S. cerevisae. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 267. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 267. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 267.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 267. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 267. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 267.
[0811] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding GOT2: Aspartate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 268. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 268. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 268.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 268. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 268. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 268.
[0812] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AADAT: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 269. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 269. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 269. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 269. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 269. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 269.
[0813] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CCLB1: Kynurenine--oxoglutarate transaminase 1, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 270. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 270. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 270.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 270. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 270. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 270.
[0814] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding CCLB2: kynurenine--oxoglutarate transaminase 3, e.g., from homo sapiens. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 271. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 271. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 271.
Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 271. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 271. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 271.
[0815] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding TnaA: tryptophanase, e.g., from E. coli. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 80% identity with SEQ ID NO: 272. In some embodiments, the gene sequence encodes a polypeptide, which has at least about 90% identity with SEQ ID NO: 272. In another embodiment, the gene sequence encodes a polypeptide, which has at least about 95% identity with SEQ ID NO: 272. Accordingly, In some embodiments, the gene sequence encodes a polypeptide, which has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 272. In another embodiment, the gene sequence encodes a polypeptide, which comprises a sequence which encodes SEQ ID NO: 272. In yet another embodiment, the gene sequence encodes a polypeptide, which consists of a sequence which encodes SEQ ID NO: 272.
[0816] In some embodiments, TNA (e.g., SEQ ID NO: 272) is mutated or deleted.
[0817] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.
[0818] Table C depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by indole-3-propionate producing bacteria.
Table C. Non-limiting Examples of Sequences for indole-3-propionate Production
Figure imgf000376_0001
Figure imgf000377_0001
[0819] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from Clostridium sporogenes. In some embodiments, FldA has at least about 80% identity with SEQ ID NO: 273. In some embodiments, FldA has at least about 85% identity with one or more of SEQ ID NO: 273. In some embodiments, FldA has at least about 90% identity with SEQ ID NO: 273. In some embodiments, FldA has at least about 95% identity with SEQ ID NO: 273. In some embodiments, FldA has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273.
Accordingly, In some embodiments, FldA has at least about 80%, 81%, 82%, 83%, 127%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. In some embodiments, FldA comprises the sequence of SEQ ID NO: 273. In some embodiments, FldA consists of the sequence of one or more of SEQ ID NO: 84.
[0820] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldB: subunit of indole-3-lactate dehydratase, e.g., from Clostridium sporogenes. In some embodiments, FldB has at least about 80% identity with SEQ ID NO: 274. In some embodiments, FldB has at least about 85% identity with one or more of SEQ ID NO: 274. In some embodiments, FldB has at least about 90% identity with SEQ ID NO: 274. In some embodiments, FldB has at least about 95% identity with SEQ ID NO: 274. In some embodiments, FldB has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 274. Accordingly, In some embodiments, FldB has at least about 80%, 81%, 82%, 83%, 128%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 274. In some embodiments, FldB comprises the sequence of SEQ ID NO: 274. In some embodiments, FldB consists of the sequence of one or more of SEQ ID NO: 274.
[0821] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldC: subunit of indole-3-lactate dehydratase from Clostridium sporogenes. In some embodiments, FldC has at least about 80% identity with SEQ ID NO: 275. In some embodiments, FldC has at least about 85% identity with one or more of SEQ ID NO: 275. In some embodiments, FldC has at least about 90% identity with SEQ ID NO: 275. In some embodiments, FldC has at least about 95% identity with SEQ ID NO: 275. In some embodiments, FldC has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 275. Accordingly, In some embodiments, FldC has at least about 80%, 81%, 82%, 83%, 129%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 275. In some embodiments, FldC comprises the sequence of SEQ ID NO: 275. In some embodiments, FldC consists of the sequence of one or more of SEQ ID NO: 275.
[0822] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldD: indole-3-acrylyl-CoA reductase, e.g., from Clostridium sporogenes. In some embodiments, FldD has at least about 80% identity with SEQ ID NO: 276. In some embodiments, FldD has at least about 85% identity with one or more of SEQ ID NO: 276. In some embodiments, FldD has at least about 90% identity with SEQ ID NO: 276. In some embodiments, FldD has at least about 95% identity with SEQ ID NO: 276. In some embodiments, FldD has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 276. Accordingly, In some embodiments, FldD has at least about 80%, 81%, 82%, 83%, 130%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 276. In some embodiments, FldD comprises the sequence of SEQ ID NO: 276. In some embodiments, FldD consists of the sequence of one or more of SEQ ID NO: 276.
[0823] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldH1: indole-3-lactate dehydrogenase, e.g., from Clostridium sporogenes. In some embodiments, FldH1 has at least about 80% identity with SEQ ID NO: 277. In some embodiments, FldH1 has at least about 85% identity with one or more of SEQ ID NO: 277. In some embodiments, FldH1 has at least about 90% identity with SEQ ID NO: 277. In some embodiments, FldH1 has at least about 95% identity with SEQ ID NO: 277. In some embodiments, FldH1 has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 277. Accordingly, In some embodiments, FldH1 has at least about 80%, 81%, 82%, 83%, 131%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 277. In some embodiments, FldH1 comprises the sequence of SEQ ID NO: 277. In some embodiments, FldH1 consists of the sequence of one or more of SEQ ID NO: 277. [0824] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding FldH2: indole-3-lactate dehydrogenase from Clostridium sporogenes. In some embodiments, FldH2 has at least about 80% identity with SEQ ID NO: 278. In some embodiments, FldH2 has at least about 85% identity with one or more of SEQ ID NO: 278. In some embodiments, FldH2 has at least about 90% identity with SEQ ID NO: 278. In some embodiments, FldH2 has at least about 95% identity with SEQ ID NO: 278. In some embodiments, FldH2 has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 278. Accordingly, In some embodiments, FldH2 has at least about 80%, 81%, 82%, 83%, 132%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 278. In some embodiments, FldH2 comprises the sequence of SEQ ID NO: 278. In some embodiments, FldH2 consists of the sequence of one or more of SEQ ID NO: 278.
[0825] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding AcuI: acrylyl-CoA reductase from Rhodobacter sphaeroides. In some embodiments, AcuI has at least about 80% identity with SEQ ID NO: 279. In some embodiments, AcuI has at least about 85% identity with one or more of SEQ ID NO: 279. In some embodiments, AcuI has at least about 90% identity with SEQ ID NO: 279. In some embodiments, AcuI has at least about 95% identity with SEQ ID NO: 279. In some embodiments, AcuI has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 279. Accordingly, In some embodiments, AcuI has at least about 80%, 81%, 82%, 83%, 133%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 279. In some embodiments, AcuI comprises the sequence of SEQ ID NO: 279. In some
embodiments, AcuI consists of the sequence of one or more of SEQ ID NO: 279.
[0826] In some embodiments, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 273 through SEQ ID NO: 279. In another embodiment, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. In some embodiments, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. In some embodiments, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. In another embodiment, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. Accordingly, In some embodiments, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which 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 the entire sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. In another embodiment, the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding tryptophan pathway catabolic enzyme which comprises the sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279. In yet another embodiment the genetically engineered bacterium comprises a gene sequence or nucleic acid sequence encoding the tryptophan pathway catabolic enzyme which consists of the sequence of one or more SEQ ID NO: 273 through SEQ ID NO: 279.
[0827] In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g. FIG. 90, FIG. 94A and FIG. 94B. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced.
[0828] In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the at least one sequence(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.
[0829] Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits.
Tryptophan Repressor (TrpR)
[0830] In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC.
[0831] In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut.
Tryptophan and Tryptophan Metabolite Transport
[0832] Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell.
[0833] The inner membrane protein YddG of E. coli, encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene.
[0834] In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mtr gene.
[0835] In some embodiments the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP.
[0836] In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transporter gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.
[0837] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.
[0838] In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut.
[0839] In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.
[0840] In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut.
[0841] In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome.
[0842] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter.
[0843] In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible promoter. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by a constitutive promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut.
[0844] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native transporter gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload , e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in E. coli Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter.
[0845] In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by a constitutive promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. IL-22
[0846] In some embodiments, the genetically engineered bacteria are capable of producing IL-22. Interleukin 22 (IL-22) cytokine can be produced by dendritic cells, lymphoid tissue inducer-like cells, natural killer cells and expressed on adaptive lymphocytes. Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of IL-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function. IL-22’s association with IBD susceptibility genes may modulate phenotypic expression of disease as well. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 280 or a functional fragment thereof. In some embodiments, 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 a nucleic acid sequence encoding SEQ ID NO: 280 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 in low-oxygen conditions.
Table D1. SEQ ID NO: 280
Figure imgf000388_0001
[0847] In some embodiments, the construct comprising IL-22 further comprises a secretion tag, such as any secretion tag described herein or known in the art. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22.
[0848] In some embodiments, the IL-22 construct comprises a membrane anchor for display of IL-22 on the cell surface.
[0849] In some embodiments, 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 tryptophan metabolites bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine- fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions.
[0850] Non-limiting examples of such membrane anchors are described herein. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0851] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0852] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in
Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 is modified and/or mutated, e.g., to enhance stability, or increase IL-22 production, secretion or display.
[0853] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 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.
[0854] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences described herein for the consumption of ammonia.
[0855] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 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. [0856] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0857] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0858] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 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. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences for the secretion of GLP2. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[0859] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of IL-22 further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0860] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
GLP2
[0861] In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 or proglucagon. Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function. GLP-2 administration has therapeutic potential in treating IBD, short bowel syndrome, and small bowel enteritis (Yazbeck et al., 2009). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency under inducing conditions. In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-2 or proglucagon under inducing conditions. GLP-2 administration in a murine model of IBD is associated with reduced mucosal damage and inflammation, as well as a reduction in inflammatory mediators, such as TNF-α and IFN-y. Further, GLP-2 supplementation may also lead to reduced mucosal myeloperoxidase in colitis/ileitis models. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 181 or a functional fragment thereof. In some embodiments, 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 a nucleic acid sequence encoding SEQ ID NO: 181 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 in low-oxygen conditions. In some embodiments, the construct comprising GLP-2 further comprises a secretion tag, such as any secretion tag described herein or known in the art. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2.
[0862] In some embodiments, the GLP-2 construct comprises a membrane anchor for display of GLP-2 on the cell surface. Non-limiting examples of such membrane anchors are described herein.
Table I. SEQ ID NO: 181 GLP-2 SEQ ID NO: 181
HADGSFSDEMNTILDNLAARDFINWLIQTKITD
[0863] In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 analogs, including but not limited to, Gattex and teduglutide.
Teduglutide is a protease resistant analog of GLP-2. It is made up of 33 amino acids and differs from GLP-2 by one amino acid (alanine is substituted by glycine). The significance of this substitution is that teduglutide is longer acting than endogenous GLP-2 as it is more resistant to proteolysis from dipeptidyl peptidase-4.
Table II. SEQ ID NO: 182 Teduglutide SEQ ID NO: 182
HGDGSFSDEMNTILDNLAARDFINWLIQTKITD [0864] In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 182 or a functional fragment thereof. In some embodiments, 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 a nucleic acid sequence encoding SEQ ID NO: 182 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide in low-oxygen conditions.
[0865] In some embodiments, 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 tryptophan metabolites bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, 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 GLP-2 or one of its analogs than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight- fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more GLP-2 or one of its analogs than unmodified bacteria of the same bacterial subtype under the same conditions.
[0866] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In some embodiments, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[0867] In some embodiments, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[0868] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the
embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof is modified and/or mutated, e.g., to enhance stability, or increase GLP-2 production, secretion, or display. [0869] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof 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.
[0870] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences described herein for the consumption of ammonia.
[0871] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof 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.
[0872] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[0873] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[0874] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of acetate. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof 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. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences for the secretion of an anti-inflammatory cytokine. In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more gene sequences for the secretion of IL-22.
[0875] In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise a GABA transport circuit and/or a GABA metabolic circuit.. In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more polypeptides for the secretion or surface display of GLP-2 or an analog thereof further comprise one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[0876] In any of the embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Inducible Promoters
[0877] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes.
[0878] Herein the terms“polypeptide of interest” or“polypeptides of interest”, “protein of interest”,“proteins of interest”,“payloads”“effector molecule”,“effector” refers to one or more effector molecules described herein and/or one or more enzyme(s) or polypeptides, which function as enzymes for the production of such effector molecules. Effectors or effector molecules can be expressed or produced by the genetically engineered bacteria for preventing, treating or managing a disease, condition, or disorder, e.g., a disease condition, or disorder associated with
hyperammonemia. Non-limiting examples of effectors include ArgAfbr, mutated Arg Boxes, mutated ArgR, mutated ArgG, butyrate, propionate, GABA-metabolizing enzymes, GABA-transporter, Mn-transporter, tryptophan or any of its metabolites, e.g., kynurenine, kynurenic acid, and indole metabolites described herein, or secreted or surface displayed polypeptides, e.g., GLP-2 or IL-22.
[0879] Non-limiting examples of payloads, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, include gene sequences and/or polypeptides encoded by the gene sequences for the production of ArgAfbr, mutated Arg Boxes, mutated ArgR, mutated ArgG, butyrate, propionate, GABA-metabolizing enzymes, GABA-transporter, Mn-transporter, tryptophan or any of its metabolites, e.g., kynurenine, kynurenic acid, and indole metabolites described herein, or secreted or surface displayed polypeptides, e.g., GLP-2 or IL-22. As used herein, the term“gene of interest” or“gene sequence of interest” includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more effectors (and/or enzymes for the production of effectors for the treatment described herein, e.g., effectors useful for preventing, treating or managing a disease associated with hyperammonemia.
[0880] In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced, e.g., under low-oxygen or anaerobic conditions. In some
embodiments, the gene encoding the payload is present on plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.
[0881] In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein.
[0882] In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on the chromosome. In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on one or more same or different plasmids. In some embodiments, the genetically engineered bacteria comprise two or more payloads, some of which are present on the chromosome and some of which are present on one or more same or different plasmids.
[0883] In any of the embodiments described above, the one or more gene sequence(s) for producing the payloads useful for preventing, treating or managing a disease associated with hyperammonemia, combinations are operably linked to one or more directly or indirectly inducible promoter(s). In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under exogenous environmental conditions, e.g., conditions found in the gut. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced by metabolites found in the gut or other specific conditions, e.g., conditions associated with hyperammonemia. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under inflammatory conditions (e.g., RNS, ROS), as described herein. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under certain conditions, e.g., as found during hyperammonemia and/or one of the diseases, disorders or conditions associated with hyperammonemia, as described herein.
[0884] In some embodiments, the two or more gene sequence(s) are linked to a directly or indirectly inducible promoter that is induced by exposure a chemical or nutritional inducer, which may or may not be present under in vivo conditions and which may be present during in vitro conditions (such as strain culture, expansion, manufacture), such as tetracycline or arabinose, or others described herein. In some embodiments, the two or more payloads are all linked to a constitutive promoter. Such constitutive promoters are described in Table IX and Table X herein.
[0885] Subjects with hepatic encephalopathy (HE) and other liver disease or disorders have chronic liver damage that results in high ammonia levels in their blood and intestines. In addition to ammonia, these subjects also have elevated levels of 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, and manganese in their blood and intestines. Promoters that respond to one of these HE– related molecules or their metabolites may be used in the genetically engineered bacteria to induce expression of one or more payloads, effectors or proteins of interest, e.g., in the gut. These promoters would not be expected to be induced in non-HE patients. Thus, in some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced by one or more of metabolites and other molecules elevated in diseases, disorders, conditions associated with hyperammoniemia, e.g., HE and other conditions described herein, which include but are not limited to, 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, and manganese.
[0886] In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is induced under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.
[0887] In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions).
[0888] In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous
environmental conditions specific to the anaerobic environment of the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, such molecules or metabolites are 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.
[0889] In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell.
FNR dependent Regulation
[0890] The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia (e.g., an effector metabolite, one or more enzymes for producing such a metabolite, or an effector polypeptide for secretion or display on the cell surface), wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the inducible promoter is an oxygen level-dependent promoter and the payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level-sensing transcription factor, thereby driving production of the payload.
[0891] 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 is 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. In one embodiment, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter. In a more specific aspect, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut.
[0892] In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In E. coli, FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed Table III below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
Table III. FNR Promoter sequences FNR-responsive
regulatory region 12345678901234567890123456789012345678901234567890
ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTAGAGC SEQ ID NO: 18 GTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCC
GACAGGCTCCCACAGGAGAAAACCG CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGC SEQ ID NO: 19 CCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCC
CACAGGAGAAAACCG GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC
nirB1 TATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGA
AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC SEQ ID NO: 20 AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA
ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAG AAATCGAGGCAAAA CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTAC AGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGTTA GGTTTCGTCAGCCGTCACCGTCAGCATAACACCCTGACCTCTCATT
nirB2 AATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCTTTTCCTCT
CTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTC SEQ ID NO: 21 TATTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAG
AAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAAA GGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAG GTAGGCGGTAATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaa ggagatatacat GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATC
nirB3 TATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGA SEQ ID NO: 22 AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC
AATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCA ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAG AAATCGAGGCAAAA ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTAT
ydfZ GGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAA SEQ ID NO: 23 AAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTG
GGCTTCGACTGTAAATCAGAAAGGAGAAAACACCT GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC
nirB+RBS TATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGA
AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC SEQ ID NO: 24 AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA
ATAAGCGGGGTTGCTGAATCGTTAAGGATCCCTCTAGAAATAATT TTGTTTAACTTTAAGAAGGAGATATACAT CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTA ydfZ+RBS TGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACA SEQ ID NO: 25 AAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCC
CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT
fnrS1 GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC
CGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC SEQ ID NO: 26 AATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAA
GAAGGAGATATACAT AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT
fnrS2 GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC
CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC SEQ ID NO: 27 AATATCTCTCTTGGATCCAAAGTGAACTCTAGAAATAATTTTGTTT
AACTTTAAGAAGGAGATATACAT TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGT CAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCA
nirB+crp CTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTA
TTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAA SEQ ID NO: 28 TATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAAT
ATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATA AGCGGGGTTGCTGAATCGTTAAGGTAGaaatgtgatctagttcacatttGCGGTA ATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaaggagatatacat AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT
fnrS+crp GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC SEQ ID NO: 29 CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC
AATATCTCTCaaatgtgatctagttcacattttttgtttaactttaagaaggagatatacat
[0893] FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload.
[0894] Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria comprise one or more of: SEQ ID NO: 18, SEQ ID NO: 19, nirB1 promoter (SEQ ID NO: 20), nirB2 promoter (SEQ ID NO: 21), nirB3 promoter (SEQ ID NO: 22), ydfZ promoter (SEQ ID NO: 23), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 24), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 25), fnrS, an anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 26 or fnrS2 promoter SEQ ID NO: 27), nirB promoter fused to a crp binding site (SEQ ID NO: 28), and fnrS fused to a crp binding site (SEQ ID NO: 29). In some embodiments, the FNR-responsive promoter 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 sequence of any one of SEQ ID NO: 20-29.
[0895] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut.
[0896] In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, expressed under the control of anaerobic regulation of arginine deiminiase and nitrate reduction
transcriptional regulator (ANR). In P. aeruginosa, ANR is“required for the expression of physiological functions which are inducible under oxygen-limiting or anaerobic conditions” (Winteler et al., 1996; Sawers 1991). P. aeruginosa ANR is homologous with E. coli FNR, and“the consensus FNR site (TTGAT----ATCAA) was recognized efficiently by ANR and FNR” (Winteler et al., 1996). Like FNR, in the anaerobic state, ANR activates numerous genes responsible for adapting to anaerobic growth. In the aerobic state, ANR is inactive. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all have functional analogs of ANR (Zimmermann et al., 1991). Promoters that are regulated by ANR are known in the art, e.g., the promoter of the arcDABC operon (see, e.g., Hasegawa et al., 1998).
[0897] ln other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Görke and Stülke, 2008). In some embodiments, the gene or gene cassette for producing the payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g., an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing a payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro.
[0898] In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen-level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic
environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N. gonorrhoeae (see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity.
[0899] In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic
environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen- level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006). In some embodiments, both the oxygen level-sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions.
[0900] In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid.
[0901] In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be
advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region.
RNS-dependent regulation
[0902] In some embodiments, the genetically engineered bacteria comprise a gene encoding a payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter.
[0903] As used herein,“reactive nitrogen species” and“RNS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO•), peroxynitrite or peroxynitrite anion (ONOO-), nitrogen dioxide (•NO2), dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO2-) (unpaired electrons denoted by•). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics.
[0904] As used herein,“RNS-inducible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s), e.g., any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences.
[0905] As used herein,“RNS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS- derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes.
[0906] As used herein,“RNS-repressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the
transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences.
[0907] As used herein, a“RNS-responsive regulatory region” refers to a RNS- inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS- derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table IV.
Table IV. Examples of RNS-sensing transcription factors and RNS-responsive genes
Figure imgf000410_0001
[0908] In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS- sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated.
[0909] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression.
[0910] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR“is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide” (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload(s), e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia,.
[0911] In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR
(dissimilatory nitrate respiration regulator)“promotes the expression of the nir, the nor and the nos genes” in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is Pseudomonas aeruginosa DNR.
[0912] In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing a payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, expressed under the control of the dissimilatory nitrate respiration regulator (DNR). DNR is a member of the FNR family (Arai et al., 1995) and is a transcriptional regulator that is required in conjunction with ANR for“anaerobic nitrate respiration of Pseudomonas aeruginosa” (Hasegawa et al., 1998). For certain genes, the FNR-binding motifs“are probably recognized only by DNR” (Hasegawa et al., 1998). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used. Non-limiting examples include ArcA/B, ResD/E, NreA/B/C, and AirSR, and others are known in the art.
[0913] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.
[0914] In some embodiments, the tunable regulatory region is a RNS- derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is“an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS- responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is Neisseria gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s).
[0915] In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some
embodiments, the genetically engineered bacterium of the invention expresses a RNS- sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the RNS-sensing transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae, wherein the
Escherichia coli does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
[0916] In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
[0917] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a payload. In one aspect of these embodiments, the RNS- sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments, include, but are not limited to, TetR, C1, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a payload gene or genes is expressed.
[0918] A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS- responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012).
[0919] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS- sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS- sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.
[0920] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions.
[0921] In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from Neisseria gonorrhoeae. In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity.
[0922] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome. [0923] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS.
[0924] In some embodiments, the gene or gene cassette for producing the payload, e.g., the payload useful for preventing, treating or managing a disease associated with hyperammonemia, is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, 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.
[0925] In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions.
[0926] In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500- fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory
biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen).
[0927] In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of payload in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS.
ROS-dependent regulation
[0928] In some embodiments, the genetically engineered bacteria comprise a gene for producing a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter.
[0929] As used herein,“reactive oxygen species” and“ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal- catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H2O2), organic peroxide (ROOH), hydroxyl ion (OH-), hydroxyl radical (•OH), superoxide or superoxide anion (•O2-), singlet oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl radical (•O2-2), hypochlorous acid (HOCl), hypochlorite ion (OCl-), sodium hypochlorite (NaOCl), nitric oxide (NO•), and peroxynitrite or peroxynitrite anion (ONOO-) (unpaired electrons denoted by•). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014).
[0930] As used herein,“ROS-inducible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS- inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS- inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes.
[0931] As used herein,“ROS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS- derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette.
[0932] As used herein,“ROS-repressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes.
[0933] As used herein, a“ROS-responsive regulatory region” refers to a ROS- inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS- derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table V.
Table V. Examples of ROS-sensing transcription factors and ROS-responsive genes ROS-sensing Primarily capable Examples of responsive genes, transcription factor: of sensing: promoters, and/or regulatory
regions:
OxyR H2O2 ahpC; ahpF; dps; dsbG; fhuF; flu;
fur; gor; grxA; hemH; katG; oxyS; ROS-sensing Primarily capable Examples of responsive genes, transcription factor: of sensing: promoters, and/or regulatory
regions:
sufA; sufB; sufC; sufD; sufE; sufS; trxC; uxuA; yaaA; yaeH; yaiA; ybjM; ydcH; ydeN; ygaQ; yljA; ytfK
PerR H2O2 katA; ahpCF; mrgA; zoaA; fur;
hemAXCDBL; srfA
OhrR Organic peroxides ohrA
NaOCl
SoxR •O - 2 soxS
NO•
(also capable of
sensing H2O2)
RosR H2O2 rbtT; tnp16a; rluC1; tnp5a; mscL;
tnp2d; phoD; tnp15b; pstA; tnp5b; xylC; gabD1; rluC2; cgtS9; azlC; narKGHJI; rosR [0934] In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated.
[0935] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression.
[0936] In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR “functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g.,“genes involved in H2O2 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe-S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)” and“OxyS, a small regulatory RNA” (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene. In the presence of ROS, e.g., H2O2, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload. In some embodiments, OxyR is encoded by an E. coli oxyR gene. In some embodiments, the oxyS regulatory region is an E. coli oxyS regulatory region. In some embodiments, the ROS-inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC.
[0937] In alternate embodiments, the tunable regulatory region is a ROS- inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is“activated by oxidation of its [2Fe–2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression” (Koo et al., 2003). “SoxR is known to respond primarily to superoxide and nitric oxide” (Koo et al., 2003), and is also capable of responding to H2O2. The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al., 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload.
[0938] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette.
[0939] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR“binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event,” but oxidized OhrR is“unable to bind its DNA target” (Duarte et al., 2010). OhrR is a“transcriptional repressor [that]… senses both organic peroxides and NaOCl” (Dubbs et al., 2012) and is“weakly activated by H2O2 but it shows much higher reactivity for organic hydroperoxides” (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a payload gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload.
[0940] OhrR is a member of the MarR family of ROS-responsive regulators. “Most members of the MarR family are transcriptional repressors and often bind to the - 10 or -35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some
embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012). [0941] In some embodiments, the tunable regulatory region is a ROS- derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is“a MarR-type transcriptional regulator” that binds to an“18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is “reversibly inhibited by the oxidant H2O2” (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to“a putative polyisoprenoid-binding protein (cg1322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cg1426), two putative FMN reductases (cg1150 and cg1850), and four putative monooxygenases (cg0823, cg1848, cg2329, and cg3084)” (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload. In the presence of ROS, e.g., H2O2, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload.
[0942] In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some
embodiments, the genetically engineered bacterium of the invention expresses a ROS- sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is Escherichia coli, and the ROS-sensing transcription factor is RosR, e.g., from Corynebacterium glutamicum, wherein the Escherichia coli does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
[0943] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence.
[0944] In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In Bacillus subtilis, PerR“when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis
(hemAXCDBL, fur, and zoaA) and its own synthesis (perR)” (Marinho et al., 2014). PerR is a“global regulator that responds primarily to H2O2” (Dubbs et al., 2012) and “interacts with DNA at the per box, a specific palindromic consensus sequence
(TTATAATNATTATAA) residing within and near the promoter sequences of PerR- controlled genes” (Marinho et al., 2014). PerR is capable of binding a regulatory region that“overlaps part of the promoter or is immediately downstream from it” (Dubbs et al., 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al., 2012).
[0945] In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these
embodiments include, but are not limited to, TetR, C1, and LexA. In some
embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed.
[0946] A ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although“OxyR is primarily thought of as a transcriptional activator under oxidizing conditions ... OxyR can function as either a repressor or activator under both oxidizing and reducing conditions” (Dubbs et al., 2012), and OxyR“has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)” (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by RosR. In addition,“PerR-mediated positive regulation has also been observed…and appears to involve PerR binding to distant upstream sites” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by PerR.
[0947] One or more types of ROS-sensing transcription factors and
corresponding regulatory region sequences may be present in genetically engineered bacteria. For example,“OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS- sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS- sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS- responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence.
[0948] Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table VI. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 580, SEQ ID NO: 581, SEQ ID NO: 582, or SEQ ID NO: 583, or a functional fragment thereof. Table VI. Nucleotide sequences of exemplary OxyR-regulated regulatory regions
Figure imgf000426_0001
Regulatory
sequence Sequence
CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGCG
oxyS ATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTCTG (SEQ ID ACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGTA NO: 66) CC [0949] In some embodiments, the regulatory region sequence 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 sequence of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and/or SEQ ID NO: 66.
[0950] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS- sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS- sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region.
[0951] In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions. [0952] In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from Escherichia coli. In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate
embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wild-type activity.
[0953] In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome.
[0954] In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS-responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS.
[0955] In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, 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.
[0956] In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some
embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS-responsive regulatory region.
[0957] Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a
corresponding transcription factor.
[0958] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome.
Propionate and other promoters
[0959] In some embodiments, the genetically engineered bacteria comprise the gene or gene cassette for producing a payload, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia, is expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., the mammalian gut. In some embodiments, such molecules or metabolites are 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.
[0960] For example, the short-chain fatty acid propionate is a major microbial fermentation metabolite localized to the gut (Hosseini et al., 2011). In one embodiment, the gene or gene cassette for producing a payload useful for preventing, treating or managing a disease associated with hyperammonemia is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the payload useful for preventing, treating or managing a disease associated with hyperammonemia is under the control of a propionate-inducible promoter that is activated by the presence of propionate in the mammalian gut. Any molecule or metabolite found in the mammalian gut, in a healthy and/or disease state, may be used to induce payload expression. Non-limiting examples of inducers include propionate, 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, and manganese. In alternate embodiments, the gene or gene cassette for producing the payload useful for preventing, treating or managing a disease associated with hyperammonemia is under the control of a pBAD promoter, which is activated in the presence of the sugar arabinose.
[0961] In some embodiments, the gene or gene cassette for producing the payload useful for preventing, treating or managing a disease associated with hyperammonemia is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut and/or specific metabolites present under conditions of hyperammonemia, e.g., HE-specific molecules. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut and/or to certain conditions encountered during hyperammonemia, e.g. HE-specific metabolites. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, 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.
[0962] In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the payload useful for preventing, treating or managing a disease associated with hyperammonemia, such that the gene or gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing the payload. In some embodiments, gene or gene cassette for producing the payload is expressed on a low- copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, gene or gene cassette for producing the payload is expressed on a high- copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette for producing the payload is expressed on a chromosome.
[0963] Table VII lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut. In some embodiments, the propionate promoter sequence 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 sequence of SEQ ID NO: 183.
Table VII. Propionate promoter sequence Description Sequence
Prp (Propionate) TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCA promoter CAGCGTGGTACGGCTGATCCCCAAATAACGTGCGGCGGCGCG Bold: prpR CTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG Lower case: CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCAC ribosome binding CAGTTCAGGTAATAATTGCCGCATAAATTGTCTGTCCAGTGT site TGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT ATG underlined: CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTT start of gene of CAGTAGAAGCGGCTGACACTGCGTCAGCCCATGACGCACCGA interest TTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA SEQ ID NO: 183 AAAGTTTTCCGCCAGAGGCAGAATATCAGGCTGTCGCTCGCG
CAAGGGGGGAAGCGGCAGACGCAGAATGCTCAAACGGTAAAA CAGATCGGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG ATCGCAATGCGTGGCGCTGATCACCCGGACATCTACCGGGAT CGGCTGATGCCCGCCAACGCGGGTGACGGCTTTTTCCTCCAG TACGCGTAGAAGGCGGGTTTGTAACGGCAGCGGCATTTCGCC AATTTCGTCAAGAAACAGCGTGCCGCCGTGGGCGACCTCAAA CAGCCCCGCACGTCCACCTCGTCTTGAGCCGGTAAACGCTCC CTCCTCATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGGT AATCGCGCCGCAATTAACGGCGACAAAGGGCGGAGAAGGCTT GTTCTGACGGTGGGGCTGACGGTTAAACAACGCCTGATGAAT CGCTTGCGCCGCCAGCTCTTTCCCGGTCCCTGTTTCCCCCTG AATCAGCACTGCCGCGCGGGAACGGGCATAGAGTGTAATCGT ATGGCGAACCTGCTCCATTTGTGGTGAATCGCCGAGGATATC GCTCAGCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTATG CTGGCTATACTGACGCCGTGTCAGGCGGGTCATATCCAGCGC ATCATGGAAAGCCTGACGTACGGTGGCCGCTGAATAAATAAA GATGGCGGTCATTCCTGCCTCTTCCGCCAGGTCGGTAATTAG TCCTGCCCCAATTACAGCCTCAATGCCGTTAGCTTTGAGCTC GTTAATTTGCCCGCGAGCATCCTCTTCAGTGATATAGCTTCG CTGTTCAAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGA AGTCAGCTTTCCCGCTTTTGCCAGAGCCTGTAATACATCGAA TCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTC GCAGCGTTCGGTTGCCAGTTTTTTGCGAATGTAGGCTACTGC CTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCG CGTTACCGAGACCGTCCAGATCACCGGTTTATCGCTATTATC GCGCGAAGCGCTATGCACAGTAACCATCGTCGTAGATTCATG TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGT TGCAATGAAACACAGGCCTCCGTTTCATGAAACGTTAGCTGA CTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATG GTTTCTCGGTTTTTGGGTCTGGCATATCCCTTGCTTTAATGA GTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag taatttcaacaaaataacgagcattcgaatg
Other Inducible Promoters
[0964] In some embodiments, the gene encoding the payload useful for preventing, treating or managing a disease associated with hyperammonemia is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).
[0965] In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the one or more gene sequences(s), inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), encoding the payload useful for preventing, treating or managing a disease associated with hyperammonemia, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct copies of the one or more gene sequences(s) encoding the payload, which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same one or more gene sequences(s) encoding the payload, which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding the payload(s), is present on a plasmid and operably linked to a directly or indirectly inducible promoter inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding the payloads, is present on a chromosome and operably linked to a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).
[0966] In some embodiments, one or more gene sequence(s) encoding polypeptides of interest described herein is present on a plasmid and operably linked to promoter a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene encoding the payload useful for preventing, treating or managing a disease associated with hyperammonemia, which is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), such that the payloads can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., under culture conditions, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same gene sequence(s) for the production of a polypeptide of interest which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of different gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).
[0967] In some embodiments, the gene sequence(s) for the production of a polypeptide of interest, e.g., a payload useful for preventing, treating or managing a disease associated with hyperammonemia is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, gene sequence(s) for the production of a polypeptide of interest is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or
metabolite(s).
[0968] In some embodiments, the promoter that is operably linked to the gene encoding the polypeptide of interest is directly or indirectly induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s).
[0969] In some embodiments, one or more inducible promoter(s) are useful for or induced during in vivo expression of the one or more protein(s) of interest, e.g. a payload useful for preventing, treating or managing a disease associated with hyperammonemia. In some embodiments, the promoters are induced during in vivo expression of one or more payloads and/or polypeptide(s) of interest. In some embodiments, expression of one or more payloads(s) and/or other polypeptide(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a chemical and/or nutritional inducer and/or metabolite which is co-administered with the genetically engineered bacteria of the invention.
[0970] In some embodiments, expression of one or more payloads useful for preventing, treating or managing a disease associated with hyperammonemia and/or other polypeptide(s) of interest, is driven directly or indirectly by one or more promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite during in vitro growth, preparation, or manufacturing of the strain prior to in vivo
administration. In some embodiments, the promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with payloads (s) and/or other polypeptide(s) of interest prior to administration. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown aerobically. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown anaerobically.
[0971] The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub-saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L- arabinose transporter (Zhang et al., Development and Application of an Arabinose- Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum; Appl. Environ. Microbiol. August 2012 vol. 78 no. 165831-5838).
Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in FIG. 88C.
[0972] In one embodiment, expression of one or more payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more arabinose inducible promoter(s).
[0973] In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more payloads useful for preventing, treating or managing a disease associated with hyperammonemia is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., arabinose.
[0974] In some embodiments, expression of one or more protein(s) of interest, e.g., one or more payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown aerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically.
[0975] In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more payloads and/or protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, such molecules or metabolites are 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.
[0976] In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s).
[0977] In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.
[0978] In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter
[0979] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 283. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 284. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 285.
[0980] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system 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. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein.
[0981] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter.
[0982] In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co- administered with the genetically engineered bacteria of the invention, e.g., rhamnose.
[0983] In some embodiments, expression of one or more payloads and/or protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown arerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically.
[0984] In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some
embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, such molecules or metabolites are 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. [0985] In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s).
[0986] In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest , from a construct which is integrated into the bacterial chromosome.
Exemplary insertion sites are described herein.
[0987] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 286.
[0988] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-1- thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydrolyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacI) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by E. coli, its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate LacI, can be used instead of IPTG in a similar manner.
[0989] In one embodiment, expression of one or more payloads or protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more IPTG inducible promoter(s).
[0990] In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the payload and/or one or more protein(s) of interest. In some embodiments, expression of one or more payload and/or protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., IPTG.
[0991] In some embodiments, expression of one or more payloads and/or protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown arerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically.
[0992] In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more payloads and/or protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non- limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, such molecules or metabolites are 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.
[0993] In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).
[0994] In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.
[0995] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 287. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacI, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 288. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 289.
[0996] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M & Bujard H.PNAS, 1992 Jun 15;89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline- controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline- controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.
[0997] In one embodiment, expression of one or more protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more tetracycline inducible promoter(s).
[0998] In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., tetracycline.
[0999] In some embodiments, expression of one or more payloads or protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically.
[1000] In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, such molecules or metabolites are 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.
[1001] In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).
[1002] In some embodiments, the tetracycline inducible promoter drives the expression of one or more payloads and/or protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. [1003] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 294 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 294 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 294 in italics (Tet repressor is in italics).
[1004] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy; J Biotechnol. 2015 Jun 10; 203: 32–40, and references therein). Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the λ promoters can then be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage λ. At temperatures below 37 °C, cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in FIG. 88A. Inducible expression from the ParaBad can be controlled or further fine- tuned through the optimization of the ribosome binding site (RBS), as described herein.
[1005] In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). [1006] In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature.
[1007] In some embodiments, expression of one or more protein(s) of interest, e.g,. payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a
thermoregulated system by growing the strain at lower temperatures, e.g., 30 C.
Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically.
[1008] In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest, e.g., payloads useful for preventing, treating or managing a disease associated with hyperammonemia, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, such molecules or metabolites are 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.
[1009] In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s).
[1010] In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.
[1011] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 290. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest . In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 291. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 293.
[1012] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions.
[1013] 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 conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. FIG. 89A depicts a schematic of the gene organization of a PssB promoter.
[1014] In one embodiment, expression of one or more protein(s) of interest, e.g,. payloads useful for preventing, treating or managing a disease associated with hyperammonemia, is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s).
[1015] In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest. In some
embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.
[1016] In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein.
[1017] In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The
chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA -as the case may be- are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, 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 conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 298.
[1018] Sequences useful for expression from inducible promoters are listed in Table VIII.
Table VIII. Inducible promoter construct sequences
Figure imgf000449_0001
polypeptide TPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFV CRPGDILLFPPGEIHHYGRHPEAHEWYHQWVYFRPRAYWHE SEQ ID NO: WLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYS 285 ELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHL
ADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRIS QAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSE FRAGCE*
Region CGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAAC comprising ATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTT rhamnose TCCTGTCAGTAACGAGAAGGTCGCGAATCAGGCGCTTTTT inducible AGACTGGTCGTAATGAAATTCAGCTGTCACCGGATGTGCT promoter TTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTAC
AAATAATTTTGTTTAAAACAACACCCACTAAGATAACTCT SEQ ID NO: AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 286
Lac Promoter ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG region CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG
CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA SEQ ID NO: TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG 287 TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG
TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA CAT
LacO GGAATTGTGAGCGCTCACAATT
LacI (in reverse TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC orientation) TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT
GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA SEQ ID NO: GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA 288 GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA
GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT TCAT
LacI MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEA polypeptide AMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAA sequence IKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLI
INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT SEQ ID NO: RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN 289 QIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQ
MALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIK QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA PNTQTASPRALADSLMQLARQVSRLESGQ
Region ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC comprising GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG Temperature CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCC sensitive GTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAA promoter CAACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAA
CTTTAAGAAGGAGATATACAT SEQ ID NO:
290
mutant cI857 TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT repressor TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG
GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT SEQ ID NO: ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCA 291 AGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCT
CAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGG CTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACC TCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGC TTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGC TTAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACA GGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAA CCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGG GCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCA ATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAA TAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCT GCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTT TTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGC TCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT RBS and leader CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA region CAT SEQ ID NO:
292
mutant cI857 MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM repressor GMGQSGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAR polypeptide EIYEMYEAVSMQPSLRSEYEYPVFSHVQAGMFSPKLRTFTKG sequence DAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGML
ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP SEQ ID NO: QYPMIPCNESCSVVGKVIASQWPEETFG
293
TetR-Tet Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataa promoter gaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcata construct ctatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacct SEQ ID NO: aaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttgg 294 cataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgta cttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaat cttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggct aaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacaccta gcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcg ctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgttgacactctatcattg atagagttattttaccactccctatcagtgatagagaaaagtgaactctagaaataattttgttt aactttaagaaggagatatacat PssB promoter tcacctttcccggattaaacgcttttttgcccggtggcatggtgctaccggcgatcacaaacggtta attatgacacaaattgacctgaatgaatatacagtattggaatgcattacccggagtgttgtgtaac SEQ ID NO: aatgtctggccaggtttgtttcccggaaccgaggtcacaacatagtaaaagcgctattggtaatgg 298 tacaatcgcgcgtttacacttattc
Constitutive promoters
[1019] In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter.
[1020] In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., conditions encountered in the gut and/or certain conditions encountered during hyperammonemia, e.g., in the presence of certain metabolites, e.g., HE-specific metabolites, as described herein. In some embodiments, the promoters is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoter is active under in vivo conditions, e.g conditions encountered in the gut and/or certain conditions encountered during hyperammonemia, e.g., in the presence of certain metabolites, e.g., HE-specific metabolites, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein.
[1021] In some embodiments, the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). [1022] In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal and/or certain conditions encountered during hyperammonemia, e.g., in the presence of certain metabolites, e.g., HE-specific metabolites, as described herein.. In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoter is active in low-oxygen or anaerobic conditions such as the environment of the mammalian gut and/or certain conditions encountered during hyperammonemia, e.g., in the presence of certain metabolites, e.g.. HE-specific metabolites, as described herein.. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal and/or certain conditions encountered during
hyperammonemia, e.g., in the presence of certain metabolites, e.g., HE-specific metabolites, as described herein. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions.
[1023] Bacterial constitutive promoters are known in the art. Exemplary constitutive promoters are listed in the following Tables. Table IX. Constitutive promoters Name Source Description Promoter Lengt SEQ
Sequence h ID
NO
Constitutive E. ...
BBa_I14018 coli σ70 P(Bla) gtttatacataggcgagta 35 299 promoter ctctgttatgg
Constitutive E. ...
BBa_I14033 coli σ70 P(Cat) agaggttccaactttcacc 38 300 promoter ataatgaaaca
Constitutive E. ...
BBa_I14034 coli σ70 P(Kat) taaacaactaacggacaa 45 301 promoter ttctacctaaca
Constitutive E. Template for ...
BBa_I732021 coli σ70 Building acatcaagccaaattaaac 159 302 promoter Primer Family
Member aggattaacac
BBa_I742126 Constitutive E. Reverse ...
coli σ70 lambda cI- gaggtaaaatagtcaaca 49 303 promoter regulated cgcacggtgtta
promoter
Constitutive E. ...
BBa_J01006 coli σ70 Key Promoter
promoter absorbs 3 caggccggaataactccc 59 304 tataatgcgcca
Constitutive E. constitutive
BBa_J23100 coli σ70 promoter ...
promoter family ggctagctcagtcctaggt 35 305 member acagtgctagc
Constitutive E. constitutive ...
BBa_J23101 coli σ70 promoter agctagctcagtcctaggt 35 306 promoter family
member attatgctagc
Constitutive E. constitutive
BBa_J23102 coli σ70 promoter ...
35 307 promoter family agctagctcagtcctaggt
member actgtgctagc
Constitutive E. constitutive
BBa_J23103 coli σ70 promoter ...
tagctcagtcctagg 35 308 promoter family agc
member gattatgctagc
Constitutive E. constitutive
BBa_J23104 coli σ70 promoter ...
promoter family agctagctcagtcctaggt 35 309 member attgtgctagc
Constitutive E. constitutive
BBa_J23105 coli σ70 promoter ...
promoter family ggctagctcagtcctaggt 35 310 member actatgctagc
Constitutive E. constitutive ...
BBa_J23106 coli σ70 promoter
promoter family ggctagctcagtcctaggt 35 311 member atagtgctagc
Constitutive E. constitutive
BBa_J23107 coli σ70 promoter ...
gg 35 312 promoter family ggctagctcagcccta
member tattatgctagc
Constitutive E. constitutive ...
BBa_J23108 coli σ70 promoter agctagctcagtcctaggt 35 313 promoter family
member ataatgctagc
Constitutive E. constitutive
BBa_J23109 coli σ70 promoter ...
promoter family agctagctcagtcctagg 35 314 member gactgtgctagc
Constitutive E. constitutive
BBa_J23110 coli σ70 promoter ...
tagctcagtcctaggt 35 315 promoter family ggc
member acaatgctagc
Constitutive E. constitutive ...
BBa_J23111 coli σ70 promoter ggctagctcagtcctaggt 35 316 promoter family atagtgctagc member
Constitutive E. constitutive
BBa_J23112 coli σ70 promoter ...
tcagtcctagg 35 317 promoter family agctagc
member gattatgctagc
Constitutive E. constitutive ...
BBa_J23113 coli σ70 promoter
promoter family ggctagctcagtcctagg 35 318 member gattatgctagc
Constitutive E. constitutive
BBa_J23114 coli σ70 promoter ...
ctcagtcctaggt 35 319 promoter family ggctag
member acaatgctagc
Constitutive E. constitutive
BBa_J23115 coli σ70 promoter ...
35 320 promoter family agctagctcagcccttggt
member acaatgctagc
Constitutive E. constitutive
BBa_J23116 coli σ70 promoter ...
ctcagtcctagg 35 321 promoter family agctag
member gactatgctagc
Constitutive E. constitutive
BBa_J23117 coli σ70 promoter ...
promoter family agctagctcagtcctagg 35 322 member gattgtgctagc
Constitutive E. constitutive
BBa_J23118 coli σ70 promoter ...
gctcagtcctaggt 35 323 promoter family ggcta
member attgtgctagc
Constitutive E. constitutive
BBa_J23119 coli σ70 promoter ...
35 324 promoter family agctagctcagtcctaggt
member ataatgctagc
Constitutive E.
BBa_J23150 coli σ70 1bp mutant ...
promoter from J23107 ggctagctcagtcctaggt 35 325 attatgctagc
Constitutive E. ...
BBa_J23151 coli σ70 1bp mutant
promoter from J23114 ggctagctcagtcctaggt 35 326 acaatgctagc
Constitutive E. ...
BBa_J44002 coli σ70 pBAD reverse aaagtgtgacgccgtgca 130 327 promoter aataatcaatgt
NikR
promoter, a
Constitutive E. protein of the
BBa_J48104 coli σ70 ribbon helix- ...
promoter helix family of gacgaatacttaaaatcgt 40 328 trancription catacttattt
factors that
repress expre
BBa_J54200 Constitutive E. ...
coli σ70 lacq_Promoter aaacctttcgcggtatggc 50 329 promoter atgatagcgcc Constitutive E. lacIQ - ...
BBa_J56015 coli σ70 promoter tgatagcgcccggaaga 57 330 promoter sequence gagtcaattcagg
E. coli
Constitutive E. CreABCD ...
BBa_J64951 coli σ70 phosphate ttatttaccgtgacgaacta 81 331 promoter sensing operon attgctcgtg
promoter
Constitutive E. ...
BBa_K088007 coli σ70 GlnRS catacgccgttatacgttgt
promoter promoter 38 332 ttacgctttg
Constitutive E. Constitutive ...
BBa_K119000 coli σ70 weak promoter ttatgcttccggctcgtatg 38 333 promoter of lacZ ttgtgtggac Constitutive E. ...
BBa_K119001 coli σ70 Mutated LacZ
promoter promoter ttatgcttccggctcgtatg 38 334 gtgtgtggac
BBa_K133000 Constitutive E. Constitutive ...
coli σ70 promoter ggctagctcagtcctaggt 35 335 2 promoter (J23105) actatgctagc
constitutive
Constitutive E. promoter with ...
BBa_K137029 coli σ70 (TA)10
promoter between -10 atatatatatatatataatgg 39 336 and -35 aagcgtttt
elements
constitutive
Constitutive E. promoter with ...
BBa_K137030 coli σ70 (TA)9 between atatatatatatatataatgg 37 337 promoter -10 and -35 aagcgtttt
elements
constitutive
Constitutive E. promoter with ...
BBa_K137031 coli σ70 (C)10 between ccccgaaagcttaagaat 62 338 promoter -10 and -35 ataattgtaagc
elements
constitutive
Constitutive E. promoter with ...
BBa_K137032 coli σ70 (C)12 between ccccgaaagcttaagaat 64 339 promoter -10 and -35 ataattgtaagc
elements
optimized
(TA) repeat
Constitutive E. constitutive ...
BBa_K137085 coli σ70 promoter with tgacaatatatatatatatat 31 340 promoter 13 bp between aatgctagc
-10 and -35
elements
Constitutive E. optimized ...
BBa_K137086 coli σ70 (TA) repeat acaatatatatatatatatat 33 341 promoter constitutive
promoter with aatgctagc 15 bp between
-10 and -35
elements
optimized
(TA) repeat
Constitutive E. constitutive ...
BBa_K137087 coli σ70 promoter with aatatatatatatatatatat 35 342 promoter 17 bp between aatgctagc
-10 and -35
elements
optimized
(TA) repeat
Constitutive E. constitutive ...
BBa_K137088 coli σ70 promoter with tatatatatatatatatatata 37 343 promoter 19 bp between atgctagc
-10 and -35
elements
optimized
(TA) repeat
Constitutive E. constitutive ...
BBa_K137089 coli σ70 promoter with tatatatatatatatatatata 39 344 promoter 21 bp between atgctagc
-10 and -35
elements
optimized (A)
repeat
Constitutive E. constitutive ...
BBa_K137090 coli σ70 promoter with aaaaaaaaaaaaaaaaaa 35 345 promoter 17 bp between tataatgctagc
-10 and -35
elements
optimized (A)
repeat
Constitutive E. constitutive ...
BBa_K137091 coli σ70 promoter with aaaaaaaaaaaaaaaaaa 36 346 promoter 18 bp between tataatgctagc
-10 and -35
elements
Anderson
BBa_K158510 Constitutive E. ...
0 coli σ70 Promoter with
promoter lacI binding ggaattgtgagcggataa 78 347 site caatttcacaca
Anderson
BBa_K158510 Constitutive E. ...
1 coli σ70 Promoter with 78 348 promoter lacI binding ggaattgtgagcggataa
site caatttcacaca
Anderson
BBa_K158510 Constitutive E. ...
2 coli σ70 Promoter with
promoter lacI binding ggaattgtgagcggataa 78 349 site caatttcacaca
BBa_K158510 Constitutive E. Anderson ...
coli σ70 Promoter with ggaattgtgagcggataa 78 350 3 promoter lacI binding caatttcacaca site
nderson
BBa_K158510 Constitutive E. A
Promoter with ...
4 coli σ70 agcggataa 78 351 promoter lacI binding ggaattgtg
site caatttcacaca BBa_K158510 Constitutive E. Anderson ...
5 coli σ70 Promoter with
promoter lacI binding ggaattgtgagcggataa 78 352 site caatttcacaca
rson
BBa_K158510 Constitutive E. Ande
Promoter with ...
6 coli σ70
promoter lacI binding ggaattgtgagcggataa 78 353 site caatttcacaca BBa_K158511 Constitutive E. Anderson ...
0 coli σ70 Promoter with
promoter lacI binding ggaattgtgagcggataa 78 354 site caatttcacaca
n
BBa_K158511 Constitutive E. Anderso
Promoter with ...
3 coli σ70 aa 78 355 promoter lacI binding ggaattgtgagcggat
site caatttcacaca
Anderson
BBa_K158511 Constitutive E. ...
5 coli σ70 Promoter with ggaattgtgagcggataa 78 356 promoter lacI binding
site caatttcacaca
Anderson
BBa_K158511 Constitutive E. oter with ...
6 coli σ70 Prom
promoter lacI binding ggaattgtgagcggataa 78 357 site caatttcacaca
Anderson
BBa_K158511 Constitutive E. ..
7 coli σ70 Promoter with .
ggaattgtgagcggataa 78 358 promoter lacI binding
site caatttcacaca
Anderson
BBa_K158511 Constitutive E. Promoter with ...
8 coli σ70 78 359 promoter lacI binding ggaattgtgagcggataa
site caatttcacaca
Anderson
BBa_K158511 Constitutive E. Promoter with ...
9 coli σ70 aattgtgagcggataa 78 360 promoter lacI binding gg
site caatttcacaca
BBa_K182489 Constitutive E. ...
coli σ70 J23100 + RBS gattaaagaggagaaata 88 361 6 promoter ctagagtactag
Constitutive E. ...
BBa_K256002 coli σ70 J23101:GFP caccttcgggtgggccttt 918 362 promoter ctgcgtttata Constitutive E. ...
BBa_K256018 coli σ70 J23119:IFP caccttcgggtgggccttt 1167 363 promoter ctgcgtttata BBa_K256020 Constitutive E. ...
coli σ70 J23119:HO1 caccttcgggtgggccttt 949 364 promoter ctgcgtttata Constitutive E. Infrared signal ...
BBa_K256033 coli σ70 reporter caccttcgggtgggccttt 2124 365 promoter (J23119:IFP:J
23119:HO1) ctgcgtttata
Constitutive E. Double
BBa_K292000 coli σ70 terminator + ...
promoter constitutive ggctagctcagtcctaggt 138 366 promoter acagtgctagc
Double
Constitutive E. terminator + ...
BBa_K292001 coli σ70 Constitutive tgctagctactagagatta 161 367 promoter promoter + aagaggagaaa
Strong RBS
Constitutive E. IPTG ...
BBa_K418000 coli σ70 inducible Lac
promoter promoter ttgtgagcggataacaag 1416 368 cassette atactgagcaca
Constitutive E. IPTG
BBa_K418002 coli σ70 inducible Lac ...
1414 369 promoter promoter ttgtgagcggataacaag
cassette atactgagcaca
Constitutive E. IPTG
BBa_K418003 coli σ70 inducible Lac ...
acaag 1416 370 promoter promoter ttgtgagcggata
cassette atactgagcaca
Constitutive E. Anderson ...
BBa_K823004 coli σ70 promoter ggctagctcagtcctaggt 35 371 promoter J23100 acagtgctagc Constitutive E. Anderson ...
BBa_K823005 coli σ70 promoter agctagctcagtcctaggt 35 372 promoter J23101 attatgctagc Constitutive E. Anderson ...
BBa_K823006 coli σ70 promoter agctagctcagtcctaggt 35 373 promoter J23102 actgtgctagc Constitutive E. Anderson ...
BBa_K823007 coli σ70 promoter agctagctcagtcctagg 35 374 promoter J23103 gattatgctagc Constitutive E. Anderson ...
BBa_K823008 coli σ70 promoter ggctagctcagtcctaggt 35 375 promoter J23106 atagtgctagc Constitutive E. Anderson ...
BBa_K823010 coli σ70 promoter ggctagctcagtcctagg 35 376 promoter J23113 gattatgctagc Constitutive E. Anderson ...
BBa_K823011 coli σ70 promoter ggctagctcagtcctaggt 35 377 promoter J23114 acaatgctagc Constitutive E. Anderson ...
BBa_K823013 coli σ70 promoter agctagctcagtcctagg 35 378 promoter J23117 gattgtgctagc BBa_K823014 Constitutive E. Anderson ...
coli σ70 promoter ggctagctcagtcctaggt 35 379
Figure imgf000460_0001
subtilis σA pro caagcttttcctttataatag moters aatgaatga Constitutive B. ...
BBa_K823002 subtilis σA pro PlepA tctaagctagtgtattttgc 157 400 moters gtttaatagt Constitutive B. ...
BBa_K823003 subtilis σA pro Pveg aatgggctcgtgttgtaca 237 401 moters ataaatgtagt Constitutive B. ...
BBa_K143010 subtilis σB pro Promoter ctc 2 moters for B. subtilis atccttatcgttatgggtatt 56 40 gtttgtaat
Constitutive B.
BBa_K143011 subtilis σB pro Promoter gsiB ...
moters for B. subtilis taaaagaattgtgagcgg 38 403 gaatacaacaac
Constitutive B. Promoter 43 a
BBa_K143013 subtilis σB pro constitutive ...
moters promoter for aaaaaaagcgcgcgatta 56 404
B. subtilis tgtaaaatataa
Constitutive
promoters Pspv2 ...
BBa_K112706 from from Salmonel tacaaaataattcccctgc 474 405 miscellaneous la aaacattatca prokaryotes
Constitutive
promoters Pspv ...
BBa_K112707 from from Salmonel tacaaaataattcccctgc 1956 406 miscellaneous la aaacattatcg prokaryotes
Constitutive T7 promoter
promoters (strong ...
BBa_I712074 from promoter from agggaatacaagctactt 46 407 bacteriophage T7 gttctttttgca T7 bacteriophage)
Constitutive
promoters
BBa_I719005 from T7 Promoter taatacgactcactatagg
gaga 23 408 bacteriophage
T7
Constitutive
promoters
BBa_J34814 from T7 Promoter gaatttaatacgactcacta
tagggaga 28 409 bacteriophage
T7
Constitutive
promoters
BBa_J64997 from T7 consensus - 10 and rest taatacgactcactatagg 19 410 bacteriophage
T7
Constitutive
BBa_K113010 promoters overlapping ...
from T7 promoter gagtcgtattaatacgact 40 411 bacteriophage cactatagggg T7
Constitutive
promoters more ...
BBa_K113011 from overlapping agtgagtcgtactacgact 37 412 bacteriophage T7 promoter cactatagggg T7
Constitutive
promoters weaken ...
BBa_K113012 from overlapping gagtcgtattaatacgact 40 413 bacteriophage T7 promoter ctctatagggg T7
Constitutive T7 promoter
BBa_K161400 promoters for expression
0 from 14 bacteriophage of functional taatacgactcactatag 18 4 T7 RNA
Constitutive
promoters T7 Consensus
BBa_R0085 from Promoter taatacgactcactatagg
gaga 23 415 bacteriophage Sequence
T7
Constitutive
promoters
BBa_R0180 from T7 RNAP ttatacgactcactatagg
gaga 23 416 bacteriophage promoter
T7
Constitutive
promoters
BBa_R0181 from T7 RNAP gaatacgactcactatagg
gaga 23 417 bacteriophage promoter
T7
Constitutive
promoters
BBa_R0182 from T7 RNAP taatacgtctcactatagg
bacteriophage promoter gaga 23 418 T7
Constitutive
promoters
BBa_R0183 from T7 RNAP tcatacgactcactatagg
gaga 23 419 bacteriophage promoter
T7
Constitutive
promoters
BBa_Z0251 from T7 strong ...
tatagg 35 420 bacteriophage promoter taatacgactcac
gagaccacaac
T7
Constitutive
promoters T7 weak ...
BBa_Z0252 from binding and taattgaactcactaaagg 35 421 bacteriophage processivity gagaccacagc T7
BBa_Z0253 Constitutive T7 weak ...
promoters binding cgaagtaatacgactcact 35 422 from promoter attagggaaga bacteriophage
T7
Constitutive
promoters consensus -10
BBa_J64998 from and rest from atttaggtgacactataga 19 423 bacteriophage SP6
SP6
Constitutive pCyc ...
BBa_I766555 promoters (Medium) acaaacacaaatacacac 244 424 from yeast Promoter actaaattaata Constitutive
BBa_I766556 promoters pAdh (Strong) ...
from yeast Promoter ccaagcatacaatcaacta 1501 425 tctcatataca
Constitutive
BBa_I766557 promoters pSte5 (Weak) ...
from yeast Promoter gatacaggatacagcgga 601 426 aacaacttttaa
Constitutive
BBa_J63005 promoters yeast ADH1 ...
from yeast promoter tttcaagctataccaagcat 1445 427 acaatcaact
Constitutive cyc100 ...
BBa_K105027 promoters minimal cctttgcagcataaattact 103 428 from yeast promoter atacttctat Constitutive
BBa_K105028 promoters cyc70 minimal ...
from yeast promoter cctttgcagcataaattact 103 429 atacttctat
Constitutive
BBa_K105029 promoters cyc43 minimal ...
cctttgcagcataaattact 103 430 from yeast promoter atacttctat Constitutive
BBa_K105030 promoters cyc28 minimal ...
from yeast promoter cctttgcagcataaattact 103 431 atacttctat
Constitutive
BBa_K105031 promoters cyc16 minimal ...
from yeast promoter cctttgcagcataaattact 103 432 atacttctat
Constitutive ...
BBa_K122000 promoters pPGK1 ttatctactttttacaacaaa 1497 433 from yeast tataaaaca Constitutive
BBa_K124000 promoters pCYC Yeast ...
from yeast Promoter acaaacacaaatacacac 288 434 actaaattaata
Constitutive Yeast GPD ...
BBa_K124002 promoters (TDH3) gtttcgaataaacacacat 681 435 from yeast Promoter aaacaaacaaa Constitutive yeast mid- ...
BBa_K319005 promoters length ADH1 ccaagcatacaatcaacta 720 436 from yeast promoter tctcatataca
Yeast CLB1
Constitutive promoter ...
BBa_M31201 promoters region, G2/M accatcaaaggaagcttta 500 437 from yeast cell cycle atcttctcata
specific Constitutive
promoters
BBa_I712004 from CMV ...
miscellaneous promoter agaacccactgcttactgg 654 438 cttatcgaaat
eukaryotes
Constitutive
promoters ...
BBa_K076017 from Ubc Promoter ggccgtttttggcttttttgtt 1219 439 miscellaneous agacgaag eukaryotes
Table X. Promoters
Figure imgf000464_0001
[1024] In some embodiments, the constitutive promoter 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 sequence of any one of SEQ ID NOs: 598-744.
Ribosome Binding Sites
[1025] In some embodiments, ribosome binding sites are added, switched out or replaced. By testing a few ribosome binding sites, expression levels can be fine- tuned to the desired level. Table XI lists a number RBS which are suitable for prokaryotic expression and can be used to achieve the desired expression levels (See, e.g., Registry of standard biological parts).
Table XI. Selected Ribosome Binding Sites Identifier Sequence a SEQ ID
NO
Master
Sequence TCTAGAGAAAGANNNGANNNACTAGATG 445 BBa_J61100 TCTAGAGAAAGAGGGGACAAACTAGATG 446 BBa_J61101 TCTAGAGAAAGACAGGACCCACTAGATG 447 BBa_J61102 TCTAGAGAAAGATCCGATGTACTAGATG 448 BBa_J61103 TCTAGAGAAAGATTAGACAAACTAGATG 449 BBa_J61104 TCTAGAGAAAGAAGGGACAGACTAGATG 450 BBa_J61105 TCTAGAGAAAGACATGACGTACTAGATG 451 BBa_J61106 TCTAGAGAAAGATAGGAGACACTAGATG 452 BBa_J61107 TCTAGAGAAAGAAGAGACTCACTAGATG 453 BBa_J61108 TCTAGAGAAAGACGAGATATACTAGATG 454 BBa_J61109 TCTAGAGAAAGACTGGAGACACTAGATG 455 BBa_J61110 TCTAGAGAAAGAGGCGAATTACTAGATG 456 BBa_J61111 TCTAGAGAAAGAGGCGATACACTAGATG 457 BBa_J61112 TCTAGAGAAAGAGGTGACATACTAGATG 458 BBa_J61113 TCTAGAGAAAGAGTGGAAAAACTAGATG 459 BBa_J61114 TCTAGAGAAAGATGAGAAGAACTAGATG 460 BBa_J61115 TCTAGAGAAAGAAGGGATACACTAGATG 461 BBa_J61116 TCTAGAGAAAGACATGAGGCACTAGATG 462 BBa_J61117 TCTAGAGAAAGACATGAGTTACTAGATG 463 BBa_J61118 TCTAGAGAAAGAGACGAATCACTAGATG 464 BBa_J61119 TCTAGAGAAAGATTTGATATACTAGATG 465 BBa_J61120 TCTAGAGAAAGACGCGAGAAACTAGATG 466 BBa_J61121 TCTAGAGAAAGAGACGAGTCACTAGATG 467 BBa_J61122 TCTAGAGAAAGAGAGGAGCCACTAGATG 468 BBa_J61123 TCTAGAGAAAGAGATGACTAACTAGATG 469 BBa_J61124 TCTAGAGAAAGAGCCGACATACTAGATG 470 BBa_J61125 TCTAGAGAAAGAGCCGAGTTACTAGATG 471 BBa_J61126 TCTAGAGAAAGAGGTGACTCACTAGATG 472 BBa_J61127 TCTAGAGAAAGAGTGGAACTACTAGATG 473 BBa_J61128 TCTAGAGAAAGATAGGACTCACTAGATG 474 BBa_J61129 TCTAGAGAAAGATTGGACGTACTAGATG 475 BBa_J61130 TCTAGAGAAAGAAACGACATACTAGATG 476 BBa_J61131 TCTAGAGAAAGAACCGAATTACTAGATG 477 BBa_J61132 TCTAGAGAAAGACAGGATTAACTAGATG 478 BBa_J61133 TCTAGAGAAAGACCCGAGACACTAGATG 479 BBa_J61134 TCTAGAGAAAGACCGGAAATACTAGATG 480 BBa_J61135 TCTAGAGAAAGACCGGAGACACTAGATG 481 BBa_J61136 TCTAGAGAAAGAGCTGAGCAACTAGATG 482 BBa_J61137 TCTAGAGAAAGAGTAGATCAACTAGATG 483 BBa_J61138 TCTAGAGAAAGATATGAATAACTAGATG 484 BBa_J61139 TCTAGAGAAAGATTAGAGTCACTAGATG 485 BBa_B0029 TCTAGAGTTCACACAGGAAACCTACTAGATG 486 BBa_B0030 TCTAGAGATTAAAGAGGAGAAATACTAGATG 487 BBa_B0031 TCTAGAGTCACACAGGAAACCTACTAGATG 488 BBa_B0032 TCTAGAGTCACACAGGAAAGTACTAGATG 489 BBa_B0033 TCTAGAGTCACACAGGACTACTAGATG 490 BBa_B0034 TCTAGAGAAAGAGGAGAAATACTAGATG 491 BBa_B0035 TCTAGAGATTAAAGAGGAGAATACTAGATG 492 BBa_B0064 TCTAGAGAAAGAGGGGAAATACTAGATG 493 Induction of Payloads During Strain Culture
[1026] In some embodiments, it is desirable to pre-induce payload or protein of interest expression and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term“bacterial culture” or bacterial cell culture” or“culture” refers to bacterial cells or
microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term“fermentation” refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like.
[1027] Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium.
[1028] In some embodiments, the one or more payloads and/or protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is“wasted”, in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites).
[1029] In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters.
[1030] In some embodiments, the strains are administered without any pre- induction protocols during strain growth prior to in vivo administration. [1031] Anaerobic induction
[1032] In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^8 to 1X10^11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) under the control of one or more FNR promoters.
[1033] In one embodiment, expression of one or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions.
[1034] In one embodiment, expression of two or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s) under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the one or more different promoters under anaerobic or low oxygen conditions.
[1035] Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut. [1036] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In some embodiments, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some
embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.
[1037] In one embodiment, expression of one or more payload gene sequence(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.
[1038] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions.
[1039] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions.
Aerobic induction [1040] In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^8 to 1X10^11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours.
[1041] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.
[1042] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions.
[1043] In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. [1044] In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions.
[1045] In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s)and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions.
[1046] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions.
[1047] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline.
[1048] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a
combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequences under the control of one or more constitutive promoter(s) active under aerobic conditions.
[1049] In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.
[1050] In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.
[1051] In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut and/or conditions of the tumor microenvironment. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. [1052] In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut.
[1053] As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with
thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions.
Microaerobic Induction
[1054] In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to“strike a balance” between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) are driven by an anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^8 to 1X10^11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours.
[1055] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.
[1056] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions.
[1057] Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut.
[1058] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein.
[1059] In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions.
[1060] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions.
[1061] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions.
[1062] Induction of Strains using Phasing, Pulsing and/or Cycling
[1063] In some embodiments, cycling, phasing, or pulsing techniques are employed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficiently induce and grow the strains prior to in vivo administration. This method is used to“strike a balance” between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g., for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1X10^8 to 1X10^11, and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g., reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle.
[1064] In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed.
[1065] In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non-limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g., for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1X10^8 to 1X10^11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed.
[1066] In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g., 37 C). In some embodiments, the cells are then induced, e.g., by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload.
[1067] In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other
embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer.
[1068] In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques.
[1069] In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques. [1070] In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques.
[1071] Any of the strains described herein can be grown through the
employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions.
[1072] In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques.
[1073] In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques.
Aerobic induction of the FNR promoter
[1074] 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 O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, an oxygen bypass system shown and described in FIG. 85A is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of the protein of interest (e.g., one or more anti-cancer effector(s) described herein) by binding and activating the FNR promoter under aerobic conditions. Thus, 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 the protein of interest. 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 protein of interest, e.g., one or more anti-cancer effector(s) described herein.
[1075] In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non-limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g., from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest (e.g., one or more anti-cancer effector(s) described herein) are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter.
[1076] In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y.
Multiple Mechanisms of Action
[1077] In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MoAs), e.g., circuits producing multiple copies of the same product or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in FIG. 18. For example, the genetically engineered bacteria may include four copies of argAfbr inserted at four different insertion sites, e.g., malE/K, insB/I, araC/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of argAfbr inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three mutant arginine regulons, e.g., two producing citrulline and one producing arginine, inserted at three different insertion sites dapA, cea, and araC/BAD. In some embodiments, the genetically engineered bacteria may include one or more ammonia conversion circuit(s) inserted at one or more different insertion sites and one or more additional circuits inserted at one or more other insertion sites. For example, the genetically engineered bacteria may include one or more copies of argAfbr (and/or other ammonia conversion circuit(s)) inserted at one or more different insertion sites, and one or more gut barrier enhancing circuits, e.g., one or more butyrate production circuit(s) (or other gut barrier enhancing circuit(s)) at other insertion sites. In other exemplary embodiments, the genetically engineered bacteria may include one or more copies of argAfbr and/or other ammonia conversion circuit(s)) inserted at one or more different insertion sites, and one or more GABA reducing circuits, e.g., GABA transport and/or GABA metabolic circuit(s), inserted at other insertion site(s). In other exemplary embodiments, the genetically engineered bacteria may include one or more copies of argAfbr (and/or other ammonia conversion circuit(s)) inserted at one or more different insertion sites, and one or more manganese transport circuit(s) inserted at other insertion sites (FIG. 47A). In some embodiments, one or more ammonia conversion circuit(s) (e.g., argAfbr and/or other ammonia conversion circuit(s)), one or more gut barrier enhancing circuit (e.g., butyrate, propionate, acetate biosynthetic circuit(s)), one or more GABA reducing circuit(s) (e.g., GABA transport and/or GABA metabolic circuit), and one or more manganese transport circuit(s) are inserted at four or more different chromosomal insertion sites (e.g., FIG. 45).In some embodiments, an ammonia conversion circuit, a gut barrier enhancing circuit, a GABA transport and/or GABA metabolic circuit are inserted at three different chromosomal insertion sites. In some embodiments, an ammonia conversion circuit, a GABA transport/metabolic circuit, and a manganese transport circuit are inserted at three different chromosomal insertion sites (FIG. 46B). In other embodiments, an ammonia conversion circuit, and a manganese transport circuit are inserted at two different chromosomal insertion sites (FIG. 47A). In still other embodiments, an ammonia conversion circuit, and a GABA transport and/or GABA metabolic circuit are inserted at two different chromosomal insertion sites. In still other embodiments, an ammonia conversion circuit, and a gut barrier enhancing circuit are inserted at two different chromosomal insertion sites. In still other embodiments, an ammonia conversion circuit, and a manganese reducing circuit are inserted at two different chromosomal insertion sites.
[1078] Table D2 lists non-limiting examples of embodiments of the disclosure. Table D2. Non-limiting examples of embodiments of the disclosure
Figure imgf000483_0001
SYN- ∆ARG box Wild type tetracycline- Wild Amp constitutively UCD105 ArgR inducible type expressed argAfbr on a ThyA argG low copy (BBa_J23100 plasmid (Amp) constitutive promoter)
∆ArgR
SYN- Wild type ∆ArgR none ∆ThyA Cam none UCD106 ARG Box
SYN- Wild type ∆ArgR none Wild none none UCD201/ ARG Box type
SYN- ThyA
UCD312
SYN- Wild type ∆ArgR tetracycline- Wild Amp none UCD202 ARG Box inducible type
argAfbr on a ThyA
high-copy
plasmid (Amp)
SYN- Wild type ∆ArgR tetracycline- Wild Amp none UCD203 ARG Box inducible type
argAfbr on a ThyA
low-copy
plasmid (Amp)
SYN- Wild type ∆ArgR tet-ArgAfbr on Wild Amp none UCD204 ARG Box a low-copy type
plasmid (Amp) ThyA SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild Amp none UCD205 ARG Box on a low-copy type
plasmid (Amp) ThyA
SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA Amp, none UCD206 ARG Box on a low-copy Cam
plasmid (Amp)
Integrated FNRS-argAfbr
SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild Cam none UCD301 ARG Box integrated into type
the ThyA
chromosome
at the malEK
locus
SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA Cam none UCD302 ARG Box integrated into
the
chromosome
at the malEK
locus
SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA Kan none UCD303 ARG Box integrated into
the
chromosome
at the malEK
Figure imgf000485_0001
Figure imgf000486_0001
SYN- Wild type ∆ArgR none ∆ThyA none Rifaximin UCD414 ARG Box resistance Butyrate Circuits
SYN- Wild type Wild type none Wild Amp Logic156 UCD500 ARG Box ArgR type (pSC101
ThyA PydfZ-Bcd butyrate plasmid; amp resistance
Figure imgf000487_0001
Figure imgf000487_0002
SYN- Wild type Wild type none Wild Amp tesB-butyrate UCD509 ARG Box ArgR type cassette
ThyA integrated into chromosome under control of FNR promoter SYN- Wild type Wild type none Wild None pLogic031 UCD510 ARG Box ArgR type (bdc2 butyrate
ThyA cassette
under control of tet promoter on a plasmid); nuoB deletion SYN- Wild type Wild type none Wild None pLogic046 (ter UCD511 ARG Box ArgR type butyrate
ThyA cassette
under control of tet promoter on a plasmid); nuoB deletion Butyrate and Ammonium Circuits
Figure imgf000488_0001
chromosome integrated on at the malEK the locus chromosome SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA none PydfZ-ter UCD608 ARG Box ArgR integrated into butyrate
the cassette chromosome integrated on at the malEK the locus chromosome SYN- Wild type Wild type PfnrS- ArgAfbr Wild Kan PydfZ-ter UCD609 ARG Box ArgR integrated into type butyrate
the ThyA cassette chromosome integrated on at the malEK the locus chromosome SYN- Wild type Wild type PfnrS- ArgAfbr Wild none PydfZ-ter UCD610 ARG Box ArgR integrated into type butyrate
the ThyA cassette chromosome integrated on at the malEK the locus chromosome SYN- Wild type ∆ArgR none Wild Kan PydfZ-ter UCD611 ARG Box type butyrate
ThyA cassette
integrated on the chromosome SYN- Wild type ∆ArgR none Wild none PydfZ-ter UCD612 ARG Box type butyrate
ThyA cassette
integrated on the chromosome SYN- Wild type ∆ArgR none ∆ThyA Kan PydfZ-ter UCD613 ARG Box butyrate
cassette integrated on the chromosome SYN- Wild type ∆ArgR none ∆ThyA none PydfZ-ter UCD614 ARG Box butyrate
cassette integrated on the chromosome SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA Kan PydfZ-ter UCD703 ARG Box integrated into butyrate
the cassette chromosome integrated on at the malEK the locus chromosome ;
Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA None PydfZ-ter UCD705 ARG Box integrated into butyrate
the cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild None PydfZ-ter UCD704 ARG Box integrated into type butyrate the ThyA cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild Kan PydfZ-ter UCD706 ARG Box integrated into type butyrate the ThyA cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA Kan PydfZ-ter UCD707 ARG Box ArgR integrated into butyrate the cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA none PydfZ-ter UCD708 ARG Box ArgR integrated into butyrate the cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr Wild Kan PydfZ-ter UCD709 ARG Box ArgR integrated into type butyrate the ThyA cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr Wild none PydfZ-ter UCD710 ARG Box ArgR integrated into type butyrate the ThyA cassette chromosome integrated on at the malEK the locus chromosome;
Rifaximin resistance SYN- Wild type ∆ArgR none Wild Kan PydfZ-ter UCD711 ARG Box type butyrate
ThyA cassette integrated on the chromosome;
Rifaximin resistance SYN- Wild type ∆ArgR none Wild none PydfZ-ter UCD712 ARG Box type butyrate
ThyA cassette integrated on the chromosome; Rifaximin resistance SYN- Wild type ∆ArgR none ∆ThyA Kan PydfZ-ter UCD713 ARG Box butyrate cassette integrated on the chromosome; Rifaximin resistance SYN- Wild type ∆ArgR none ∆ThyA none PydfZ-ter UCD714 ARG Box butyrate cassette integrated on the chromosome; Rifaximin r i n
Figure imgf000491_0001
chromosome chromosome at malEK locus under control of FNR promoter SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA none tesB-butyrate UCD720 ARG Box ArgR integrated into cassette the integrated into chromosome chromosome at malEK locus under control of FNR promoter SYN- Wild type Wild type PfnrS- ArgAfbr Wild Kan tesB-butyrate UCD721 ARG Box ArgR integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter SYN- Wild type Wild type PfnrS- ArgAfbr Wild none tesB-butyrate UCD722 ARG Box ArgR integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter SYN- Wild type ∆ArgR none Wild Kan tesB-butyrate UCD723 ARG Box type cassette
ThyA integrated into chromosome under control of FNR promoter SYN- Wild type ∆ArgR none Wild none tesB-butyrate UCD724 ARG Box type cassette
ThyA integrated into chromosome under control of FNR promoter SYN- Wild type ∆ArgR none ∆ThyA Kan tesB-butyrate UCD725 ARG Box cassette integrated into chromosome under control of FNR promoter SYN- Wild type ∆ArgR none ∆ThyA none tesB-butyrate UCD726 ARG Box cassette integrated into chromosome under control of FNR promoter SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA Kan tesB-butyrate UCD727 ARG Box integrated into cassette the integrated into chromosome chromosome at malEK locus under control of FNR promoter;
Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr ∆ThyA None tesB-butyrate UCD728 ARG Box integrated into cassette the integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild None tesB-butyrate UCD729 ARG Box integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type ∆ArgR PfnrS- ArgAfbr Wild Kan tesB-butyrate UCD730 ARG Box integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA Kan tesB-butyrate UCD731 ARG Box ArgR integrated into cassette the integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr ∆ThyA none tesB-butyrate UCD732 ARG Box ArgR integrated into cassette the integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr Wild Kan tesB-butyrate UCD733 ARG Box ArgR integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter; Rifaximin resistance SYN- Wild type Wild type PfnrS- ArgAfbr Wild none tesB-butyrate UCD734 ARG Box ArgR integrated into type cassette the ThyA integrated into chromosome chromosome at malEK locus under control of FNR promoter;
Rifaximin resistance SYN- Wild type ∆ArgR none Wild Kan tesB-butyrate UCD735 ARG Box type cassette
ThyA integrated into chromosome under control of FNR promoter;
Rifaximin resistance SYN- Wild type ∆ArgR none Wild none tesB-butyrate UCD736 ARG Box type cassette
ThyA integrated into chromosome under control of FNR promoter;
Rifaximin resistance SYN- Wild type ∆ArgR none ∆ThyA Kan tesB-butyrate UCD737 ARG Box cassette
integrated into chromosome under control of FNR promoter;
Rifaximin resistance SYN- Wild type ∆ArgR none ∆ThyA none tesB-butyrate UCD38 ARG Box cassette
integrated into chromosome under control of FNR promoter;
Rifaximin resistance [1079] In some embodiments, butyrate production by the genetically engineered bacteria can be further enhanced by additional modifications. Butyrate production under anaerobic conditions depends on endogenous NADH pools. In some embodiments, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. A non-limiting example is the mutation/deletion of frdA, which utilizes NADH to catalyze the conversion of phosphoenolpyruvate to succinate. In some embodiments, any of the genetically engineered bacteria described herein further comprise a mutation, which eliminates competing routes for NADH utilization, e.g., a mutation/deletion of frdA. Other non-limiting examples include AdhE and LdhA, described herein.
[1080] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene), further comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of butyrate.
[1081] In some embodiments, the gene sequences for the production of butyrate comprise ter, thiA1, hbd, crt2, pbt, and buk genes. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in the endogenous pta gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in the endogenous adhE gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in the endogenous ldhA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in the endogenous frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA). In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes further comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous adhE gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous ldhA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA). In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, pbt, and buk genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene.
[1082] In some embodiments, the genetically engineered bacteria comprising gene sequence encoding one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) further comprise gene sequences for the production of butyrate comprising the ter, thiA1, hbd, crt2, and tesB genes. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in the endogenous pta gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in the endogenous adhE gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in the endogenous ldhA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in the endogenous frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA). In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes further comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous adhE gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous ldhA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in the endogenous frdA gene. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA). In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit comprising ter, thiA1, hbd, crt2, and tesB genes and a deletion or mutation in the endogenous pta gene further comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene.
[1083] In any of the embodiments described above and elsewhere herein, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, furthers comprise one or more gene sequences and/or deletions of endogenous genes described herein, for the production of propionate.
[1084] In any of the embodiments described above and elsewhere herein, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, further comprises one or more gene sequences and/or deletions or mutations of endogenous genes described herein, for the production of acetate. Such deletions or mutations include deletions in one or more of the ldhA, frdA, and/or adhE genes described herein.
[1085] In any of the embodiments described above and elsewhere herein, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, further comprises one or more gene sequences for the secretion of an anti-inflammatory cytokine. the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, further comprises one or more gene sequences for the secretion of IL-22. the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, further comprises one or more gene sequences for the secretion of GLP2. the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit, further comprises one or more gene sequences for the secretion of a satiety effector, e.g., GLP1.
[1086] In any of the embodiments described above and elsewhere herein, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit further comprises a GABA transport circuit and/or a GABA metabolic circuit. In some embodiments, the bacterium comprising one or more of any of the ammonia conversion circuits described herein (e.g., an ArgAfbr circuit combined with a mutation or deletion in the endogenous ArgR gene) and a butyrate production circuit further comprises one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and is capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[1087] In any of the combination embodiments described above and elsewhere herein, any gene sequence encoding one or more polypeptides for any of the circuits in the combination, is operably linked to an inducible promoter. In any of the combination embodiments described above and elsewhere herein, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In any of the combination embodiments described above and elsewhere herein, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In any of the combination embodiments described above and elsewhere herein, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In any of the combination embodiments described above and elsewhere herein, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In any of the combination embodiments described above and elsewhere herein, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[1088] In any of the combination embodiments described above and elsewhere herein, 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. In any of the combination embodiments described above and elsewhere herein, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In any of the combination embodiments described above and elsewhere herein, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In any of the combination embodiments described above and elsewhere herein, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[1089] In any of the combination embodiments described above and elsewhere herein, any gene sequences encoding one or more polypeptides for any of the circuits in the combination, are operably linked to a constitutive promoter. In any of the combination embodiments described above and elsewhere herein, 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. In any of the combination embodiments described above and elsewhere herein, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the combination embodiments described above and elsewhere herein, 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. In any of the combination embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the reduction of ammonia levels, e.g., ArgAfbr, is operably linked to a RBS, enhancer or other regulatory sequence. In any of the combination embodiments described above and elsewhere herein, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI.
[1090] In any of the combination embodiments described above and elsewhere herein, the circuits in the combination are all under the control of the same constitutive or inducible promoter. In any of the combination embodiments described above and elsewhere herein, the circuits in the combination are under the control of one or more different constitutive and/or inducible promoters.
[1091] In any of the combination embodiments described above and elsewhere herein, any gene sequence encoding one or more polypeptides for any of the circuits in the combination is modified and/or mutated, e.g., to enhance stability, or increase tryptophan/tryptophan metabolite production or catalysis.
[1092] In any of the combination embodiments described above and elsewhere herein, any gene sequence encoding one or more polypeptides for any of the circuits in the combination may be codon optimized, e.g., to improve expression in the host microorganism. In any of the combination embodiments described above and elsewhere herein, a gene sequence encoding one or more polypeptides for any of the circuits in the combination, is 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.
[1093] In any of the combination embodiments described above and elsewhere herein, the genetically engineered bacteria may further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, alone or in combination with mutations/deletions in endogenous genes, allowing for a“leaky” membrane (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art, alone or in combination with mutations/deletions in endogenous genes, allowing for a“leaky” phenotype (7) one or more circuits for the degradation of ammonia described herein (8) one or more circuits for the production and/or secretion of a gut barrier enhancer molecule and (9) one or more circuits for the production or degradation of one or more metabolites (e.g., tryptophan, tryptophan metabolites, arginine or another amino acid, butyrate, acetate, and/or propionate) described herein, (10) one or more polypeptides for secretion (11) one or more mutations in endogenous genes, e.g., allowing for improved flux through a desired pathway (12) combinations of one or more of such additional circuits.
[1094] In any of the combination embodiments described herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
Nucleic Acids
[1095] In some embodiments, the disclosure provides novel nucleic acids for reducing ammonia levels. In some embodiments, the nucleic acid comprises gene sequence encoding feedback resistant ArgA. In some embodiments, the nucleic acid comprises gene sequence encoding ArgAfbr. In some embodiments, the nucleic acid comprises a ArgAfbr gene sequence. In certain embodiments, the nucleic acid comprising the ArgAfbr gene sequence has at least about 80% identity with SEQ ID NO: 30. In certain embodiments, the nucleic acid comprising the ArgAfbr gene sequence has at least about 90% identity with SEQ ID NO: 30. In certain embodiments, the nucleic acid comprising the ArgAfbr gene sequence has at least about 95% identity with SEQ ID NO: 30. In some embodiments, the nucleic acid comprising the ArgAfbr gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 30. In some specific embodiments, the nucleic acid comprising the ArgAfbr gene sequence comprises SEQ ID NO: 30. In other specific embodiments the nucleic acid comprising the ArgAfbr gene sequence consists of SEQ ID NO: 30.
[1096] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an Bcd2 (part of bcd2, etfA3, and etfB3 complex, e.g., in Clostridium difficile). In some embodiments, the nucleic acid comprises gene sequence encoding bcd2. In some embodiments, the nucleic acid comprises a bcd2 gene sequence. In certain embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 80% identity with SEQ ID NO: 39. In certain embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 90% identity with SEQ ID NO: 39. In certain embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 95% identity with SEQ ID NO: 39. In some embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In some specific embodiments, the nucleic acid comprising the bcd2 gene sequence comprises SEQ ID NO: 39. In other specific embodiments the nucleic acid comprising the bcd2 gene sequence consists of SEQ ID NO: 39.
[1097] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an EtfB3 (part of bcd2, etfA3, and etfB3 complex, e.g., in Clostridium difficile). In some embodiments, the nucleic acid comprises gene sequence encoding etfB3. In some embodiments, the nucleic acid comprises a etfB3 gene sequence. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 80% identity with SEQ ID NO: 40. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 90% identity with SEQ ID NO: 40. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 95% identity with SEQ ID NO: 40. In some embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. In some specific embodiments, the nucleic acid comprising the etfB3 gene sequence comprises SEQ ID NO: 40. In other specific embodiments the nucleic acid comprising the etfB3 gene sequence consists of SEQ ID NO: 40.
[1098] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an EtfA3 (part of bcd2, etfA3, and etfB3 complex, e.g., in Clostridium difficile). In some embodiments, the nucleic acid comprises gene sequence encoding etfA3. In some embodiments, the nucleic acid comprises a etfA3 gene sequence. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 80% identity with SEQ ID NO: 41. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 90% identity with SEQ ID NO: 41. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 95% identity with SEQ ID NO: 41. In some embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. In some specific embodiments, the nucleic acid comprising the etfA3 gene sequence comprises SEQ ID NO: 41. In other specific embodiments the nucleic acid comprising the etfA3 gene sequence consists of SEQ ID NO: 41.
[1099] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an ThiA1. In some embodiments, the nucleic acid comprises gene sequence encoding thiA1. In some embodiments, the nucleic acid comprises a thiA1 gene sequence. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 80% identity with SEQ ID NO: 42. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 90% identity with SEQ ID NO: 42. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 95% identity with SEQ ID NO: 42. In some embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42. In some specific embodiments, the nucleic acid comprising the thiA1 gene sequence comprises SEQ ID NO: 42. In other specific embodiments the nucleic acid comprising the thiA1 gene sequence consists of SEQ ID NO: 42.
[1100] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an hbd. In some embodiments, the nucleic acid comprises gene sequence encoding hbd. In some embodiments, the nucleic acid comprises a hbd gene sequence. In certain embodiments, the nucleic acid comprising the hbd gene sequence has at least about 80% identity with SEQ ID NO: 43. In certain embodiments, the nucleic acid comprising the hbd gene sequence has at least about 90% identity with SEQ ID NO: 43. In certain embodiments, the nucleic acid comprising the hbd gene sequence has at least about 95% identity with SEQ ID NO: 43. In some embodiments, the nucleic acid comprising the hbd gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43. In some specific embodiments, the nucleic acid comprising the hbd gene sequence comprises SEQ ID NO: 43. In other specific embodiments the nucleic acid comprising the hbd gene sequence consists of SEQ ID NO: 43.
[1101] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an Crt2. In some embodiments, the nucleic acid comprises gene sequence encoding crt2. In some embodiments, the nucleic acid comprises a crt2 gene sequence. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 80% identity with SEQ ID NO: 44. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 90% identity with SEQ ID NO: 44. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 95% identity with SEQ ID NO: 44. In some embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. In some specific embodiments, the nucleic acid comprising the crt2 gene sequence comprises SEQ ID NO: 44. In other specific embodiments the nucleic acid comprising the crt2 gene sequence consists of SEQ ID NO: 44.
[1102] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an Pbt. In some embodiments, the nucleic acid comprises gene sequence encoding pbt. In some embodiments, the nucleic acid comprises a pbt gene sequence. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 80% identity with SEQ ID NO: 45. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 90% identity with SEQ ID NO: 45. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 95% identity with SEQ ID NO: 45. In some embodiments, the nucleic acid comprising the pbt gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. In some specific embodiments, the nucleic acid comprising the pbt gene sequence comprises SEQ ID NO: 45. In other specific embodiments the nucleic acid comprising the pbt gene sequence consists of SEQ ID NO: 45.
[1103] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an Buk. In some embodiments, the nucleic acid comprises gene sequence encoding buk. In some embodiments, the nucleic acid comprises a buk gene sequence. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 80% identity with SEQ ID NO: 46. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 90% identity with SEQ ID NO: 46. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 95% identity with SEQ ID NO: 46. In some embodiments, the nucleic acid comprising the buk gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46. In some specific embodiments, the nucleic acid comprising the buk gene sequence comprises SEQ ID NO: 46. In other specific embodiments the nucleic acid comprising the buk gene sequence consists of SEQ ID NO: 46.
[1104] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an Ter (trans-2-enoynl-CoA reductase, e.g., from Treponema denticola). In some embodiments, the nucleic acid comprises gene sequence encoding ter. In some embodiments, the nucleic acid comprises a ter gene sequence. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 80% identity with SEQ ID NO: 47. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 90% identity with SEQ ID NO: 47. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 95% identity with SEQ ID NO: 47. In some embodiments, the nucleic acid comprising the ter gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47. In some specific embodiments, the nucleic acid comprising the ter gene sequence comprises SEQ ID NO: 47. In other specific embodiments the nucleic acid comprising the ter gene sequence consists of SEQ ID NO: 47.
[1105] In some embodiments, the disclosure provides novel nucleic acids for the production of butyrate. In some embodiments, the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme(s). In some embodiments, the nucleic acid comprises gene sequence encoding an TesB. In some embodiments, the nucleic acid comprises gene sequence encoding tesB. In some embodiments, the nucleic acid comprises a tesB gene sequence. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 80% identity with SEQ ID NO: 48. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 90% identity with SEQ ID NO: 48. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 95% identity with SEQ ID NO: 48. In some embodiments, the nucleic acid comprising the tesB gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48. In some specific embodiments, the nucleic acid comprising the tesB gene sequence comprises SEQ ID NO: 48. In other specific embodiments the nucleic acid comprising the tesB gene sequence consists of SEQ ID NO: 48.
[1106] In other embodiments, the disclosure provides novel nucleic acids for producing butyrate in which the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme cassette(s). In some embodiments, the nucleic acid comprises gene sequence encoding a butyrate production cassette comprising ter-thiA1- hbd-ctr2-tesB. In some embodiments, the nucleic acid comprises a butyrate production cassette comprising ter-thiA1-hbd-ctr2-tesB gene sequence. In certain embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2-tesB sequence has at least about 80% identity with SEQ ID NO: 511. In certain embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2-tesB gene sequence has at least about 90% identity with SEQ ID NO: 37. In certain embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2-tesB sequence has at least about 95% identity with SEQ ID NO: 511. In some embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2-tesB gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 511. In some specific embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2-tesB gene sequence comprises the sequence of SEQ ID NO: 511. In other specific embodiments, the nucleic acid comprising ter-thiA1-hbd- ctr2-tesB gene sequence consists of the sequence of SEQ ID NO: 511.
[1107] In other embodiments, the disclosure provides novel nucleic acids for producing butyrate in which the nucleic acid comprises gene sequence encoding one or more butyrate production enzyme cassette(s). In some embodiments, the nucleic acid comprises gene sequence encoding a butyrate production cassette comprising ter-thiA1- hbd-ctr2- pbt-buk. In some embodiments, the nucleic acid comprises a butyrate production cassette comprising ter-thiA1-hbd-ctr2- pbt-bukgene sequence. In certain embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2- pbt-buksequence has at least about 80% identity with SEQ ID NO: 512. In certain embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2- pbt-buk gene sequence has at least about 90% identity with SEQ ID NO: 37. In certain embodiments, the nucleic acid comprising ter- thiA1-hbd-ctr2- pbt-buksequence has at least about 95% identity with SEQ ID NO: 512. In some embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2- pbt-bukgene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 512. In some specific embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2- pbt-bukgene sequence comprises the sequence of SEQ ID NO: 512. In other specific embodiments, the nucleic acid comprising ter-thiA1-hbd-ctr2- pbt-bukgene sequence consists of the sequence of SEQ ID NO: 512.
[1108] In some embodiments, the nucleic acid comprises gene sequence encoding Bcd2. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 164. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 164. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 164. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 164. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 164. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 164.
[1109] In some embodiments, the nucleic acid comprises gene sequence encoding EtfB3. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 165. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 165. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 165. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 165. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 165. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 165.
[1110] In some embodiments, the nucleic acid comprises gene sequence encoding EtfA3. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 166. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 166. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 166. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 166. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 166. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 166.
[1111] In some embodiments, the nucleic acid comprises gene sequence encoding Ter. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 167. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 167. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 167. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 167. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 167. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 167.
[1112] In some embodiments, the nucleic acid comprises gene sequence encoding ThiA. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 168. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 168. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 168. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 168. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 168. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 168.
[1113] In some embodiments, the nucleic acid comprises gene sequence encoding Hbd. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 169. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 169. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 169. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 169. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 169.
[1114] In some embodiments, the nucleic acid comprises gene sequence encoding Crt2. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 170. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 170. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 170. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 170. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 170. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 170.
[1115] In some embodiments, the nucleic acid comprises gene sequence encoding Pbt. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 171. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 171. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 171. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 171. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 171. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 171.
[1116] In some embodiments, the nucleic acid comprises gene sequence encoding Buk. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 172. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 172. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 172. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 172. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 172. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 172.
[1117] In some embodiments, the nucleic acid comprises gene sequence encoding TesB. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 80% identity with SEQ ID NO: 173. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 90% identity with SEQ ID NO: 173. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide having at least about 95% identity with SEQ ID NO: 173. In some embodiments, the nucleic acid comprises gene sequences encoding a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 173. In some embodiments, the nucleic acid comprises gene sequence encoding a polypeptide comprising SEQ ID NO: 173. In other embodiments, the nucleic acid comprises gene sequence encoding a polypeptide consisting of SEQ ID NO: 173.
[1118] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any of the one or more polypeptides described herein is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with liver disease,
hyperammonemia, or 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. In some embodiments the promoter is induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the promoter is induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein.
[1119] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is active under exogenous in vivo conditions, e.g., such as those found in the gut, or under conditions of hyperammonemia and/or liver disease. In some
embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX and X.
[1120] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX, X, or XI. In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein is operably linked to a sequence that targets the gene sequence or its resultant polypeptide to a particular cellular location, including but not limited to a signal sequence, a secretion sequence, or an display anchor sequence. [1121] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein is modified and/or mutated (for example, by deletion, insertion, and/or substitution of nucleotide(s))e.g., to enhance stability, or increase metabolite production or catalysis.
[1122] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein may be codon optimized, e.g., to improve expression in the host microorganism.
[1123] In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding any one or more polypeptides described herein 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. Secretion
[1124] In any of the embodiments described herein, in which the genetically engineered organism, e.g., engineered bacteria or engineered virus, produces a protein, polypeptide, or peptide, DNA, RNA, small molecule or other molecule intended to be secreted from the microorganism, the engineered microorganism may comprise a secretion mechanism and corresponding gene sequence(s) encoding the secretion system.
[1125] In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting the molecule from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane.
[1126] In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Double membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in figures and examples. Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane- spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the
extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015).
[1127] In some embodiments in which the one or more proteins of interest or therapeutic proteins are secreted or exported from the microorganism, the engineered microorganism comprises gene sequence(s) that includes a secretion tag. In some embodiments, the one or more proteins of interest or therapeutic proteins include a “secretion tag” of either RNA or peptide origin to direct the one or more proteins of interest or therapeutic proteins to specific secretion systems. For example, a secretion tag for the Type I Hemolysin secretion system is encoded in the C-terminal 53 amino acids of the alpha hemolysin protein (HlyA).
[1128] In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types. FIG. 69C shows the alpha-hemolysin (HlyA) of uropathogenic Escherichia coli. This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. HlyB inserts into inner membrane to form a pore, HlyD aligns HlyB with TolC (outer membrane pore) thereby forming a channel through inner and outer membrane. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide. The C- terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the one or more proteins of interest or therapeutic proteins into the extracellular milieu. In some embodiments the one or more proteins of interest or therapeutic proteins contain expressed as fusion protein with the 53 amino acids of the C termini of alpha-hemolysin (hlyA) of E. coli CFT073 (C terminal secretion tag).
[1129] In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. The Type V Auto- secretion System utilizes an N-terminal Sec-dependent peptide tag (inner membrane) and C-terminal tag (outer-membrane). This system uses the Sec-system to get from the cytoplasm to the periplasm. The C-terminal tag then inserts into the outer membrane forming a pore through which the“passenger protein” threads through. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in FIG 69B, 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, Sec-dependent 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 (‘Beta-barrel assembly machinery’) where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once across the outer membrane, the passenger is released from the membrane-embedded C-terminal tag by either an autocatalytic, intein-like mechanism (left side of Bam complex) or via a membrane-bound protease (black scissors; right side of Bam complex) (i.e., OmpT). For example, a membrane-associated peptidase to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria.
[1130] The N-terminal tag is removed by the Sec system. Thus, in some embodiments, the secretion system is able to remove this tag before secreting the one or more proteins of interest or therapeutic proteins, from the engineered bacteria. In the Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the“passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the molecule(s) into the extracellular milieu.
[1131] In some embodiments, the genetically engineered bacteria of the invention comprise a type III or a type III-like secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The traditional T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. In the Type III traditional secretion system, the basal body closely resembles the flagella, however, instead of a“tail”/whip, the traditional T3SS has a syringe to inject the passenger proteins into host cells. The secretion tag is encoded by an N-terminal peptide (lengths vary and there are several different tags, see PCT/US14/020972). The N-terminal tag is not removed from the polypeptides in this secretion system.
[1132] The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm (FIG. 70). Thus, the molecule is secreted into the gut lumen, tumor microenvironment, or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises a type III secretion sequence that allows the molecule of interest to be secreted from the bacteria.
[1133] In the Flagellar modified Type III Secretion, the tag is encoded in 5’untranslated region of the mRNA and thus there is no peptide tag to cleave/remove. This modified system does not contain the“syringe” portion and instead uses the basal body of the flagella structure as the pore to translocate across both membranes and out through the forming flagella. If the fliC/fliD genes (encoding the flagella“tail”/whip) are disrupted the flagella cannot fully form and this promotes overall secretion. In some embodiments, the tail portion can be removed entirely.
[1134] In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N- terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment.
[1135] For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173- bp region, is fused to the gene encoding the heterologous protein or peptide can be used to secrete polypeptides of interest (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus. Nat Biotechnol. 2005 Apr;23(4):475-81). In some cases, the untranslated region from the fliC loci may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example, by using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et al., Secretion of
Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 237437-7438).
[1136] In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane- spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in E. coli), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads.
[1137] In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides)– particularly those of eukaryotic origin – contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again.
[1138] One way to secrete properly folded proteins in gram-negative bacteria– particularly those requiring disulphide bonds– is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These“leaky” gram-negative mutants are therefore capable of secreting bioactive, properly disulphide- bonded polypeptides. In some embodiments, the genetically engineered bacteria have a “leaky” or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can 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, tolB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at ~500,000 copies per cell and functions as the primary‘staple’ of the bacterial cell wall to the peptidoglycan. 1.
Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). 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 are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpI can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases.
Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes. in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.
[1139] To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl, under the control of an inducible promoter. For example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., overexpression of colicins or the third topological domain of TolA, wherein peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter.
[05] Table E and Table F below lists secretion systems for Gram positive bacteria and Gram negative bacteria.
Table E Secretion systems for gram positive bacteria
Figure imgf000521_0001
Table F. Secretion Systems for Gram negative bacteria
Figure imgf000521_0002
Oxa1 Cytochrome 2.A.9 + + + 1 None (YidC) oxidase (mitochon or biogenesis dria PMF family chloroplast
s)
MscL Large 1.A.2 + + + 1 None conductance 2
mechanosens
itive channel
family
Holins Holin 1.E.1 + - - 1 None functional •21
superfamily
Eukaryotic Organelles
MPT Mitochondria 3.A. - - + ?20 ATP l protein B (mitochon
translocase drial)
CEPT Chloroplast 3.A.9 (+) - + ≥3 GTP envelope (chloroplas
protein ts)
translocase
Bcl-2 Eukaryotic 1.A.2 - - + 1? None
Bcl-2 family 1
(programmed
cell death)
Gram-negative bacterial outer membrane channel-forming translocases MTB Main 3.A.1 +b - - ∼14 ATP; (IISP) terminal 5 PMF branch of the
general
secretory
translocase
FUP AT-1 Fimbrial 1.B.1 +b - - 1 None usher protein 1 +b - 1 None Autotransport 1.B.1
er-1 2
AT-2 Autotransport 1.B.4 +b - - 1 None OMF er-2 0 +b +(?) 1 None (ISP) 1.B.1
7
TPS 1.B.2 + - + 1 None Secretin 0 +b - 1 None (IISP and 1.B.2
IISP) 2
OmpIP Outer 1.B.3 + - + ≥4 None membrane 3 (mitochon ? insertion dria;
porin chloroplast
s) [1140] The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe / Volume 1, Number 9, 2006“Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently”, the contents of which is herein incorporated by reference in its entirety.
[1141] In some embodiments, the genetically engineered bacterial comprise a native or non-native secretion system described herein for the secretion of a molecule, e.g., a cytokine, antibody (e.g., scFv), metabolic enzyme, e.g., kynureninase, an others described herein.
Table G. Polypeptide Sequences of exemplary secretion tags
Figure imgf000523_0001
SEQ ID NO: 502
HlyA secretion CTTAATCCATTAATTAATGAAATCAGCAAAATCATTTCAGCT signal GCAGGTAATTTTGATGTTAAAGAGGAAAGAGCTGCAGCTTC
TTTATTGCAGTTGTCCGGTAATGCCAGTGATTTTTCATATGG SEQ ID NO: 503 ACGGAACTCAATAACTTTGACAGCATCAGCATAA. Table H. Additionals secretion tag sequences (native to E coli.)
Figure imgf000524_0001
[1142] In some embodiments, genetically engineered bacteria comprise a
nucleic acid sequence that encodes a polypeptide which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID
NO: 506, and/or SEQ ID NO: 507.
[1143] Any secretion tag or secretion system can be combined with any
cytokine described herein, and can be used to generate a construct (plasmid based or integrated) which is driven by an directly or indirectly inducible or constitutive
promoter described herein. In some embodiments, the secretion system is used in
combination with one or more genomic mutations, which leads to the leaky or diffusible outer membrane phenotype (DOM), including but not limited to, lpp, nlP, tolA, PAL.
[1144] In some embodiments, the secretion system is selected from the type
III flagellar, modified type III flagellar, type I (e.g., hemolysin system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, a single membrane secretion system, Sec and, TAT secretion systems.
[1145] Any of the secretion systems described herein may according to the disclosure be employed to secrete the polypeptides of interest. In some embodiments, the therapeutic proteins secreted by the genetically engineered bacteria are modified to increase resistance to proteases, e.g. intestinal proteases.
[1146] Non-limiting examples of proteins of interest include GLP-2 peptides, GLP-2 analogs, IL-22, tryptophan synthesis enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, e.g., those in the indole pathway as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity.
Table I. Comparison of Secretion systems for secretion of polypeptide from engineered bacteria
Figure imgf000525_0001
[1147] In some embodiments, the therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, such as, gut barrier enhancer molecules, such as GLP-2 peptides, GLP-2 analogs, or IL-22, is assembled behind a fliC-5’UTR (e.g., 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of the therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by IBD specific molecules or 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 is used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In some embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g., the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency.
[1148] In some embodiments, the therapeutic polypeptides of interest, e.g., gut barrier enhancer molecules, such as GLP-2 peptides, GLP-2 analogs, or IL-22, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the therapeutic polypeptide of interest is fused to a Tat-dependent secretion signal. Exemplary Tat- dependent tags include TorA, FdnG, and DmsA.
[1149] In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include lpp, pal, tolA, and/or nlpI. In some embodiments, lpp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, tolA is deleted or mutated. In other embodiments, nlpI is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non-limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated.
[1150] In some embodiments, the therapeutic polypeptides of interest, e.g., gut barrier enhancer molecules, such as GLP-2 peptides, GLP-2 analogs, or IL-22, are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodiments, the therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter E. coli_01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest.
[1151] In some embodiments, the therapeutic polypeptides of interest, gut barrier enhancer molecules, such as GLP-2 peptides, GLP-2 analogs, or IL-22 are secreted via Type I Hemolysin Secretion. In one embodiment, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of E. coli CFT073.
[1152] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with hepatic encephalopathy, UCD or another hyperammonemia disorder, or 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. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some
embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein. [1153] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space is operably linked to a RBS, enhancer or other regulatory sequence. In some embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space is modified and/or mutated, e.g., to enhance stability, or increase secretion, potency or stability.
[1154] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for the secretion into the extracellular space 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.
[1155] In any of the embodiments, above and described elsewhere herein, the genetically engineered bacteria may further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art, (7) one or more circuits for the degradation of ammonia described herein (8) one or more circuits for the production and/or secretion of a gut barrier enhancer molecule and (9) one or more circuits for the production or degradation of one or more metabolites (e.g., tryptophan, tryptophan metabolites, arginine or another amino acid, butyrate, acetate, and/or propionate) described herein (10) combinations of one or more of such additional circuits.
Surface Display [1156] In some embodiments, the genetically engineered bacteria and/or microorganisms encode one or more gene(s) and/or gene cassette(s) encoding an anti- cancer molecule which is anchored or displayed on the surface of the bacteria and/or microorganisms. Examples of the anti-cancer molecules which are displayed or anchored to the bacteria and/or microorganism, are any of the anti-cancer molecules described herein, and include but are not limited to antibodies, e.g., scFv fragments, and tumor-specific antigens or neoantigens. In a non-limiting example, the antibodies or scFv fragments which are anchored or displayed on the bacterial cell surface are directed against checkpoint inhibitors described herein, including, but not limited to, CLTLA4, PD-1, PD-L1, and others described herein.
[1157] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding therapeutic polypeptide or effector molecule, e.g., a ScFv, which is anchored or displayed on the surface of the bacteria, and which remains anchored while exerting its effector function. In other embodiments, the genetically engineered bacteria encoding the surface-displayed therapeutic polypeptide, e.g., the antibodies or scFv fragments, lyse before, during or after exerting their effector function. In some embodiments, the genetically engineered bacteria encode a therapeutic peptide that is temporarily attached to the cell surface and which dissociates from the bacterium before, during, or after exerting its function.
[1158] In some embodiments, shorter peptides or polypeptides, e.g. peptides or polypeptides of less than 60 amino acids of length, are displayed on the cell surface of the genetically engineered bacteria. In some embodiments, such shorter peptides or polypeptides comprise a immune modulatory effector molecule. Non-limiting examples of such therapeutic polypeptides are described herein. [1159] Several strategies for the display of shorter peptides or polypeptides on the surface of gram negative bacteria are known in the art, and are for example described in Georgiou et al., Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines: Nat Biotechnol. 1997 Jan;15(1):29-34, the contents of which is herein incorporated by reference in its entirety. These systems all share a common theme, targeting recombinant proteins to the cell surface by the construction of gene fusions using sequences from membrane-anchoring domains of surface proteins.
[1160] Non-limiting examples of such strategies are described in Table 63A and Table 63B.
Table J. Exemplary Cell Surface Display Strategies
Figure imgf000530_0001
FimH (type I E. coli Sandwich Cell surface pili) fusion
PapA (Pap E. coli Sandwich Cell surface pili) fusion
PulA Klebsiella C-terminal Cell
fusion surface/extracellular fluid
Table K. Exemplary Cell Surface Display Strategies
Figure imgf000531_0001
Fimbriae 7-52 aa
S-layer proteins
RsaA 12 aa
Table L. Exemplary Cell Surface Strategies
Figure imgf000532_0001
[1161] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more short therapeutic peptides or polypeptides fused into surface exposed loops of outer membrane proteins (OMPs), e.g., from enteric bacteria. In a non-limiting example, the short therapeutic peptides or polypeptides expressed by the genetically engineered bacteria are inserted into the outer membrane protein LamB, e.g., from E. coli, and displayed on the bacterial cell surface. Extracellular display of peptides through Insertion of peptides into surface exposed loops of LamB is for example described in Hofnung et al., Expression of foreign polypeptides at the Escherichia coli cell surface; Methods Cell Biol. 34:77-105, and Charbit, A. et al., 1987. Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria, J. Immunol. 139:1658–1664.
[1162] In another non-limiting example, the short therapeutic peptides or polypeptides encoded by one or more gene sequence(s) comprised in the genetically engineered bacteria are inserted into the outer membrane protein PhoE, e.g., from E. coli, and displayed on the bacterial cell surface. The PhoE protein is another abundant outer membrane protein of E. coli K-12, which has a trimeric structure and functions as a pore for small molecules. Analysis of the primary structure of PhoE revealed 16 beta sheets which traverse through the membranes, and eight hypervariable regions exposed at the surface of the cell. One or more of these cell surface exposed regions of PhoE protein can be used to insert heterologous peptides. For example, antigenic determinants of pathogenic organisms have been presented in one or more cell surface exposed regions of PhoE protein (e.g., as described in Aterberg et al., 1990; Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants:
immunogenicity of epitopes of foot-and-mouth disease virus; Vaccine. 1990
Feb;8(1):85-91).
[1163] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more short therapeutic peptides or polypeptides fused to protein components of extracellular appendages. Several systems have been described, in which extracellular appendages, such as pili and flagella are used to display peptides of interest at the bacterial cell surface. Examples of flagellar and pilar proteins used include FliC, a major structural component of the E. coli flagellum, and PapA, the major subunit of the Pap pilus. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more components of a FLITRX system. The FLITRX system is an E. coli display system based on the use of fusion protein of FliC and thioredoxin, a small redox protein which represents a highly versatile scaffold that allows peptide inserts to assume a confirmation compatible with binding to other proteins. In the FLITRX system, thioredoxin is fused into a dispensable region of FliC. Then, heterologous peptides can be inserted within the thioredoxin domain in the FliC fusion, and are surface exposed. Other scaffolding proteins are known in the art, some of which may replace thioredoxin as a scaffolding protein in this system.
[1164] In some embodiments, the genetically engineered bacteria comprise a FimH fusion protein, in which the therapeutic peptide of interest is fused to FimH, an adhesin of type 1 fimbriae, e.g., from E. coli. FimH adhesin chimeras containing as many as 56 foreign amino acids in certain positions are transported to the bacterial surface as components of the fimbrial organelles (Pallesen et al., Chimeric FimH adhesion of type I fimbriae: a bacterial surface display system for heterologous sequences. Microbiology 141: 2839-2848).
[1165] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a fusion protein in which the therapeutic peptide of interest is fused to the major subunit of F11 fimbriae, e.g., from E. coli. Hypervariable regions of the major subunit of F11 fimbriae can be used for insertion of heterologous peptides, e.g., antigenic epitopes (Van Die et al., Expression of foreign epitopes in P- fimbriae of Escherichia coli. Mol. Gen. Genet. 222: 297-303).
[1166] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a papA fusion protein, in which the therapeutic peptide of interest is fused to papA. In some embodiments, peptides of interest are inserted following either codon 7 or 68 of the coding sequence for the mature portion of PapA, as peptides in the area of amino acids 7 and 68 of PapA are localized at the external side of the pilus (Steidler et al., Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in
Escherichia coli; J Bacteriol. 1993 Dec;175(23):7639-43).
[1167] In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which encode polypeptides larger than 60 amino acids, e.g., immune modulatory effector, and which are displayed on the bacterial cell surface. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s), which encode a fusion protein, in which a therapeutic peptide of interest, e.g., a polypeptide greater than 60 amino acids in length, is fused to a lipoprotein from a gram negative bacterium, or one or more fragments thereof.
[1168] In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s), which encode a fusion protein, in which a therapeutic protein of interest is fused to peptidoglycan associated lipoprotein (PAL) or a fragment thereof. The fusion protein in located in the periplasm and can be displayed externally upon permeablization of the outer membrane. For example, a PAL-scFv fusion protein was shown to bind its antigen and to be tightly bound to the murein layer of the cell envelope (Fuchs et al., Targeting recombinant antibodies to the surface of Escherichia coli fusion to a peptidoglycan-associated lipoprotein; Biotechnology (N Y).1991 Dec;9(12):1369-72). The PAL-scFv fusion was located in the periplasm and bound to the murein layer, and after permeabilization of the outer membrane, the scFv became accessible to externally added antigen. In some embodiments, the genetically engineered bacteria comprising a fusion protein for surface display further have a permeable outer membrane. Mutations and/or deletions resulting in a leaky outer membrane are described elsewhere herein.
[1169] In one embodiment, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, e.g., an immune modulatory effector, is fused to residues of the major lipoprotein of a gram negative bacterium, e.g., E. coli. In one embodiment, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, is fused to the signal peptide and the nine N- terminal amino acid residues of the major lipoprotein of a gram negative bacterium, e.g., E. coli. These residues of the E. coli major lipoprotein function as a hydrophobic membrane anchor. For example, a fusion construct of these residues with a therapeutic polypeptide, in this case a scFv fragment, resulted in specific accumulation of an immunoreactive and cell-bound polypeptide in E. coli (Laukkanen et al., Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein-single-chain antibody fusion protein. Mol. Microbiol. 4:1259-1268).
[1170] In one embodiment, the genetically engineered bacteria encode a fusion protein, in which a therapeutic protein of interest, is inserted into the TraT protein of a gram negative bacterium, e.g., E. coli, e.g. at position 180. The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. Taylor et al. showed that insertion of the C3 epitope of polio virus, e.g., at position 180, allowed exposure of the antigen to the cell surface, while the oligomeric conformation of the wild-type protein was maintained (Taylor et al., The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationship in the TraT protein. Mol Microbiol. 1990 Aug;4(8):1259-68).
[1171] In one embodiment, the genetically engineered bacteria comprise one or more genes and/or gene cassettes encoding a fusion protein comprising a Lpp-OmpA display vehicle comprising the N terminal outer membrane signal from the major lipoprotein (Lpp) fused to a domain from the outer membrane protein OmpA, fused to the therapeutic polypeptide of interest. In this system, the Lpp signal peptide mediates localization, and OmpA provides the framework for the display of the therapeutic protein of interest. Lpp-OmpA fusions have been used to display several proteins between 20 and 54 kDa in size on the surface of E. coli (see, e.g., Staphopoulos et al., Characterization of Escherichia coli expressing and Lpp-OpmA (46-159)-PhoA fusion protein localized in the outer membrane). For example, Fransco et al fused beta - lactamase to the N-terminal targeting sequence of Lpp and an OmpA fragment containing 5 of the 8 membrane spanning loops of the native protein. This fusion protein was assembled on the cell surface and the beta-lactamase domain was stably anchored in the cell wall (Fransisco et al., Transport ansd anchoring of beta-lactamase to the external surface of Escherichia coli; Proc. Natl. Acad. Sci. USA Vol 89, pp.2713- 2717, 1992).
[1172] In one embodiment, the Type II secretion pathway or a variation thereof is used to for transient or longer duration display of therapeutic proteins of interest on the bacterial cell surface, e.g., the IgA protease secretion pathway of Neisseria or the VirG protein pathway of Shigella. In one embodiment, the IgA protease secretion pathway is used to export and display therapeutic peptides of interest on the cell surface of gram negative bacteria. The IgA proteases of Neisseria gonorrhoeae and Hemophilus influenza use a variation of the most common, Type II secretion pathway, to achieve extracellular export independent of any other gene products. The IgA genes of Neisseria species encode extracellular proteins that cleave human IgA1 antibody. The iga gene alone is sufficient to direct selected extracellular secretion of IgA protease in Neisseria, Salmonella, and E. coli species (Klauser et al., 1993, Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J 9:1991-1999, and references therein). The mature IgA protease is processed in several steps from a large precursor by signal peptidase and autoproteolytic cleavage. The precursor consists of four domains: (1) an aminoterminal signal peptide which mediates inner membrane transport; (2) the protease domain (3) the alpha domain, a basic alpha helical region which is secreted with the protease and (4) the autotransporter beta domain which harbors the essential function for outer membrane transport. Essentially, the C-terminal beta autotransporter domain of the IgA protease forms a channel in the outer membrane that mediates the export of the N terminal domain across the membrane, which in turn becomes transiently displayed on the external surface of the bacteria. The alpha domain and protease domain are then released through proteolytic cleavage. Klauser et al. (1993), showed that replacement of the native N-terminal domains of IgA protease of N.
gonorrhoeae with the cholera toxin B resulted in the surface presentation of the passenger polypeptide in S. typhymurium. In another study, the signal sequence and the C-terminal beta autotransporter domain of the IgA protease of Neisseria gonorrhoeae was used to translocate and display a scFv directed against a porcine epidemic diarrhea virus epitope on the bacterial cell surface of E. coli (Pyo et al., Escherichia coli expressing single chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus; Vaccine (27) (2009) 2030-2036.).
[1173] Thus, in one embodiment, the genetically engineered bacteria encode a IgA protease fragment in which the alpha domain is substituted with a therapeutic protein of interest, and fused to a functional IgA protease beta-domain, which mediates export through the outer membrane. Without wishing to be bound by theory, IgA protease activity is eliminated in such a fusion protein, and therefore the autoproteolytic release of the fusion protein into the medium does not occur, resulting in the display of the therapeutic protein of interest on the cell surface of the gram-negative host bacterium.
[1174] The secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria (see., e.g., Suzuki et al., 1995;
Extracellular transport of VirG protein in Shigella J Biol. Chem 270:30874-30880, and references therein). Thus, in some embodiments, the genetically engineered bacteria encode a fusion protein comprising a therapeutic protein of interest fused to the membrane spanning region of VirG, resulting in surface display of the therapeutic protein of interest. The VirG gene on the large plasmid of Shigella has been shown to be responsible for the localized deposition of filamentous actin (F-actin) trailing from one pole of invading bacterial cells and extending in a filament through the host epithelial cytoplasm. VirG is a surface-exposed outer membrane protein consisting of three distinctive domains, the N-terminal signal sequence (amino acids 1-52), the id α-domain (amino acids 53-758), and the dC-terminal β-core (amino acids 759-1102) (see, e.g., Suzuki et al., 1996; Functional Analysis of Shigella VirG Domains Essential for Interaction with Vinculin and Actin-based Motility; J. Biol. Chem., 271, 21878-21885, and references therein). Suzuki et al. (1995); showed that the fusion of a foreign protein such as MalE or PhoA protein to the N terminus 37-kDa VirG portion resulted in the transport of the passenger polypeptides from the periplasm to the external side of the outer membrane, indicating that the C-terminal 37-kDa VirG portion embedded in the outer membrane is involved in the translocation of the preceding VirG portion or the heterologous or passenger polypeptide from the periplasmic space to the external side of the outer membrane, in a manner homologous to the IgA protease beta-domain. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein, in which a C-terminal 37-kDa VirG protein fragment is fused to a therapeutic protein of interest.
[1175] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein, in which a therapeutic protein of interest is fused to pullulanase for temporary surface display. Pullulanase is specifically released into the medium by Klebsiella pneumonieae, and exists as a fully exposed, cell surface-bound intermediate before it is released into the medium from early stationary growth phase onwards. Cell-surface anchoring is accomplished by an N-terminal fatty acyl modification whose chemical composition is identical to that of other bacterial protein.
[1176] Unlike the IgA protease, the lipoprotein pullulanase (PulA) of Klebsiella pneumoniae, which is also exported via a type II secretion mechanism, requires 14 genes for its translocation across the outer membrane. For example, Pugsley and coworkers have shown that the lipoprotein pullulanase (PulA) can facilitate
translocation of the periplasmic enzyme beta-lactamase across the outer membrane. In particular, in E. coli strains expressing all pullulanase secretion genes, pullulanase-beta- lactamase hybrid protein molecules containing an N-terminal 834-amino-acid pullulanase segment were efficiently transported to the cell surface. Of note, pullulanase hybrids remain only temporarily attached to the bacterial surface and are subsequently released into the medium (Kornacker and Pugsley: The normally periplasmic enzyme beta-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell surface enzyme pullulanase. Mol.
Microbiol. 4:1101-1109, and references therein). Accordingly, in some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising a complete set of pullulanase genes required for secretion and fusion protein comprising a therapeutic protein of interest fused to a N-terminal pullulanase polypeptide fragment, e.g., as described by Kornacker and Pugsley. In some embodiments, the fusion proteins comprising N-terminal pullulanase polypeptide fused to the therapeutic protein of interest, are transiently displayed on the surface of the bacterial cell, and subsequently released into the media or extracellular space.
[1177] In one embodiment, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a fusion protein in which the ice nucleation protein (INP) from Pseudomonas syringae anchors a therapeutic protein of interest in the cell wall. INP is a secretory protein that catalyzes extracellular ice formation as the ice nuclei. INP has been found in a number of Gram-negative species, including P. syringae, Erwinia herbicola, Xanthomonas campestris, and Pseudomonas fluorescens. Four genes in P. syringae strains , inaK, inaV , and inaZ , and inaQ exhibit high similarities in sequences and in primary organization (Li et al., Molecular
Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli Int J Biol Sci. 2012; 8(8): 1097–1108). All INPs (1200 aa to 1500 aa) comprise of three distinct structural domains: (1) the N-terminal domain (approximately 15% of the total sequence), which is relatively hydrophobic and which is are potentially capable of being coupled to the mannan-phosphatidylinositol group in the outer membrane through N- glycan (Asp) or O-glycan (Ser, Thr) linkages; (2) the C-terminal domain (approximately 4%), which is a relatively hydrophilic terminus; and (3) the central repeating domain (CRD) (approximately 81%), which constitutes contiguous repeats given by 16-residue (or 48-residue) periodicities with a consensus octapeptide (Ala-Gly-Tyr-Gly-Ser-Thr- Leu-Thr). INPs have been employed in various bacterial cell-surface display systems including E. coli, Zymomonas mobilis, Salmonellas sp., Vibrio anguillarum,
Pseudomonas putida, and cyanobacteria, in all of which INPs were able to target a heterologous protein onto the surface of the host cell. Moreover, the N-terminal region alone was shown to direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif (Li et al., 2004 Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein; Biotechnol Bioeng. 2004 Jan 20;85(2):214-21). Accordingly, in some embodiments, the genetically engineered bacteria comprise IMP fusions for surface display of a therapeutic peptide of interest. In some embodiments, the N-terminal region of the INP protein is fused to the polypeptide of interest for surface display.
[1178] IMP proteins further have modifiable internal repeating units, ie., CRD length is adjustable, which is allows flexibility in protein fusion length (Jung et al., 1998), and also can accommodate larger polypeptides. For example, the INP-based display systems were used to successfully express a 90 kDA protein on the cell surface of E. coli (Wu et al., 2006; Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity; FEMS Bicrobiology Letters, Volume 256, Issue 1; Pages 119–125).
[1179] It is understood by those skilled in the art that translocation of such fusion or hybrid proteins described herein requires a“translocation-competent” conformation, e.g., the formation of disulfide bonds, e.g., in the periplasmic space, may be undesirable and inhibit translocation through the outer membrane (see, e.g., Klauser et al., 1990), or alternatively may be required for, (or at least not impede) translocation through the outer membrane (see, e.g., Puggsley, 1992; Translocation of a folded protein across the outer membrane in Escherichia coli; Proc Natl Acad Sci U S A. 1992 Dec 15; 89(24): 12058–12062). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding for a fusion protein in which disulfide bonds are prevented from forming prior to the translocation to the cell surface. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding for a fusion protein in which disulfide bonds are formed prior to translocation to the cell surface.
[1180] Expression systems for the display of proteins in Gram-positive bacteria have also been developed. Consequently, in some embodiments, gram positive bacteria are engineered to display therapeutic proteins of interest on their cell surface. Uhlen et al. used fusions to the cell-wall bound, X-domain of protein A, for the display of foreign peptides up to 88 amino acids long to the surface of Staphylococcus strains. For example one study describes an expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram- positive bacterium (Hansson et al., Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus; J Bacteriol. 1992
Jul;174(13):4239-45).
[1181] The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. Accordingly, in some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a therapeutic polypeptide fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A
[1182] E. coli-staphylococcus shuttle vectors have been constructed by taking advantage of the promoter, signal sequence, and propeptide region from the lipase gene construct derived from S. hyicus and the cell surface attachment part of staphylococcal protein A. This system has been investigated for the surface display of heterologous polypeptides on S. carnosus (Samuelson et al., Cell surface display of recombinant proteins on Staphylococcus carnosus; J Bacteriol. 1995 Mar;177(6):1470-6). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a therapeutic polypeptide fusion protein comprising promoter, signal sequence, and propeptide region from the lipase gene construct derived from S. hyicus and the cell surface attachment part of staphylococcal protein A.
[1183] In other studies, the fibrillary M6 proteins of Streptococcus pyrogenes was employed as a carrier for antigen delivery in Streptococcus cells. (Pozzi et al., 1992; Delivery and expression of a heterologous antigen on the surface of streptococci. Infect. Immunm. 60: 1902-1907). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising therapeutic polypeptide fusion proteins comprising the fibrillary M6 proteins of Streptococcus pyrogenes for cell surface display of the therapeutic polypeptide. [1184] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an intimin or invasin. Intimins and invasins belong to a family of bacterial adhesins which specifically interact with various eukaryotic cell surface receptors, thereby mediating bacterial adherence and invasion. Both intimins and invasins provide a structural scaffold ideally suited to the cell surface display.
[1185] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an intimin, e.g., with the
Enterohemorragic E. coli Intimin EaeA protein or a carboxy-terminal truncation thereof (e.g., as described in Wentzel et al, Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA J Bacteriol. 2001 Dec; 183(24): 7273–7284). For example, N-terminal 489 amino acids of invasin are sufficient to promote the localization of a fusion protein to the cell surface. [030] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with an invasin, e.g. Enterohemorrhagic E. coli invasion, or a carboxyterminal truncation thereof. For example, N-terminal 539 amino acids of intimin were sufficient to promote outer membrane localization of a fusion protein (Liu et al., The Tir-binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial-induced host cell signaling; Mol Microbiol. 1999 Oct;34(1):67-81).
[1186] In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with Bacillus anthracis exosporal protein (BclA) as an anchoring motif. The BclA is an exosporium protein, a hair-like protein surrounding the B. anthracis spore. In a nonlimiting example, a polypeptide of interest is linked to the C-terminus of N-terminal domain (21 amino acids) of BclA, e.g., as described in Park et al. (Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis).
[1187] Various other anchoring motifs have been developed including OprF, OmpC, and OmpX. In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) encoding a polypeptide of interest which is displayed on the cell surface through a fusion with OprF, OmpC, and OmpX.
[1188] In some embodiments, the therapeutic polypeptides of interest are permanently displayed on the cell surface of the genetically engineered bacterium. In some embodiments, the therapeutic polypeptides of interest are transiently displayed on the cell surface of the genetically engineered bacterium.
[1189] In some embodiments, the therapeutic polypeptides are displayed in strains, e.g., described herein which display a leaky phenotype. Such strains have deactivating mutations in one or more of genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl.
[1190] In some embodiments, one or more ScFvs are displayed on the bacterial cell surface, alone or in combination with other therapeutic polypeptides of interest.
[1191] In some embodiments, a cell surface display strategy or circuit is combined with a secretion strategy or circuit in one bacterium. In some embodiments, the same polypeptide is both displayed and secreted. In some embodiments, a first polypeptide is displayed and a second is secreted. In some embodiments, a display strategy or circuit strategy is combined with a circuit for the intracellular production of an enzyme and consequentially intracellular catabolism of its substrate. In some embodiments, a display strategy or display circuit is combined with a circuit for the intracellular production of a gut barrier enhancer molecule and/or an anti-inflammatory effector molecule.
Table M. Selected display anchors
Figure imgf000543_0001
509
IntiminN ITHGCYTRTRHKHKLKKTLIMLSAGLGLFFYVNQNSFANGENYFKLGSDSKLLTHDSYQN display tag RLFYTLKTGETVADLSKSQDINLSTIWSLNKHLYSSESEMMKAAPGQQIILPLKKLPFEY
SEQ ID NO: SALPLLGSAPLVAAGGVAGHTNKLTKMSPDVTKSNMTDDKALNYAAQQAASLGSQLQSRS 510 LNGDYAKDTALGIAGNQASSQLQAWLQHYGTAEVNLQSGNNFDGSSLDFLLPFYDSEKML
AFGQVGARYIDSRFTANLGAGQRFFLPANMLGYNVFIDQDFSGDNTRLGIGGEYWRDYFK SSVNGYFRMSGWHESYNKKDYDERPANGFDIRFNGYLPSYPALGAKLIYEQYYGDNVALF NSDKLQSNPGAATVGVNYTPIPLVTMGIDYRHGTGNENDLLYSMQFRYQFDKSWSQQIEP QYVNELRTLSGSRYDLVQRNNNIILEYKKQDILSLNIPHDINGTEHSTQKIQLIVKSKYG LDRIVWDDSALRSQGGQIQHSGSQSAQDYQAILPAYVQGGSNIYKVTARAYYRNGNSSNN VQLTITVLSNGQVVDQVGVTDFTADKTSAKADNADTITYTATVKKNGVAQANVPVSFNIV SGTATLGANSAKTDANGKATVTLKSSTPGQVVVSAKTAEMTSALNASAVIFFDQTKAS [1192] In some embodiments, the scFv Display Construct Sequence 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 sequence of SEQ ID NO: 508, SEQ ID NO: 509, and/or SEQ ID NO: 510.
[1193] In some embodiments, the display anchor 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 sequence of SEQ ID NO: 990, SEQ ID NO: 991, and/or SEQ ID NO: 992.
[1194] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface is operably linked to an inducible promoter. In some embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In some
embodiments, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut, or in the presence of molecules or metabolites associated with hepatic encephalopathy, UCD or another hyperammonemia disorder, or 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. In some embodiments, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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. In some embodiments, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In some
embodiments, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In some embodiments, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[1195] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface is operably linked to a constitutive promoter. In some embodiments, 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. In some embodiments, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the constitutive promoter is selected from a promoter provided in Table IX or Table X. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface is modified and/or mutated, e.g., to enhance stability, or increase secretion, potency or stability.
[1196] In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface may be codon optimized, e.g., to improve expression in the host microorganism. In any of the embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides for display on the bacterial surface 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.
[1197] In any of the embodiments, above and described elsewhere herein, the genetically engineered bacteria may further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art, (7) one or more circuits for the degradation of ammonia described herein (8) one or more circuits for the production and/or secretion of a gut barrier enhancer molecule and (9) one or more circuits for the production or degradation of one or more metabolites (e.g., tryptophan, tryptophan metabolites, arginine or another amino acid, butyrate, acetate, and/or propionate) described herein (10) combinations of one or more of such additional circuits.
Essential Genes and Auxotrophs
[1198] As used herein, the term“essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin.
Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).
[1199] An“essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. [1200] Exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain are shown below. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.
[1201] n auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria. Table XII lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis.
Table XII. Non-limiting Examples of Bacterial Genes Useful for Generation of an
Auxotroph
Figure imgf000547_0001
pheA
proA
thrC
trpC
tyrA [1202] Table XIII shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of E. coli.
Table XIII. Survival of amino acid auxotrophs in the mouse gut
Figure imgf000548_0001
[1203] For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
[1204] Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
[1205] In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
[1206] In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non- auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth.
[1207] Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, lspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB ,nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsI, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art.
[1208] In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain,”ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference). [1209] In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some
embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS
comprising one or more of the following mutations: L36V, C38A, and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A, and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I, and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I, and L6G.
[1210] In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2- aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I, and L6G) are complemented by benzothiazole or indole. [1211] In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).
[1212] In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system, e.g., as shown in FIG. 66.
[1213] In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the recombinant bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more
transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein and in FIG. 62-65. Other embodiments are described in Wright et al.,“GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-316, the entire contents of which are expressly incorporated herein by reference). In some
embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et al., 2015).
[1214] In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that consume excess ammonia. In a more specific aspect, auxotrophic modifications may be used to screen for mutant bacteria that consume excess ammonia by overproducing arginine. As described herein, many genes involved in arginine metabolism are subject to repression by arginine via its interaction with ArgR. The astC gene promoter is unique in that the arginine-ArgR complex acts as a transcriptional activator, as opposed to a transcriptional repressor. AstC encodes succinylornithine aminotransferase, the third enzyme of the ammonia-producing arginine succinyltransferase (AST) pathway and the first of the astCADBE operon in E. coli (Schneider et al., 1998). In certain embodiments, the genetically engineered bacteria are auxotrophic for a gene, and express the auxotrophic gene product under the control of an astC promoter. In these embodiments, the auxotrophy is subject to a positive feedback mechanism and used to select for mutant bacteria which consume excess ammonia by overproducing arginine. A non-limiting example of a positive feedback auxotroph is shown in FIG. 60A and 60B.
Genetic Regulatory Circuits
[1215] In some embodiments, the genetically engineered bacteria comprise multi-layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811, incorporated herein by reference in its entirety).
[1216] In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that overproduce arginine. In some embodiments, the invention provides methods for selecting genetically engineered bacteria that consume excess ammonia via an alternative metabolic pathway, e.g., a histidine biosynthesis pathway, a methionine biosynthesis pathway, a lysine biosynthesis pathway, an asparagine biosynthesis pathway, a glutamine biosynthesis pathway, and a tryptophan biosynthesis pathway. In some embodiments, the invention provides genetically engineered bacteria comprising a mutant arginine regulon and an ArgR-regulated two- repressor activation genetic regulatory circuit. The two-repressor activation genetic regulatory circuit is useful to screen for mutant bacteria that reduce ammonia or rescue an auxotroph. In some constructs, high levels of arginine and the resultant activation of ArgR by arginine can cause expression of a detectable label or an essential gene that is required for cell survival.
[1217] The two-repressor activation regulatory circuit comprises a first ArgR and a second repressor, e.g., the Tet repressor. In one aspect of these embodiments, ArgR inhibits transcription of a second repressor, which inhibits the transcription of a particular gene of interest, e.g., a detectable product, which may be used to screen for mutants that consume excess ammonia, and/or an essential gene that is required for cell survival. Any detectable product may be used, including but not limited to, luciferase, β-galactosidase, and fluorescent proteins such as GFP. In some embodiments, the second repressor is a Tet repressor protein (TetR). In this embodiment, an ArgR- repressible promoter comprising wild-type ARG boxes drives the expression of TetR, and a TetR-repressible promoter drives the expression of at least one gene of interest, e.g., GFP. In the absence of ArgR binding (which occurs at low arginine
concentrations), tetR is transcribed, and TetR represses GFP expression. In the presence of ArgR binding (which occurs at high arginine concentrations), tetR expression is repressed, and GFP is generated. Examples of other second repressors useful in these embodiments include, but are not limited to, ArsR, AscG, LacI, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191). In some embodiments, the mutant arginine regulon comprising a switch is subjected to mutagenesis, and mutants that reduce ammonia by overproducing arginine are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence- activated cell sorting (FACS) when the detectable product fluoresces.
[1218] In some embodiments, the gene of interest is one required for survival and/or growth of the bacteria. Any such gene may be used, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene has been removed or mutated so as not to produce the gene product except under control of ArgR. In some embodiments, an ArgR- repressible promoter comprising wild-type ARG boxes drives the expression of a TetR protein, and a TetR-repressible promoter drives the expression of at least one gene required for survival and/or growth of the bacteria, e.g., thyA, uraA (Sat et al., 2003). In some embodiments, the genetically engineered bacterium is auxotrophic in a gene that is not complemented when the bacterium is present in the mammalian gut, wherein said gene is complemented by a second inducible gene present in the bacterium;
transcription of the second gene is ArgR-repressible and induced in the presence of sufficiently high concentrations of arginine (thus complementing the auxotrophic gene). In some embodiments, the mutant arginine regulon comprising a two-repressor activation circuit is subjected to mutagenesis, and mutants that reduce excess ammonia are selected by growth in the absence of the gene product required for survival and/or growth. In some embodiments, the mutant arginine regulon comprising a two-repressor activation circuit is used to ensure that the bacteria do not survive in the absence of high levels of arginine (e.g., outside of the gut).
Host-Plasmid Mutual Dependency
[1219] In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad-spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention.
[1220] The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies.
[1221] Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a gut enhancer molecule and further comprise a toxin-anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015; FIG. 68). In some embodiments, the genetically engineered bacteria further comprise alp7 from B. subtilis plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015).
Kill Switch
[1222] In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos.
62/183,935, 62/263,329, and 62/277,654, each of which is incorporated herein by reference in their entireties). The kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.
[1223] Bacteria engineered with kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a therapeutic gene(s) or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of argAfbr. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of argAfbr, for example, after the production of arginine or citrulline. Alternatively, the bacteria may be engineered to die after the bacteria has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl β-D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010). In some embodiments, the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of argAfbr. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of argAfbr.
[1224] Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.
[1225] Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in a low-oxygen environment. In some embodiments, the genetically engineered bacteria of the present disclosure, e.g., bacteria expressing argAfbr and repressor ArgR, comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable.
[1226] In another embodiment in which the genetically engineered bacteria of the present disclosure, e.g., bacteria expressing argAfbr and repressor ArgR, express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse
recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.
[1227] In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present.
[1228] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase.
[1229] In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised.
[1230] In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the wherein the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase.
[1231] In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs.
[1232] In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: BxbI, PhiC31, TP901, BxbI, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, Int10, Int11, Int12, Int13, Int14, Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof.
[1233] In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. An exemplary kill-switch in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in FIG. 66- 68. The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment.
[1234] Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter. In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an anti- toxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide.
fbr
[1235] In some embodiments, the argA gene is directly or indirectly under the control of the araBAD promoter. FIG. 13 depicts a schematic diagram of an exemplary fbr fbr
BAD promoter-driven argA construct. In this embodiment, the argA gene is
fbr
inserted between the araC and araD genes. ArgA is flanked by a ribosome binding site, a FRT site, and one or more transcription terminator sequences. The nucleic acid sequence of an exemplary BAD promoter-driven argAfbr construct is shown in Table XIV. All bolded sequences are Nissle genomic DNA. A portion of the araC gene is bolded and underlined, the argAfbr gene is boxed, and the bolded sequence in between is the promoter that is activated by the presence of arabinose. The ribosome binding site is in italics, the terminator sequences are highlighted, and the FRT site is boxed . A portion of the araD gene is boxed in dashes. In some embodiments, 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 BAD promoter sequence of SEQ ID NO: 67 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise the BAD promoter sequence of SEQ ID NO: 67 or a functional fragment thereof.
Table XIV
Figure imgf000561_0001
catgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgtgag cctccggatgacgaccgtagtgatgaatctctccaggcgggaacagcaaaatat cacccggtcggcagacaaattctcgtccctgatttttcaccaccccctgaccgc gaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaa aatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcgt taaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttca tactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgcc gtcactgcgtcttttactggctcttctcgctaacccaaccggtaaccccgctta ttaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaaca aaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtca cactttgctatgccatagcatttttatccataagattagcggatccagcctgac gctttttttcgcaactctctactgtttctccatacccgtttttttggatggagt gaaacgATGGTAAAGGAACGTAAAACCGAGTTGGTCGAGGGATTCCGCCATTC GGTTCCCTGTATCAATACCCACCGGGGAAAAACGTTTGTCATCATGCTCGGCG GTGAAGCCATTGAGCATGAGAATTTCTCCAGTATCGTTAATGATATCGGGTTG TTGCACAGCCTCGGCATCCGTCTGGTGGTGGTCTATGGCGCACGTCCGCAGAT CGACGCAAATCTGGCTGCGCATCACCACGAACCGCTGTATCACAAGAATATAC GTGTGACCGACGCCAAAACACTGGAACTGGTGAAGCAGGCTGCGGGAACATTG CAACTGGATATTACTGCTCGCCTGTCGATGAGTCTCAATAACACGCCGCTGCA GGGCGCGCATATCAACGTCGTCAGTGGCAATTTTATTATTGCCCAGCCGCTGG GCGTCGATGACGGCGTGGATTACTGCCATAGCGGGCGTATCCGGCGGATTGAT GAAGACGCGATCCATCGTCAACTGGACAGCGGTGCAATAGTGCTAATGGGGCC GGTCGCTGTTTCAGTCACTGGCGAGAGCTTTAACCTGACCTCGGAAGAGATTG CCACTCAACTGGCCATCAAACTGAAAGCTGAAAAGATGATTGGTTTTTGCTCT TCCCAGGGCGTCACTAATGACGACGGTGATATTGTCTCCGAACTTTTCCCTAA CGAAGCGCAAGCGCGGGTAGAAGCCCAGGAAGAGAAAGGCGATTACAACTCCG GTACGGTGCGCTTTTTGCGTGGCGCAGTGAAAGCCTGCCGCAGCGGCGTGCGT CGCTGTCATTTAATCAGTTATCAGGAAGATGGCGCGCTGTTGCAAGAGTTGTT CTCACGCGACGGTATCGGTACGCAGATTGTGATGGAAAGCGCCGAGCAGATTC GTCGCGCAACAATCAACGATATTGGCGGTATTCTGGAGTTGATTCGCCCACTG GAGCAGCAAGGTATTCTGGTACGCCGTTCTCGCGAGCAGCTGGAGATGGAAAT CGACAAATTCACCATTATTCAGCGCGATAACACGACTATTGCCTGCGCCGCGC TCTATCCGTTCCCGGAAGAGAAGATTGGGGAAATGGCCTGTGTGGCAGTTCAC CCGGATTACCGCAGTTCATCAAGGGGTGAAGTTCTGCTGGAACGCATTGCCGC TCAGGCTAAGCAGAGCGGCTTAAGCAAATTGTTTGTGCTGACCACGCGCAGTA TTCACTGGTTCCAGGAACGTGGATTTACCCCAGTGGATATTGATTTACTGCCC GAGAGCAAAAAGCAGTTGTACAACTACCAGCGTAAATCCAAAGTGTTGATGGC GGATTTAGGGTAATGGGAATTAGCCATGGTCCATATGAATATCCTCCTTAGTT CCTATTCC gaagttcctattccgaagttcctattctctagaaagtataggaac
Figure imgf000563_0001
GCATGCAAGCTTGGCACTGGCCACGCAAAAAGGCCATCCGTCAGGATGGCCTTC TGCTTAATTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCG GGCCGTTGCTTCGCAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGG AGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGA GCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGctcgagcca tgggacgtccaggtattagaagccaacctggcgctgccaaaacacaacctggt cacgctcacctggggcaatgtcagcgccgttgatcgcgggcgcggcgtcctgg tgatcaaaccttccggcgtcgactacagcatcatgaccgctgacgatatggtc gtggtcagcatcgaaaccggtgaagtggttgaaggtacgaaaaagccctcctc cgacacgccaactcaccggctgctctatcaggcattcccgtctattggcggca ttgtgcacacacactcgcgccacgccaccatctgggcgcaggcgggccagtcg attccagcagccggcaccacccacgccgactatttctacggcaccattccctg cacccgcaaaatgaccgacgcagaaatcaacggtgaatatgagtgggaaaccg gtaacgtcatcgtagaaaccttcgaaaaacagggtatcaatgcagcgcaaatg cccggcgtgctggtccattctcacggcccatttgcatggggaaaaaacgccga agatgcggtgcataacgccatcgtgctggaagaagtcgcttatatggggatat tctgccgtcagttagcgccgcagttaccggatatgcagcaaacgctgctggat aaacactatctgcgtaagcatggcgcgaaggcatattacgggcagtaa [1236] Arabinose inducible promoters are known in the art, including
Figure imgf000563_0002
ParaB, ParaC, and ParaBAD. In one embodiment, the arabinose inducible promoter is from E. coli. In some embodiments, the ParaC promoter and the ParaBAD promoter operate as a bidirectional promoter, with the ParaBAD promoter controlling expression of a heterologous gene(s) in one direction, and the ParaC (in close proximity to, and on the opposite strand from the ParaBAD promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced.
[1237] In one exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding a tetracycline repressor protein (TetR), a ParaC promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the tetracycline repressor protein (PTetR). In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed.
[1238] In one embodiment of the disclosure, the recombinant bacterial cell further comprises an anti-toxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the anti-toxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin.
[1239] In another embodiment of the disclosure, the recombinant bacterial cell further comprises an anti-toxin under the control of the ParaBAD promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti- toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin.
[1240] In another exemplary embodiment of the disclosure, the engineered bacteria of the present disclosure contain a kill-switch having at least the following sequences: a ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a ParaC promoter operably linked to a heterologous gene encoding the AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the ParaBAD promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the ParaBAD promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of ParaBAD promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anti- toxin kill-switch system described directly above.
[1241] In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti- toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it.
[1242] In some embodiments, the engineered bacteria of the present disclosure, for example, bacteria expressing argAfbr and repressor ArgR further comprise the gene(s) encoding the components of any of the above-described kill-switch circuits.
[1243] In any of the above-described embodiments, the bacterial toxin is selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin I47, microcin M, colicin A, colicin E1, colicin K, colicin N, colicin U, colicin B, colicin Ia, colicin Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof.
[1244] In any of the above-described embodiments, the anti-toxin is selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccECTD, MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof.
[1245] In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium.
[1246] In some embodiments, the engineered bacteria provided herein have an arginine regulon comprising one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of each of the operons that encode the enzymes responsible for converting glutamate to arginine and/or an intermediate byproduct, e.g., citrulline, in the arginine biosynthesis pathway, such that the mutant arginine regulon produces more arginine and/or intermediate byproduct than an unmodified regulon from the same bacterial subtype under the same conditions. In some embodiments, the genetically engineered bacteria comprise an arginine feedback resistant N- acetylglutamate synthase mutant, e.g., argAfbr. In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, and carbamoylphosphate synthase, thereby derepressing the regulon and enhancing arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria further comprise an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by an oxygen level-dependent promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter. In some embodiments, the arginine feedback resistant N- acetylglutamate synthase mutant is argAfbr.
[1247] In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the genetically engineered bacteria comprise a mutant arginine regulon, wherein the bacterium comprises a gene encoding a functional N-acetylglutamate synthetase that is mutated to reduce arginine feedback inhibition as compared to a wild-type N- acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding the mutated N-acetylglutamate synthetase is controlled by a promoter that is induced under low-oxygen or anaerobic conditions, wherein the mutant arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N-acetylglutamate kinase, N-acetylglutamate phosphate reductase, acetylornithine aminotransferase, N-acetylornithinase,
carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase, and wherein each operon comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine- mediated repression of the operon via ArgR repressor binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the genes in the operon.
[1248] In some embodiments, the genetically engineered bacteria is an auxotroph comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant. In one embodiment, the genetically engineered bacteria comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant 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 thi1 auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a∆thyA and∆dapA auxotroph.
[1249] In some embodiments, the genetically engineered bacteria comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant further comprises a kill- switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin.
[1250] In some embodiments, the genetically engineered bacteria is an auxotroph comprising a mutant arginine regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons that encode the arginine biosynthesis enzymes and an arginine feedback resistant N-acetylglutamate synthase mutant and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
[1251] In some embodiments of the above described genetically engineered bacteria, the gene encoding the arginine feedback resistant N-acetylglutamate synthetase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding the arginine feedback resistant N-acetylglutamate synthetase 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.
[1252] In some embodiments, the genetically engineered bacteria comprise a mutant arginine repressor comprising one or more nucleic acid mutations such that arginine repressor function is decreased or inactive, or the genetically engineered bacteria do not have an arginine repressor (e.g., the arginine repressor gene has been deleted), resulting in derepression of the regulon and enhancement of arginine and/or intermediate byproduct biosynthesis. In some embodiments, the genetically engineered bacteria further comprise an arginine feedback resistant N-acetylglutamate synthase mutant. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by an oxygen level-dependent promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is controlled by a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from the fumarate and nitrate reductase regulator (FNR) promoter, arginine deiminiase and nitrate reduction (ANR) promoter, and dissimilatory nitrate respiration regulator (DNR) promoter. In some embodiments, the arginine feedback resistant N-acetylglutamate synthase mutant is argAfbr.
[1253] In some embodiments, the genetically engineered bacteria comprise a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant. In some embodiments, the genetically engineered bacterium comprise an arginine regulon, wherein the bacterium comprises a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions and wherein the bacterium has been genetically engineered to lack a functional ArgR repressor.
[1254] In some embodiments, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant is an auxotroph. In one embodiment, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant 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 thi1 auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a∆thyA and∆dapA auxotroph.
[1255] In some embodiments, the genetically engineered bacteria comprising a mutant or deleted arginine repressor and an arginine feedback resistant N- acetylglutamate synthase mutant further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti- toxin.
[1256] In some embodiments, the genetically engineered bacterium is an auxotroph comprising a mutant or deleted arginine repressor and an arginine feedback resistant N-acetylglutamate synthase mutant and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein.
[1257] In some embodiments of the above described genetically engineered bacteria, the gene encoding the arginine feedback resistant N-acetylglutamate synthetase is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene encoding the arginine feedback resistant N-acetylglutamate synthetase 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. Ammonia Transport
[1258] Ammonia transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance ammonia transport into the cell. AmtB is a membrane transport protein that transports ammonia into bacterial cells. In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the native amtB gene. In some
embodiments, the genetically engineered bacteria of the invention also comprise an amtB gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of an amtB gene from a different bacterial species. In some embodiments, the native amtB gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise an amtB gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter.
[1259] In some embodiments, the native amtB gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native amtB gene are inserted into the genome under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter. In alternate embodiments, the native amtB gene is not modified, and a copy of a non-native amtB gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter.
[1260] In some embodiments, the native amtB gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native amtB gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter. In alternate embodiments, the native amtB gene is not modified, and a copy of a non-native amtB gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter.
[1261] In some embodiments, the native amtB gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized amtB gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native amtB gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The ammonia transporter modifications described herein may be present on a plasmid or chromosome.
[1262] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native amtB gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle amtB genes are inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter. In an alternate embodiment, the native amtB gene in E. coli Nissle is not modified, and a copy of a non-native amtB gene from a different bacterium, e.g., Lactobacillus plantarum, is inserted into the E. coli Nissle genome under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr or a constitutive promoter.
[1263] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and the native amtB gene in E. coli Nissle is not modified; one or more additional copies the native E. coli Nissle amtB genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr, or a constitutive promoter. In an alternate embodiment, the native amtB gene in E. coli Nissle is not modified, and a copy of a non-native amtB gene from a different bacterium, e.g., Lactobacillus plantarum, are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of argAfbr, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of argAfbr, or a constitutive promoter. Pharmaceutical Compositions and Formulations
[1264] Pharmaceutical compositions comprising the genetically engineered bacteria described herein may be used to treat, manage, ameliorate, and/or prevent a disorder associated with hyperammonemia or symptom(s) associated with
hyperammonemia. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.
[1265] In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein.
[1266] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.
[1267] The genetically engineered bacteria described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate- release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 105 to 1012 bacteria, e.g., approximately 105 bacteria, approximately 106 bacteria, approximately 107 bacteria, approximately 108 bacteria, approximately 109 bacteria, approximately 1010 bacteria, approximately 1011 bacteria, or approximately 1011 bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.
[1268] The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
[1269] The genetically engineered bacteria disclosed herein may be
administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well- known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.
[1270] The genetically engineered bacteria disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
[1271] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose,
carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid,
polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate- polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG- A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA- MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly
pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch
polymethacrylates, polyamino acids, and enteric coating polymers.
[1272] In some embodiments, the genetically engineered bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.
[1273] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered bacteria described herein. [1274] In one embodiment, the genetically engineered bacteria of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.
[1275] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.
[1276] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.
[1277] In certain embodiments, the genetically engineered bacteria may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject’s diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. [1278] In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S.2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.
[1279] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via
nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.
[1280] The genetically engineered bacteria described herein may be
administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[1281] The genetically engineered bacteria may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
[1282] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.
[1283] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.
[1284] In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see, e.g., U.S. Patent No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.
[1285] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.
[1286] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[1287] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.
[1288] In some embodiments, the disclosure provides a composition comprising a composition (e.g., two or more) of different genetically engineered bacteria, each bacteria encoding and producing a different effector molecule.
[1289] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more gut barrier enhancer molecules and/or anti-inflammatory molecules known in the art or described herein.
[1290] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more enzymes for the production of a short chain fatty acid (SCFA). In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more enzymes for the production of butyrate and optionally one or more deletions of endogenous genes described herein (e.g., pta, ldhA, adhE, frdA). In some embodiments, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) for the production of butyrate comprising ter, thiA1, hbd, crt2, pbt, and buk genes. In some embodiments, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) for the production of butyrate comprising ter, thiA1, hbd, crt2, and tesB genes. In some embodiments, the bacterium comprises an endogenous pta gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises an endogenous adhE gene, and ldhA gene and/or an frdA gene which is knocked down via mutation or deletion. In some embodiments, the bacterium comprises a mutation or deletion in the adhE gene. In some embodiments, the bacterium comprises a mutation or deletion in the frd gene. In some embodiments, the bacterium comprises a mutation or deletion in the ldhA gene. In some embodiments, the bacterium comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA). In some embodiments, the bacterium comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene. In some embodiments, the bacterium comprises a mutation or deletion in the adhE gene and in the pta gene. In some embodiments, the bacterium comprises a mutation or deletion in the frd gene and in the pta gene. In some embodiments, the bacterium comprises a mutation or deletion in the ldhA gene and in the pta gene. In some embodiments, the bacterium comprises a mutation or deletion in two or more of the endogenous adhE gene, ldhA gene and/or frdA gene (e.g., deletion in adhE and ldhA; adhE and frdA; ldhA and frdA) and in the pta gene. In some embodiments, the bacterium comprises a mutation or deletion in all three of adhE gene, ldhA gene and frdA gene and in the pta gene.
[1291] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the production of butyrate and/or deletions of endogenous genes described herein.
[1292] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the production of acetate and/or deletions of endogenous genes described herein.
[1293] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the secretion of an anti-inflammatory cytokine. In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the secretion of IL-22. In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and
deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the secretion of GLP2. In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the secretion of a satiety effector, e.g., GLP1.
[1294] In one embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding GABA transport circuit and/or a GABA metabolic circuit. In one
embodiment, the composition comprises a genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more circuits for producing a manganese membrane transport protein, e.g., MntH, and are capable of transporting manganese ions into the cell (a“manganese transport circuit”).
[1295] In any of the composition embodiments, the composition comprises two or more different genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR), and nucleic acid sequence(s) encoding polypeptides for the production of SCFA and another additional circuit. In any of the composition embodiments, the composition comprises two or more genetically engineered bacteria comprising nucleic acid sequence(s) encoding one or more polypeptides for the reduction of ammonia levels (e.g., ArgAfbr and deletion/mutation of ArgR) and further comprises another genetically engineered bacteria comprising nucleic acid sequence(s) encoding polypeptides for the production of butyrate (e.g., butyrate cassettes alone or in combination with endogenous mutations described herein) and another genetically engineered bacteria comprising an additional circuit. Such additional circuits include circuits for production of a SCFA, e.g., acetate or proprionate described herein, circuits for the secretion of cytokines, e.g., IL-22, circuits for secretion of GLP-1 and/or GLP-2, circuits for the production and/or catabolism of tryptophan and/or one of its metabolites as described herein, circuits for GABA transport and/or metabolism, and circuits for manganese transport.
[1296] In any of the composition embodiments described above and elsewhere herein, a gene sequence encoding one or more polypeptides of any one of the circuits in the composition, is operably linked to an inducible promoter. In any of the composition embodiments described above and elsewhere herein, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In any of the composition embodiments described above and elsewhere herein, the inducible promoter is directly or indirectly induced under condition(s) found in the gut in vivo, e.g., low oxygen conditions. In any of the composition embodiments described above and elsewhere herein, the promoter is induced in the presence of certain molecules or metabolites, e.g., metabolites found in the gut. In any of the composition embodiments described above and elsewhere herein, such molecules or metabolites are specific to certain conditions, e.g., conditions associated with hyperammonemia, such as HE-related molecules. In any of the composition embodiments described above and elsewhere herein, the genetically engineered bacteria comprise a promoter induced by a molecule or metabolite associated with hepatic encephalopathy, 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.
[1297] In any of the composition embodiments described above and elsewhere herein, 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. In any of the composition embodiments described above and elsewhere herein, the promoter is directly or indirectly induced under in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In some embodiments, the promoter is induced in vitro by one or more chemical and/or nutritional inducers, such as arabinose and others described herein. In any of the composition embodiments described above and elsewhere herein, the promoter is directly or indirectly induced in vitro under low oxygen conditions or other conditions described herein. In any of the composition embodiments described above and elsewhere herein, the promoter is directly or indirectly induced in vitro and/or in vivo, under certain conditions described herein.
[1298] In any of the composition embodiments described above and elsewhere herein, a gene sequence encoding one or more polypeptides of any one of the circuits in the composition, is operably linked to a constitutive promoter. In any of the composition embodiments described above and elsewhere herein, 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. In any of the composition embodiments described above and elsewhere herein, the constitutive promoter is active in in vitro conditions, e.g., during strain culture, expansion, production and/or manufacture. In any of the composition embodiments described above and elsewhere herein, the constitutive promoter is selected from a promoter provided in Table IX and Table X. In any of the embodiments described above and elsewhere herein, 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. In any of the composition embodiments described above and elsewhere herein described above and elsewhere herein, a gene sequence encoding one or more polypeptides of any one of the circuits in the composition, is operably linked to a RBS, enhancer or other regulatory sequence. In some
embodiments, the RBS is selected from a promoter provided in Table IX or Table X or is listed in Table XI. In any of the composition embodiments described above and elsewhere herein, a gene sequence encoding one or more polypeptides of any one of the circuits in the composition is modified and/or mutated.
[1299] In any of the composition embodiments described above and elsewhere herein described above and elsewhere herein, the gene sequence encoding one or more polypeptides of any one of the circuits in the composition may be codon optimized, e.g., to improve expression in the host microorganism. In any of the composition
embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides of any one of the circuits in the composition, 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.
[1300] In any of the composition embodiments described above and elsewhere herein, the genetically engineered bacteria may further comprise a resistance to rifaximin. Resistance to rifaximin is caused primarily by mutations in the rpoB gene. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
[1301]
Methods of Treatment
[1302] Another aspect of the invention provides methods of treating a disease or disorder associated with hyperammonemia. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders. In some embodiments, the disorder is a urea cycle disorder such as argininosuccinic aciduria, arginase deficiency, carbamoylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency. In alternate embodiments, the disorder is a liver disorder such as hepatic encephalopathy, acute liver failure, or chronic liver failure; organic acid disorders; isovaleric aciduria; 3- methylcrotonylglycinuria; methylmalonic acidemia; propionic aciduria; fatty acid oxidation defects; carnitine cycle defects; carnitine deficiency; β-oxidation deficiency; lysinuric protein intolerance; pyrroline-5-carboxylate synthetase deficiency; pyruvate carboxylase deficiency; ornithine aminotransferase deficiency; carbonic anhydrase deficiency; hyperinsulinism-hyperammonemia syndrome; mitochondrial disorders; valproate therapy; asparaginase therapy; total parenteral nutrition; cystoscopy with glycine-containing solutions; post-lung/bone marrow transplantation; portosystemic shunting; urinary tract infections; ureter dilation; multiple myeloma; chemotherapy; infection; neurogenic bladder; or intestinal bacterial overgrowth. In some embodiments, the symptom(s) associated thereof include, but are not limited to, seizures, ataxia, stroke-like lesions, coma, psychosis, vision loss, acute encephalopathy, cerebral edema, as well as vomiting, respiratory alkalosis, and hypothermia.
[1303] The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a
therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally, e.g., in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally,
intraduodenally, intraileally, and/or intracolically.
[1304] In certain embodiments, administering the pharmaceutical composition to the subject reduces ammonia concentrations in a subject. In some embodiments, the methods of the present disclosure may reduce the ammonia concentration in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing the ammonia concentration in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating hyperammonemia allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
[1305] Before, during, and after the administration of the pharmaceutical composition, ammonia concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to reduce ammonia concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject’s ammonia concentrations prior to treatment.
[1306] In certain embodiments, the genetically engineered bacteria comprising the mutant arginine regulon is E. coli Nissle. The genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009), or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the mutant arginine regulon may be re- administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in FIGs. 34 and 35. In alternate embodiments, the genetically engineered bacteria are not destroyed within hours or days after
administration and may propagate and colonize the gut.
[1307] The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, including but not limited to, sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate. An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria.
[1308] In one embodiment, the genetically engineered bacteria are administered for prevention, treatment or management of HE. In some embodiments, the genetically engineered bacteria are administered in combination with another therapeutic approach to prevent HE reoccurrence. In one embodiment, the genetically engineered bacteria are administered in combination with branched-chain amino acid supplementation. In one embodiment, the genetically engineered bacteria are administered in combination with acetyl-l-carnitine and/or sodium benzoate and/or zinc and/or acarbose and/or ornithine aspartate. In one embodiment, the genetically engineered bacteria are administered in combination with non-absorbable disaccharides, which are commonly applied to both treat and prevent HE in patients. In one embodiment, the genetically engineered bacteria are administered in combination with lactulose and/or lactitol.
[1309] In one embodiment, the genetically engineered bacteria are administered in combination with one or more antibiotics, for example for the treatment of HE.
Examples of such antibiotics include, but are not limited to, non-absorbable antibiotics, such as aminoglicosides, e.g., neomycin and/or paramomycin. In one embodiment, the antibiotic is rifamycin. In one embodiment, the antibiotic is a rifamycin derivative, e.g., a synthetic derivative, including but not limited to, rifaximin.
[1310] Rifaximin has been shown to significantly reduce the risk of an episode of hepatic encephalopathy, as compared with placebo, over a 6-month period (Bass et a., Rifaximin Treatment in Hepatic Encephalopathy; N Engl J Med 2010; 362:1071- 1081). Rifaximin is a semi-synthetic derivative of rifampin and acts by binding to the beta-subunit of bacterial DNA-dependent RNA polymerase, and thereby blocking transcription. As a result, bacterial protein synthesis and growth is inhibited.
[1311] Rifaximin has been shown to be active against E. coli both in vitro and in clinical studies. It therefore is understood that, for a combination treatment with rifaximin to be effective, the genetically engineered bacteria must further comprise a rifaximin resistance.
[1312] Resistance to rifaximin is caused primarily by mutations in the rpoB gene. This changes the binding site on DNA dependent RNA polymerase and decreases rifaximin binding affinity, thereby reducing efficacy. In one embodiment, the rifaximin resistance is a mutation in the rpoB gene. Non-limiting examples of such mutations are described in e.g., Rodríguez-Verdugo, Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress. BMC Evol Biol. 2013 Feb 22;13:50. Of note, mutations in the same three codons of the rpoB consensus sequence occur repeatedly in unrelated rifaximin-resistant clinical isolates of several different bacterial species (as reviewed in Goldstein, Resistance to rifampicin: a review; The Journal of Antibiotics (2014), 1–6, the contents of which is herein incorporated by reference in its entirety. In some embodiments, the genetically engineered bacteria comprise a known rifaximin resistance mutation, e.g., in the rpoB gene. In other embodiments, a screen can be employed, exposing the genetically engineered bacteria to increasing amounts of rifaximin, to identify a useful mutation which confers rifaximin resistance.
[1313] In some embodiments, the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food. The pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet and amino acid supplementation. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.
[1314] Table 20 shows non-limiting examples of target degradation rates, based on levels of phenylalanine on average in ) in hyperammonemic patients (UCD<HE).
Table 20. Target Ammonia Degradation/Arginine Production Rates
Figure imgf000590_0001
Treatment In Vivo
[1315] The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with hyperammonemia may be used (see, e.g., Deignan et al., 2008; Nicaise et al., 2008), for example, a mouse model of acute liver failure and hyperammonemia. This acute liver failure and hyperammonemia may be induced by treatment with thiol acetamide (TAA) (Basile et al., 1990; Nicaise et al., 2008). Alternatively, liver damage may be modeled using physical bile duct ligation (Rivera-Mancía et al., 2012).
Hyperammonemia may also be induced by oral supplementation with ammonium acetate and/or magnesium chloride (Azorín et al., 1989; Rivera-Mancía et al., 2012).
[1316] Additionally, CCl4 is often used to induce hepaticfibrosis and cirrhosis in animals (Nhung et al., Establishment of a standardized mouse model of hepatic fibrosis for biomedical research; Biomedical Research and Therapy 2014, 1(2):43-49).
[1317] The genetically engineered bacteria of the invention may be administered to the animal, e.g., by oral gavage, and treatment efficacy determined, e.g., by measuring ammonia in blood samples and/or arginine, citrulline, or other byproducts in fecal samples.
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[1319] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure.
Arginine Repressor Binding Sites (ARG Boxes)
Example 1. ARG box mutations [1320] The wild-type genomic sequences comprising ArgR binding sites for each arginine biosynthesis operon in E. coli Nissle is shown in Table 3. Modifications to those sequences are designed according to the following parameters. For each wild- type sequence, the ARG boxes are shown in italics. The ARG boxes of the arginine regulon overlap with the promoter region of each operon. The underlined sequences represent RNA polymerase binding sites and those sequences were not altered. Bases that are protected from DNA methylation during ArgR binding are highlighted, and bases that are protected from hydroxyl radical attack during ArgR binding are bolded. The highlighted and bolded bases were the primary targets for mutations to disrupt ArgR binding.
Example 2. Lambda red recombination [1321] Lambda red recombination is used to make chromosomal modifications, e.g., ARG box mutations. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of E. coli. A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD600 of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.
[1322] DNA sequences comprising the desired ARG box sequences shown in Table 3 were ordered from a gene synthesis company. For the argA operon, the mutant regulatory region comprises the following nucleic acid sequence (SEQ ID NO: 2):
[1323] gcaaaaaaacaCTTtaaaaaCTTaataatttcCTTtaatcaCTTaaagaggtgtaccgtg.
[1324] The lambda enzymes are used to insert this construct into the genome of E. coli Nissle through homologous recombination. The construct is inserted into a specific site in the genome of E. coli Nissle based on its DNA sequence. To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified. The homologous sequence of DNA includes approximately 50 bases on either side of the mutated sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct is used to replace the natural sequence upstream of argA in the E. coli Nissle genome. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting mutant argA construct comprises
approximately 50 bases of homology upstream of argA, a kanamycin resistance marker that can be removed by recombination,
gcaaaaaaacaCTTtaaaaaCTTaataatttcCTTtaatcaCTTaaagaggtgtaccgtg, and
approximately 50 bases of homology to argA.
[1325] In some embodiments, the ARG boxes were mutated in the argG regulatory region as described above, and a BBa_J23100 constitutive promoter was inserted into the regulatory region using lambda red recombination (SYN-UCD105). These bacteria were capable of producing arginine. In alternate embodiments, the argG regulatory region (SEQ ID NO: 16) remained ArgR-repressible (SYN-UCD104), and the bacteria were capable of producing citrulline.
Example 3. Transforming E. coli Nissle [1326] The mutated ARG box construct is transformed into E. coli Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1:100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD600 of 0.1. 0.05 mL of 100X L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD600 of 0.4-0.6. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 0.5 µg of the mutated ARG box construct is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing kanamycin and incubated overnight.
Example 4. Verifying mutants [1327] The presence of the mutation is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 µl of cold ddH2O by pipetting up and down. 3 of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C overnight. A PCR master mix is made using 5 µL of 10X PCR buffer, 0.6 µl of 10 mM dNTPs, 0.4 µL of 50 mM Mg2SO4, 6.0 µL of 10X enhancer, and 3.0 µL of ddH2O (15 µL of master mix per PCR reaction). A 10 µM primer mix is made by mixing 2 µL of primers unique to the argA mutant construct (100 µM stock) into 16 µL of ddH2O. For each 20 µL reaction, 15µL of the PCR master mix, 2.0 µL of the colony suspension (template), 2.0 µL of the primer mix, and 1.0 µL of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C at 5:00 min., 2) 94° C at 0:15 min., 3) 55° C at 0:30 min., 4) 68° C at 2:00 min., 5) 68° C at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 µL of each amplicon and 2.5 µL 5X dye. The PCR product only forms if the mutation has inserted into the genome.
Example 5. Removing selection marker [1328] The antibiotic resistance gene is removed with pCP20. Each strain with the mutated ARG boxes is grown in LB media containing antibiotics at 37° C until it reaches an OD600 of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pCP20 plasmid DNA is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette was placed into the sample chamber, and the electric pulse was applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1-3 hrs. The cells are spread out on an LB plate containing kanamycin and incubated overnight. Colonies that do not grow to a sufficient OD600 overnight are further incubated for an additional 24 hrs. 200 µL of cells are spread on ampicillin plates, 200 µL of cells are spread on kanamycin plates, and both are grown at 37° C overnight. The ampicillin plate contains cells with pCP20. The kanamycin plate provides an indication of how many cells survived the electroporation. Transformants from the ampicillin plate are purified non-selectively at 43° C and allowed to grow overnight.
Example 6. Verifying transformants [1329] The purified transformants are tested for sensitivity to ampicillin and kanamycin. A colony from the plate grown at 43° C is picked and resuspended in 10 µL of LB media. 3 µL of the cell suspension is pipetted onto each of three plates: 1) an LB plate with kanamycin incubated at 37° C, which tests for the presence or absence of the kanR gene in the genome of the host strain; 2) an LB plate with ampicillin incubated at 30° C, which tests for the presence or absence of the ampR gene from the pCP20 plasmid; and 3) an LB plate without antibiotic incubated at 37° C. If no growth is observed on the kanamycin or ampicillin plates for a particular colony, then both the kanR gene and the pCP20 plasmid were lost, and the colony is saved for further analysis. The saved colonies are restreaked onto an LB plate to obtain single colonies and grown overnight at 37° C. The presence of the mutated genomic ARG box is confirmed by sequencing the argA region of the genome.
[1330] The methods for lambda red recombination, transforming E. coli Nissle, verifying the mutation, removing the selection marker, and verifying/sequencing the transformants are repeated for each of the ARG box mutations and operons shown in Table 3. The resulting bacteria comprise mutations in each ARG box for one or more operons encoding the arginine biosynthesis enzymes, such that ArgR binding to the ARG boxes is reduced and total ArgR binding to the regulatory region of said operons is reduced.
Example 7. Arginine feedback resistant N-acetylglutamate synthetase (argAfbr) [1331] In addition to the ARG box mutations described above, the E. coli Nissle bacteria further comprise an arginine feedback resistant N-acetylglutamate synthetase (argAfbr, SEQ ID NO: 30) gene expressed under the control of each of the following promoters: tetracycline-inducible promoter, FNR promoter selected from SEQ ID NOs: 18-29. As discussed herein, other promoters may be used.
[1332] The argAfbr gene is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. SYN-UCD101 comprises wild-type ArgR, wild-type ArgA, tetracycline-inducible argAfbr on a plasmid, and mutations in each ARG box for each arginine biosynthesis operon. The plasmid does not comprise functional ArgR binding sites, i.e., ARG boxes. SYN-UCD101 was used to generate SYN-UCD102, which comprises wild-type ArgR, wild-type ArgA, tetracycline-inducible argAfbr on a plasmid, and mutations in each ARG box for each arginine biosynthesis operon. The plasmid further comprises functional ArgR binding sites, i.e., ARG boxes. In some instances, the presence and/or build-up of functional ArgR may result in off-target binding at sites other than the ARG boxes. Introducing functional ARG boxes in this plasmid may be useful for reducing or eliminating off-target ArgR binding, i.e., by acting as an ArgR sink. SYN-UCD104 comprises wild-type ArgR, wild-type ArgA, tetracycline-inducible argAfbr on a low-copy plasmid, tetracycline-inducible argG, and mutations in each ARG box for each arginine biosynthesis operon except for argG. SYN-UCD105 comprises wild-type ArgR, wild-type ArgA, tetracycline-inducible argAfbr on a low-copy plasmid, constitutively expressed argG (SEQ ID NO: 17 comprising the BBa_J23100
constitutive promoter), and mutations in each ARG box for each arginine biosynthesis operon. SYN-UCD103 is a control Nissle construct.
[1333] The argAfbr gene 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. 18. 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. At the site of insertion, DNA primers that are homologous to the site of insertion and to the argAfbr construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described above.
[1334] The resulting E. coli Nissle bacteria are genetically engineered to include nucleic acid mutations that reduce arginine-mediated repression– via ArgR binding and arginine binding to N-acetylglutamate synthetase– of one or more of the operons that encode the arginine biosynthesis enzymes, thereby enhancing arginine and/or citrulline biosynthesis.
[1335] Arginine Repressor (ArgR) sequences The wild-type argR nucleotide sequence in E. coli Nissle and the nucleotide sequence following argR deletion are shown below in Table 21 and Table 22.
Table 21. Wild-type argR nucleotide sequence
Figure imgf000602_0001
Table 22. Nucleotide sequence following argR deletion
Figure imgf000603_0001
Example 9. Deleting ArgR [1336] A pKD46 plasmid is transformed into the E. coli Nissle host strain. E. coli Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD600 of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The E. coli cells are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the E. coli cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C.
[1337] Approximately 50 bases of homology upstream and downstream of the ArgR gene are added by PCR to the kanamycin resistance gene in the pKD4 plasmid to generate the following KanR construct: (~50 bases upstream of ArgR) (terminator) (kanR gene flanked by FRT sites from pKD4) (DNA downstream of argR).
[1338] In some embodiments, both argR and argG genes are deleted using lambda red recombination as described above, and the bacteria are capable of producing citrulline. Example 10. Bacterial Strains having Arginine feedback resistant N- acetylglutamate synthetase (argAfbr) and ArgR deletion [1339] In addition to the ArgR deletion described above, the E. coli Nissle bacteria further comprise an arginine feedback resistant N-acetylglutamate synthetase (argAfbr, SEQ ID NO: 30) gene expressed under the control of each of the following promoters: tetracycline-inducible promoter, FNR promoter selected from SEQ ID NOs: 18-29. As discussed herein, other promoters may be used.
[1340] The argAfbr gene is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. ArgR is deleted (∆ArgR) in each of SYN-UCD201, SYN- UCD202, and SYN-UCD203. SYN-UCD201 further comprises wild-type argA, but lacks inducible argAfbr. SYN-UCD202 comprises∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a high-copy plasmid. SYN-UCD203 comprises∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid. SYN-UCD204 comprises∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid. SYN-UCD205 comprises∆ArgR and argAfbr expressed under the control of a FNR- inducible promoter (fnrS2) on a low-copy plasmid.
[1341] The argAfbr gene 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. 18. 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. At the site of insertion, DNA primers that are homologous to the site of insertion and to the argAfbr construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described above. The resulting E. coli Nissle bacteria have deleted ArgR and inserted feedback resistant N-acetylglutamate synthetase, thereby increasing arginine or citrulline biosynthesis.
Example 11. Generation of∆ThyA [1342] An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in E. coli Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine.
[1343] A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a 100um concentration are found in Table 23.
Table 23. Primer Sequences
Figure imgf000605_0001
[1344] For the first PCR round, 4x50ul PCR reactions containing 1ng pKD3 as template, 25ul 2xphusion, 0.2ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions:
[1345] step1: 98c for 30s
[1346] step2: 98c for 10s
[1347] step3: 55c for 15s
[1348] step4: 72c for 20s
[1349] repeat step 2-4 for 30 cycles
[1350] step5: 72c for 5min
[1351] Subsequently, 5ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer’s instructions and eluted in 30ul nuclease free water. [1352] For the second round of PCR, 1ul purified PCR product from round 1 was used as template, in 4x50ul PCR reactions as described above except with 0.2ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30ul as described above.
[1353] For the third round of PCR, 1ul of purified PCR product from round 2 was used as template in 4x50ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2.
Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a E. coli Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1ml SOC medium containing 3mM thymidine was added, and cells were allowed to recover at 37 C for 2h with shaking. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100ul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 20ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. + cam 20ug/ml + or - thy 3mM. (thyA auxotrophs will only grow in media
supplemented with thy 3mM).
[1354] Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37°C until OD600 = 0.4 - 0.6. 1mL of cells were washed as follows: cells were pelleted at 16,000xg for 1 minute. The supernatant was discarded and the pellet was resuspended in 1mL ice-cold 10% glycerol. This wash step was repeated 3x times. The final pellet was resuspended in 70ul ice-cold 10% glycerol. Next, cells were electroporated with 1ng pCP20 plasmid DNA, and 1mL SOC supplemented with 3mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30°C for 1 hour. Cells were then pelleted at 10,000xg for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100ul LB containing 3mM thymidine and spread on LB agar plates containing 3mM thy and 100ug/ml carbenicillin and grown at 30°C for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42°C.
[1355] To test the colony-purified transformants, a colony was picked from the 42°C plate with a pipette tip and resuspended in
Figure imgf000607_0001
of the cell suspension was pipetted onto a set of 3 plates: Cam, (37°C; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30°C, tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37°C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37°C.
Example 12. Quantifying ammonia [1356] The genetically engineered bacteria described above were grown overnight in 5 mL LB. The next day, cells were pelleted and washed in M9 + glucose, pelleted, and resuspended in 3 mL M9 + glucose. Cell cultures were incubated with shaking (250 rpm) for 4 hrs and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2) at 37° C. At baseline (t=0), 2 hours, and 4 hours, the OD600 of each cell culture was measured in order to determine the relative abundance of each cell.
[1357] At t=0, 2 hrs, and 4 hrs, a 1 mL aliquot of each cell culture was analyzed on the Nova Biomedical Bioprofile Analyzer 300 in order to determine the
concentration of ammonia in the media. Both SYN-UCD101 and SYN-UCD102 were capable of consuming ammonia in vitro (FIG. 26). FIG. 25, 26, and 27 depict bar graphs of ammonia concentrations using SYN-UCD202, SYN-UCD204, SYN- UCD103, and blank controls.
Example 13. Quantifying arginine and citrulline [1358] In some embodiments, the genetically engineered bacteria described above are grown overnight in LB at 37° C with shaking. The bacteria are diluted 1:100 in 5mL LB and grown at 37° C with shaking for 1.5 hr. The bacteria cultures are induced as follows: (1) bacteria comprising FNR-inducible argAfbr are induced in LB at 37° C for up to 4 hrs in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2, and 20mM nitrate) at 37° C; (2) bacteria comprising tetracycline-inducible argAfbr are induced with anhydrotetracycline (100 ng/mL); (3) bacteria comprising arabinose-inducible argAfbr are induced with 1% arabinose in media lacking glucose. After induction, bacterial cells are removed from the incubator and spun down at maximum speed for 5 min. The cells are resuspended in 1 mL M9 glucose, and the OD600 is measured. Cells are diluted until the OD600 is between 0.6- 0.8. Resuspended cells in M9 glucose media are grown aerobically with shaking at 37C. 100 µL of the cell resuspension is removed and the OD600 is measured at time = 0. A 100 µL aliquot is frozen at -20° C in a round-bottom 96-well plate for mass spectrometry analysis (LC-MS/MS). At each subsequent time point, 100 µL of the cell suspension is removed and the OD600 is measured; a 100 µL aliquot is frozen at -20C in a round-bottom 96-well plate for mass spectrometry analysis. Samples are analyzed for arginine and/or citrulline concentrations. At each time point, normalized concentrations as determined by mass spectrometry vs. OD600 are used to determine the rate of arginine and/or citrulline production per cell per unit time.
[1359] In some embodiments, the genetically engineered bacteria described above are streaked from glycerol stocks for single colonies on agar. A colony is picked and grown in 3 mL LB for 4 hrs or overnight, then centrifuged for 5 min at 2,500 rcf. The cultures are washed in M9 media with 0.5% glucose. The cultures are resuspended in 3 mL of M9 media with 0.5% glucose, and the OD600 is measured. The cultures are diluted in M9 media with 0.5% glucose, with or without ATC (100 ng/mL), with or without 20 mM glutamine, so that all of the OD600 are between 0.4 and 0.5. A 0.5 mL aliquot of each sample is removed, centrifuged for 5 min. at 14,000 rpm, and the supernatant is removed and saved. The supernatant is frozen at -80° C, and the cell pellets are frozen at -80° C (t=0). The remaining cells are grown with shaking (250 rpm) for 4-6 hrs and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2) at 37° C. One 0.5 mL aliquot is removed from each sample every two hours and the OD600 is measured. The aliquots are centrifuged for 5 min at 14,000 rpm, and the supernatant is removed. The supernatant is frozen at - 80° C, and the cell pellets are frozen at -80° C (t=2, 4, and 6 hrs). The samples are placed on ice, and arginine and citrulline levels are determined using mass
spectrometry.
[1360] For bacterial culture supernatants, samples of 500, 100, 20, 4, and 0.8 µg/mL arginine and citrulline standards in water are prepared. In a round-bottom 96- well plate, 20 µL of sample (bacterial supernatant or standards) is added to 80 µL of water with L-Arginine-13C6,15N4 (Sigma) and L-Citrulline-2,3,3,4,4,5,5-d7 (CDN isotope) internal standards at a final 2 µg/mL concentration. The plate is heat-sealed with a PierceASeal foil and mixed well. In a V-bottom 96-well polypropylene plate, 5 µL of diluted samples is added to 95 µL of derivatization mix (85 µL 10 mM NaHCO3 pH 9.7 and 10 µL 10 mg/mL dansyl-chloride (diluted in acetonitrile). The plate is heat- sealed with a ThermASeal foil and mixed well. The samples are incubated at 60°C for 45 min for derivatization and centrifuged at 4000 rpm for 5 min. In a round-bottom 96- well plate, 20 µL of the derivatized samples are added to 180 µL of water with 0.1% formic acid. The plate is heat-sealed with a ClearASeal sheet and mixed well.
[1361] Arginine and citrulline are measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 24 below provides a summary of a LC-MS/MS method.
Table 24. a LC-MS/MS Method Summary
Figure imgf000609_0001
[1362] Intracellular arginine and secreted (supernatant) arginine production in the genetically engineered bacteria in the presence or absence an ATC or anaerobic inducer is measured and compared to control bacteria of the same strain under the same conditions.
[1363] Total arginine production over 6 hrs in the genetically engineered bacteria in the genetically engineered bacteria in the presence or absence an ATC or anaerobic inducer is measured and compared to control bacteria of the same strain under the same conditions.
Example 14. Efficacy of genetically engineered bacteria in a mouse model of hyperammonemia and acute liver failure [1364] Wild-type C57BL6/J mice are treated with thiol acetamide (TAA), which causes acute liver failure and hyperammonemia (Nicaise et al., 2008). The TAA mouse model is an industry-accepted in vivo model for HE. Mice are treated with unmodified control Nissle bacteria or Nissle bacteria engineered to produce high levels of arginine or citrulline as described above.
[1365] On day 1, 50 mL of the bacterial cultures are grown overnight and pelleted. The pellets are resuspended in 5 mL of PBS at a final concentration of approximately 1011 CFU/mL. Blood ammonia levels in mice are measured by mandibular bleed, and ammonia levels are determined by the PocketChem Ammonia Analyzer (Arkray). Mice are gavaged with 100 µL of bacteria (approximately 1010 CFU). Drinking water for the mice is changed to contain 0.1 mg/mL
anhydrotetracycline (ATC) and 5% sucrose for palatability.
[1366] On day 2, the bacterial gavage solution is prepared as described above, and mice are gavaged with 100 µL of bacteria. The mice continue to receive drinking water containing 0.1 mg/mL ATC and 5% sucrose.
[1367] On day 3, the bacterial gavage solution is prepared as described above, and mice are gavaged with 100 µL of bacteria. The mice continue to receive drinking water containing 0.1 mg/mL ATC and 5% sucrose. Mice receive an intraperitoneal (IP) injection of 100 µL of TAA (250 mg/kg body weight in 0.5% NaCl).
[1368] On day 4, the bacterial gavage solution is prepared as described above, and mice are gavaged with 100 µL of bacteria. The mice continue to receive drinking water containing 0.1 mg/mL ATC and 5% sucrose. Mice receive another IP injection of 100 µL of TAA (250 mg/kg body weight in 0.5% NaCl). Blood ammonia levels in the mice are measured by mandibular bleed, and ammonia levels are determined by the PocketChem Ammonia Analyzer (Arkray).
[1369] On day 5, blood ammonia levels in mice are measured by mandibular bleed, and ammonia levels are determined by the PocketChem Ammonia Analyzer (Arkray). Fecal pellets are collected from mice to determine arginine content by liquid chromatography-mass spectrometry (LC-MS). Ammonia levels in mice treated with genetically engineered Nissle and unmodified control Nissle are compared.
Example 15. Efficacy of genetically engineered bacteria in a mouse model of hyperammonemia and UCD [1370] Ornithine transcarbamylase is urea cycle enzyme, and mice comprising an spf-ash mutation exhibit partial ornithine transcarbamylase deficiency, which serves as a model for human UCD. Mice are treated with unmodified control Nissle bacteria or Nissle bacteria engineered to produce high levels of arginine or citrulline as described above.
[1371] 60 spf-ash mice were treated with the genetically engineered bacteria of the invention (SYN-UCD103, SYN-UCD204) or H2O control at 100ul PO QD: H2O control, normal chow (n=15); H2O control, high protein chow (n=15); SYN-UCD103, high protein chow (n=15); SYN-UCD204, high protein chow (n=15). On Day 1, mice were weighed and sorted into groups to minimize variance in mouse weight per cage. Mice were gavaged and water with 20 mg/L ATC was added to the cages. On day 2, mice were gavaged in the morning and afternoon. On day 3, mice were gavaged in the morning and weighed, and blood was drawn 4h post-dosing to obtain baseline ammonia levels. Mice were gavaged in the afternoon and chow changed to 70% protein chow. On day 4, mice were gavaged in the morning and afternoon. On day 5, mice were gavaged in the morning and weighed, and blood was drawn 4h post-dosing to obtain ammonia levels. On days 6 and 7, mice were gavaged in the morning. On day 8, mice were gavaged in the morning and weighed, and blood was drawn 4h post-dosing to obtain ammonia levels. On day 9, mice were gavaged in the morning and afternoon. On day 10, mice were gavaged in the morning and weighed, and blood was drawn 4h post-dosing to obtain ammonia levels. On day 12, mice were gavaged in the morning and afternoon. On day 13, mice were gavaged in the morning and weighed, and blood was drawn 4h post-dosing to obtain ammonia levels. Blood ammonia levels, body weight, and survival rates are analyzed (FIG. 29).
Example 16. Efficacy of genetically engineered bacteria in a mouse model of hyperammonemia and UCD (spf-ash) maintained on a high protein diet [1372] The hyperammonia /UCD (spf-ash) model described in Example 14 was used to assess the in vivo efficacy of genetically engineered bacteria encoding ArgAfbr driven by a fnr promoter on a low copy plasmid on ammonia levels upon administration of a high protein diet.
[1373] Two strains encoding ArgAfbr driven by a fnr promoter on a low copy plasmid, SYN-UCD206 (comprising∆ArgR and∆ThyA and argAfbr expressed under the control of a FNR-inducible promoter (fnrS2) on a low-copy plasmid) and SYN- UCD205 (comprising∆ArgR and argAfbr expressed under the control of a FNR- inducible promoter (fnrS2) on a low-copy plasmid) were compared to determine whether thymidine auxotrophy can influence the efficacy of ammonia removal from the blood.
[1374] Spf-ash mice were treated by oral administration with the genetically engineered bacteria (SYN-UCD205, SYN-UCD206) or H2O control. Normal or high protein chow was provided as follows: SYN-UCD205, high protein chow (n=10); SYN- UCD206, high protein chow (n=10); H2O control, normal chow (n=10); H2O control, high protein chow (n=10). For SYN-UCD205 and SYN-UCD206, a dose of 100 ul of > 1 X1010 cells/ml was administered twice a day for 12 days, with the exception of days 1, 5, 6, and 7, where bacteria were administered once. On Day 1, mice were weighed and randomized. T=0 NH4 levels were determined from mandibular bleeds using the PocketChem Ammonia Analyzer (Arkray), and mice were subsequently and gavaged. On day 2, mice were gavaged in the morning and afternoon. On day 3, mice were gavaged in the morning and afternoon and the chow was changed from normal chow to 70% protein chow. On day 4, mice were gavaged in the morning and afternoon. On day 5, mice were gavaged in the morning and weighed, and blood was drawn 4h post- dosing to obtain ammonia levels. On days 8 through 12, mice were gavaged in the morning and afternoon.
[1375] As seen in FIG. 30, ammonia levels of spf-ash mice in a high protein diet were reduced 48 hours after switch to high protein chow in the SYN-UCD205 and SYN-UCD206 groups as compared to the H2O high protein diet control group, indicating that the FNR inducible promoter can drive ArgAfbr expression, resulting in decreased ammonia levels in the blood of the mice treated with the engineered bacteria. The observed reduction in ammonia levels was similar in both SYN-UCD205 and SYN- UCD206, indicating that ThyA auxotrophy does not have a significant effect on efficacy of SYN-UCD206.
[1376] Example 17. Engineering bacterial strains using chromosomal insertions
[1377] Bacterial strains, in which ArgAfbr is integrated directly into the E. coli Nissle genome under the control of an FNR-responsive promoter at the MALEK site were constructed.
[1378] To create a vector capable of integrating the PfnrS-ArgAfbr into the chromosome at the Nissle MalE and MalK loci, Gibson assembly was used to add 1000 bp sequences of DNA homologous to the Nissle MALE/K locus to both sides of a flippase recombination target (FRT) site-flanked chloramphenicol resistance (cmR) cassette on a knock-in knock-out (KIKO) plasmid. Gibson assembly was then used to clone the PfnrS-ArgAfbr DNA sequence between these homology arms, adjacent to the FRT-cmR-FRT site. Successful insertion of the fragment was validated by sequencing. PCR is used to amplify the entire MalEK::FRT-cmR-FRT:: PfnrS-ArgAfbr:: MalK region. This knock-in PCR fragment was used to transform an electrocompetent Nissle strain that contains a temperature-sensitive plasmid encoding the lambda red
recombinase genes. After transformation, cells were grown for 2 hrs at 37 °C. Growth at 37 °C cures the temperature-sensitive plasmid. Transformants with successful chromosomal integration of the fragment were selected on chloramphenicol at 20 µg/mL.
[1379] In some embodiments, recombinase-based switches may be used to activate PfnrS-ArgAfbr expression. To construct a strain allowing recombinase-based switches to regulate ArgAfbr expression, the PfnrS-driven Int5 gene and the rrnBUP- driven, recombinase site-flanked ArgAfbrare synthesized by Genewiz (Cambridge, MA). Gibson assembly is used to add 1000 bp sequences of DNA homologous to the Nissle malE and malK loci on either side of the PfnrS-Int5, rrnBUP- ArgAfbr sequence and to clone this sequence between the homology arms. Successful insertion of the fragment into a KIKO plasmid is validated by sequencing. PCR is used to amplify the entire PfnrS-Int5, rrnBUP- ArgAfbr region. This knock-in PCR fragment is used to transform an electrocompetent Nissle strain expressing the lambda red recombinase genes. After transformation, cells are grown for 2 hrs at 37 °C. Transformants with successful integration of the PfnrS-ArgAfbr at the malEK intergenic region are selected on kanamycin at 50 µg/mL. This strategy may also be used to construct a recombinase- based strain requiring T7 polymerase activity for ArgAfbr expression.
Example 18. Comparison of in vitro efficacy of chromosomal insertion and plasmid-bearing engineered bacterial strains [1380] To compare the in vitro efficacy between engineered bacterial strains harboring a chromosomal insertion of ArgAfbr driven by an fnr inducible promoter at the malEK locus and strains with a low copy plasmid comprising ArgAfbr driven by an fnr inducible promoter, arginine levels in the media were measured at various time points post anaerobic induction. Additionally, to assess whether auxotrophy for thymidine may have an effect on arginine production efficiency, arginine production of engineered bacterial strains with or without a ThyA deletion, comprising the fnr- ArgAfbr on a low copy plasmid or integrated on the chromosome, were compared.
[1381] Overnight cultures were diluted 1:100 in LB and grown with shaking (250 rpm) at 37 °C. After 1.5 hrs of growth, the bacteria cultures were induced as follows: (1) bacteria comprising FNR-inducible argAfbr were induced in LB at 37° C for 4 hrs in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2, and 20mM nitrate) at 37° C; (2) bacteria comprising tetracycline-inducible argAfbr were induced with anhydrotetracycline (100 ng/mL). After induction, bacteria were removed from the incubator and spun down at maximum speed for 5 min. The cells were resuspended in 1 mL M9 glucose, and the OD600 was measured. Cells were diluted until the OD600 was between 0.6-0.8. Resuspended cells in M9 glucose media were grown aerobically with shaking at 37C. 100 µL of the cell resuspension was removed and the OD600 is measured at time = 0. A 100 µL aliquot was frozen at -20° C in a round-bottom 96-well plate for mass spectrometry analysis (LC-MS/MS). At each subsequent time point (e.g., 30, 60, and 120 min), 100 µL of the cell suspension was removed and the OD600 was measured; a 100 µL aliquot was frozen at -20C in a round-bottom 96-well plate for mass spectrometry analysis. Samples were analyzed for arginine concentrations. At each time point, normalized concentrations as determined by mass spectrometry vs. OD600 were used to determine the rate of arginine production per cell per unit time. A summary of the LC-MS/MS method is provided above.
[1382] Arginine production at 30, 60, and 120 min post induction was compared between (1) Syn-UCD301 (SYN825; comprising∆ArgR and argAfbr expressed under the control of a FNR-inducible promoter integrated into the chromosome at the malEK locus), (2) SYN-UCD205 (comprising∆ArgR and argAfbr expressed under the control of a FNR-inducible promoter on a low-copy plasmid), and (3) SYN-UCD206
(comprising∆ArgR and∆ThyA and argAfbr expressed under the control of a FNR- inducible promoter on a low-copy plasmid. SYN-UCD103 was used as is a control Nissle construct and results are shown in FIG. 31A.
[1383] FIG. 31A shows the levels of arginine production of SYN-UCD205, SYN-UCD206, and SYN-UCD301 measured at 0, 30, 60, and 120 minutes. Arginine production was comparable between all three strains, with the greatest arginine production seen with SYN-UCD301 at 120 minutes, indicating that chromosomal integration of FNR ArgA fbr results in similar levels of arginine production as seen with the low copy plasmid strains expressing the same construct, and may even slightly increase the rate of arginine production. SYN-UCD206 exhibited attenuated arginine production as compared to SYN-UCD205 and SYN-UCD-301 (lower arginine levels at 60 minutes), but reached comparable arginine production levels at 120 minutes, indicating that∆ThyA may have a slight attenuating effect on arginine production. No arginine production was detected for the SYN-UCD103 control.
[1384] Next, samples were prepared as described above and arginine production at 120 min post induction was compared between (1) SYN-UCD204 (comprising ∆ArgR and argAfbr expressed under the control of a tetracycline-inducible promoter on a low-copy plasmid), and (2) SYN-UCD301 (comprising∆ArgR, CmR and argAfbr expressed under the control of a FNR-inducible promoter integrated into the
chromosome at the malEK locus), (3) SYN-UCD302 (comprising∆ArgR,∆ThyA, CmR (chloramphenicol resistance) and argAfbr expressed under the control of a FNR- inducible promoter integrated into the chromosome at the malEK locus), and (4) SYN- UCD303 (comprising∆ArgR,∆ThyA, KanR (kanamycin resistance) and argAfbr expressed under the control of a FNR-inducible promoter integrated into the
chromosome at the malEK locus). [1385] SYN-UCD106, comprising∆ArgR and∆ThyA was used as is a control Nissle construct. Results are shown in FIG. 31B. As seen in FIG. 31B, arginine production was elevated to between 0.7 and 0.9 umol/1X109 cells, indicating that arginine production is at similar levels in strains bearing ArgAfbr on a plasmid and strains with integrated copies of ArgAfbr.
Example 19. Efficacy of genetically engineered bacteria in a mouse model of hyperammonemia and UCD (spf-ash) maintained on a high protein diet [1386] The hyperammonia /UCD (spf-ash) model described in Example 14 was used to assess the in vivo efficacy of genetically engineered bacteria encoding ArgAfbr driven by a fnr promoter integrated into the bacterial chromosome on ammonia levels upon administration of a high protein diet. Mice were treated with unmodified control Nissle bacteria or Nissle bacteria engineered to produce high levels of arginine or citrulline as described above.
[1387] Two strains, one with a ThyA deletion (SYN-UCD303) and one without a ThyA deletion (SYN-UCD301) were tested for efficacy and compared to determine whether∆ThyA may influence the efficacy of ammonia removal from the blood with these stains harboring chromosomal fnr-ArgAfbr.
[1388] Spf-ash mice were treated by oral administration with the genetically engineered bacteria (SYN-UCD301, SYN-UCD303) or H2O control. Normal and high protein chow was provided as follows: SYN-UCD301, high protein chow (n=10); SYN- UCD303, high protein chow (n=10); H2O control, normal chow (n=10); H2O control, high protein chow (n=10). For SYN-UCD301, SYN-UCD303, and SYN-UCD106, a dose of 100 ul of > 1 X1010 cells/ml was administered twice a day for 12 days, with the exception of days 1, 5, 6, and 7, where bacteria were administered once. Essentially the same protocol was followed as described in Example 16, with blood being drawn on day 5 to obtain ammonia levels (FIG. 32A). On day 10, survival rates were analyzed and a time course of survival is shown in FIG. 32B.
[1389] As depicted in FIG. 32A, ammonia levels of spf-ash mice in a high protein diet were reduced in the SYN-UCD301 and SYN-UCD303 groups as compared to the H2O high protein diet control group, indicating that the FNR inducible promoter can drive ArgAfbr expression when the construct is integrated into the chromosome, resulting in decreased ammonia levels in the blood of the mice treated with the engineered bacteria. The observed reduction in ammonia levels was similar in both SYN-UCD301 and SYN-UCD303, indicating that ThyA auxotrophy does not have a significant effect on efficacy of SYN-UCD303. As seen in FIG. 32B, SYN-UCD301 and SYN-UCD303 showed prolonged survival as compared to controls. Experiments were conducted twice sequentially with similar results.
Example 20. Comparison of Efficacy at Various Doses
[1390] To determine the lowest dose which can be used, while achieving optimal arginine production in the hyperammonia /UCD (spf-ash) model described in Example 14, three doses of SYN-UCD303 were administered.
[1391] Spf-ash mice were treated by gavage with the genetically engineered bacteria (SYN-UCD303) or H2O control. For SYN-UCD303, doses of 1 X107, 1X108, 1X109, and 1 X1010 CFUs were administered in a volume of 100 ul twice a day for 12 days, with the exception of days 1, 5, 6, and 7, where bacteria were administered once. Normal chow or high protein chow was provided as follows: SYN-UCD303 (1 X107 CFU), high protein chow (n=10); SYN-UCD303 (1 X108 CFU), high protein chow (n=10); SYN-UCD303 (1 X109 CFU), high protein chow (n=10); H2O control, normal chow (n=10); H2O control, high protein chow (n=10). Essentially the same protocol was followed as described in Example 16, with blood being drawn on day 5 to obtain ammonia levels. Blood ammonia levels were analyzed for each dose on day 5. Results are depicted in FIG. 33. Both doses of 1X108 and 1X109 were sufficient to result in a significant reduction of blood ammonia levels in this model.
Example 21. Nissle residence
[1392] Unmodified E. coli Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non-limiting example using a streptomycin-resistant strain of E. coli Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention.
[1393] C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 25. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 µg/mL). The plates were incubated at 37°C overnight, and colonies were counted.
Table 25: CFU administered via oral gavage
Figure imgf000618_0001
[1394] On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1- 6; Table 26). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 µg/mL). The plates were incubated at 37°C overnight, and colonies were counted.
[1395] Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 µg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 26.
[1396] FIG. 34 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. Table 26. Nissle residence in vivo
Figure imgf000618_0002
Figure imgf000619_0001
Example 22. Intestinal Residence and Survival of Bacterial Strains in vivo
[1397] Localization and intestinal residence time of SYN-UCD303 (integrated fnrS inducible promoter driving ArgAfbr, kanamycin resistance,∆ThyA, FIG. 35C) was compared to SYN-UCD106 (∆ArgR,∆ThyA, and chloramphenicol resistance, FIG. 35B) and SYN-UCD103 (streptomycin resistant Nissle, FIG. 35A). Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon.
[1398] Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 1010 CFU/mL. Mice (C57BL6/J, 10-12 weeks old) were gavaged with 100 µL of bacteria (approximately 109 CFU). Drinking water for the mice was changed to contain 0.1 mg/mL
anhydrotetracycline (ATC) and 5% sucrose for palatability. At each time point (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating.
[1399] In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37°C overnight, and colonies were counted. The amount of bacteria and residence time of SYN-UCD103, SYN-UCD106, and SYN-UCD303 seen in each compartment is shown in FIG. 35. As seen in FIG. 35, all three strains behave in a similar manner. Comparing FIG. 35A with FIG. 35B,∆ThyA auxotrophy and∆ArgR does not seem to have any substantial effect on residency and transit time.
Example 23. Effect of Auxotrophy on Intestinal Residence Time [1400] To determine if auxotrophy may have an effect on localization and time of residence, the mouse model described above is used to compare the residency of SYN-UCD303 (∆ThyA) with SYN-UCD304 (wild type ThyA). SYN-UCD103 (streptomycin resistant control Nissle) is administered in parallel to determine whether the Arg R deletion and argAfbr have an effect on residence.
[1401] Bacteria are prepared and mice are gavaged, euthanized and intestinal effluents collected at various time points as described in Example 22. To determine the CFU of bacteria in each effluent, the effluent is serially diluted and plated as described in Example 20. For SYN-UCD103 streptomycin containing plates and for SYN- UCD301 chloramphenicol containing plates are used.
Example 24. TAA model of Hyperammonemia [1402] TAA treatment of mice has previously been employed in the literature to model increased blood ammonia levels associated with UCDs, acute and chronic liver disease and HE (Wallace MC, et al., Lab Anim. 2015 Apr;49(1 Suppl):21-9. Standard operating procedures in experimental liver research: thioacetamide model in mice and rat)s. In some embodiments, a TAA-induced mouse model of hyperammonemia is employed to investigate the ability of the genetically engineered bacteria to reduce blood ammonia levels. The TAA serves as an alternative to the spf-ash model. Because spf-ash is a genetic model, the numbers of mice are limiting so developing an inducible model in wild-type mice would greatly facilitate in vivo testing of potential strains of interest.
[1403] To investigate the effects of engineered bacteria on prolonged elevations of blood ammonia, the bacteria are administered to C57BL6 mice that are also administered a dose of 300mpk thioacetamide (TAA).
[1404] C57BL6 (10 weeks old) are administered one daily dose of SYN- UCD103 or SYN-UCD303 (100 ul of > 1 X1010 cells/ml) or vehicle control.
Alternatively, mice are administered 2 daily doses of bacteria (100 ul of > 1 X1010 cells/ml) (n=5 for each treatment group), once in the AM and once in the PM. After three days of pre-dosing with the bacteria, the mice are treated intraperitoneally with thioacetamide (TAA) at 300mpk or with H2O as control. Alternatively, the mice are treated twice daily, once in the AM and once in the PM with 250mpk. The duration of the study is five days. Ammonium levels are measured and overall health survival, body weight change is monitored.
[1405] In brief, animals are acclimated for 7 days. On day 1 of the time course, animals are weighed, bled to measure baseline ammonia and collect fecal pellets (per cage), and are randomized based on initial blood ammonia levels. Animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD103 or SYN- UCD303 (100ul/dose/animal). Water is changed to H2O(+)20mg/ml ATC. On day 2, animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN- UCD103 or SYN-UCD303 (100ul/dose/animal). On day 3, animals are weighed and dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD103 or SYN-UCD303 (100ul/dose/animal). Additionally, animals are dosed intraperitoneally with 300mpk TAA (or saline control). Alternatively, animals are dosed with 250mpk TAA (or saline control) once in the AM and once in the PM. On day 4, animals are weighed, bled, and blood ammonia is measured. Fecal pellets are collected per cage. Animals are dosed per oral gavage either once or twice (AM and PM) with H2O, SYN- UCD103 or SYN-UCD303 (100ul/dose/animal). Animals may also be dosed with 250mpk TAA (or saline control). On day 5, animals are weighed, bled, and blood ammonia levels are measured. Fecal pellets are collected (per cage). Animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD103 or SYN- UCD303 (100ul/dose/animal). Ammonium levels, bacterial load in the fecal pellets, and overall health survival, and body weight changes are monitored.
Example 25. Model of Hyperammonemia using Arginase Inhibitors
[1406] As an alternative to the genetic spf-ash model, arginase inhibitors +/- high protein chow are used as an inducible model of hyperammonemia. Arginase inhibitors fall into at least 2 classes (described in Steppan et al., Front Immunol. 2013; 4: 278. Development of Novel Arginase Inhibitors for Therapy of Endothelial
Dysfunction). The first group of arginase inhibitors consisted of the boronic acid analogs of l-arginine (2)S-amino-6-hexanoic acid (ABH) and S-2-BEC both of which inhibit the catalytic activity of arginase. Another category of arginase inhibitors, that is mainly represented by N-hydroxy-l-arginine (NOHA) and N-hydroxy-nor-l-arginine (nor-NOHA), is characterized by N-hydroxy-guanidinium side chains and inhibit arginase by displacing the metal-bridging hydroxide ion of arginase with their N- hydroxy group. The model development study employs an arginase inhibitor from each group, BEC and nor-NOHA +/- high protein chow (70% protein).
[1407] To determine if engineered bacteria can change ammonium levels in the blood, inducible models using an arginase inhibitor from each group, BEC and nor- NOHA, +/- high protein chow (70% protein) (70% protein) in wild type mice are employed. C57BL6 (Female, 8 weeks) are treated by oral gavage with the genetically engineered bacteria (100 ul of > 1 X1010 cells/ml) or a vehicle control and
intraperitoneally either with BEC or norNOHA, and are kept either on a normal chow (n=5 per treatment group) or a high protein chow diet (n=5 per treatment group).
Administration groups are as follows for SYN-UCD303 and a vehicle control-treated animals: normal chow (n=5); high protein chow (70% protein chow; n=5); high protein chow (+)BEC (n=5); high protein chow (+)norNOHA (n=5).
[1408] On day 1 of the time course, animals are weighed, bled to measure baseline ammonia, and are randomized based on initial blood ammonia levels. Fecal pellets are collected (per cage). Animals are dosed by oral gavage with H2O, SYN- UCD303 or SYN-UCD103 (100ul/dose/animal). Water is changed to H2O(+)20mg/ml ATC. On day 2, animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD303 or SYN-UCD103 (100ul/dose/animal). On day 3, animals are weighed and dosed by oral gavage either once or twice (AM and PM) with H2O, SYN- UCD303 or SYN-UCD103 (100ul/dose/animal). Additionally, animals are dosed intraperitoneally with BEC (25mpk) or norNOHA (100mpk) or saline control. For the high protein diet groups, the chow is changed from normal chow to 70% protein chow. On day 4, animals are weighed, bled, and blood ammonia is measured. Fecal pellets are collected per cage. Animals are dosed per oral gavage either once or twice (AM and PM) with H2O, SYN-UCD303 or SYN-UCD103 (100ul/dose/animal). Animals are dosed intraperitoneally with BEC (25mpk) or norNOHA (100mpk). On day 4, animals are dosed per oral gavage either once or twice (AM and PM) with H2O, SYN-UCD303 or SYN-UCD103 (100ul/dose/animal). Animals are weighed, bled 1 h post dose, and blood ammonia is measured. Fecal pellets are collected per cage. Animals are also dosed intraperitoneally with BEC (25mpk) or norNOHA (100mpk). On day 5, Animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD303 or SYN-UCD103 (100ul/dose/animal). Animals are weighed, bled 1h post dose, and ammonia levels are measured. [1409] A similar study was conducted using strains SYN-UCD202, SYN- UCD204, and SYN-UCD103, and results are shown in FIG. 28.
Example 26. Effect of Proliferation Potential and Metabolic Activity on Arginine
Production
[1410] The requirement of cell division and/or active metabolism for arginine production from the genetically engineered bacteria was investigated in strain
SYNUCD-303.
[1411] SYNUCD-301 were incubated with 70% isopropanol or phosphate buffered saline (PBS) as a control for 1 hour with shaking. 70% isopropanol disrupted the cellular membrane, which prevented cell division, but should allow cell metabolism. PBS incubation had no effect on the cells (not shown). After treatment, the cells were mixed at specific ratios in M9 media supplemented with 0.5% glucose and 3mM thymidine. Cells were incubated with shaking at 37 C for 2 hours. Cells can use the ammonium chloride contained in the M9 media to form arginine. Arginine
concentration was measured in the media at time zero and again after the 2 hour incubation, and the amount of arginine produced per hour per billion cells was calculated. As seen in FIG. 36A and 36B., a greater ratio of isopropanol treated cells to untreated in a culture results in fewer CFUs as determined by plating, and lower levels of arginine production. Arginine production relative to amount of bacteria present remained constant across the various cultures (FIG. 36C). These results indicate that only viable bacteria are contributing to arginine production.
Example 27. Repeat-Dose Pharmacokinetic and Pharmacodynamic Study of SYN- UCD-303 Following Daily Nasogastric Gavage Dose Administration for 28-days in
Cynomolgus Monkeys (non-GLP)
[1412] To evaluate any potential toxicities arising from administration of the genetically engineered bacteria or E coli Nissle alone, the pharmacokinetics and pharmacodynamics of SYN-UCD303 and an E. coli Nissle with kanamycin resistance (SYN-UCD107) were studied following daily nasogastric gavage (NG) dose
administration for 28-days to female cynomolgus monkeys. Cynomolgus monkeys were selected because this species is closely related, both phylogenetically and
physiologically, to humans and is a species commonly used for nonclinical toxicity evaluations. The genetically engineered bacteria were administered by nasal gastric gavage, consistent with the proposed route of administration in humans. Animals overall well-being (clinical observations), weight clinical pathology (serum chemistry, hematology, and coagulation) were tracked. Fecal samples were examined for bacterial load. Plasma is analyzed for ammonia levels.
A. Materials, animals and dosing regimen:
[1413] The study was conducted in compliance with nonclinical Laboratory Studies Good Laboratory Practice Regulations issued by the U.S. Food and Drug Administration (Title 21 of the Code of Federal Regulations, Part 58; effective June 20, 1979) and the OECD Principles on Good Laboratory Practice (C[97]186/Final; effective 1997). The animals were individually housed based on the recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).
[1414] Animals used in the study were Female Purpose-bred, non-naive cynomolgus monkey (Macaca fascicularis) with 3 to 6 kg (at initial physical exam) 3 to 8 years (at initial physical exam) of age (SNBL USA stock, Origin: Cambodia).
[1415] For control of bias, animals were randomized to groups in a
weight-stratified manner to achieve similar group body weight distributions. The number of animals selected for randomization and their weights and ages are included in Table 27.
Table 27. Animals for randomization
Figure imgf000624_0001
Animals were identified by unique tattoo numbers.
[1416] Seventeen animals were acclimated for 7 days prior to dose initiation and fifteen animals were assigned to treatment groups. Spare animals were removed from the study after Day 1.
[1417] For the duration of the study, animals were offered PMI LabDiet® Fiber- Plus® Monkey Diet 5049 biscuits twice daily. Animal were fasted for at least 2 hours prior to dose administration and fed within 1 hour post dose. Animals also were fasted as required by specific procedures (e.g., prior to blood draws for serum chemistry, fecal collection). The diet was routinely analyzed for contaminants and found to be within manufacturer’s specifications. No contaminants were expected to be present at levels that would interfere with the outcome of the study. [1418] Fresh drinking water was provided ad libitum to all animals. The water was routinely analyzed for contaminants. No contaminants were present at levels that would interfere with the outcome of the study. Animals were given fruits, vegetables, other dietary supplements, and cage enrichment devices throughout the course of the study.
[1419] Previously quarantined animals were acclimated to the study room for 7 days prior to initiation of dosing (day 1). The last dosing occurred on day 28. A stratified randomization scheme incorporating body weights was used to assign animals to study groups. Animals were assigned to groups and treated as indicated in Table 28.
Table 28. Group Assignments
Figure imgf000625_0001
* Concentration of Sodium Bicarbonate: .36 M or 0.12 M (0.36 M for the control article)
[1420] SYN-UCD107 and SYN-UCD303 stocks were prepared at 1×109 cfu/mL and 1×1011 cfu/mL in 15% glycerol in 1X PBS with 2.2% glucose and 3 mM thymidine and were kept at 86 to -60 °C (see Table 28). PBS made in 20% glycerol with sodium bicarbonate was used as a control vehicle. Carbonate concentration was 0.36M and 0.12M for sodium bicarbonate (see Table 28). On the day of each dosing, bacteria and vehicle control were removed from the freezer and put on ice and thawed and placed on ice until dosing.
[1421] Animals were dosed at 0, 1×109, or 1×1012 cfu/animal. All animals were dosed via nasal gastric gavage (NG) followed by control/vehicle flush once daily for 28- days. The concentration of bicarbonate and volume for each group is specified in Table 28. Vials were inverted at least 3 times prior to drawing the dose in the syringe. The dose site and dose time (end of flush time) was recorded. On Day 6, Animals 19, 21, and 23 in Group 4 (SYN-UCD303, 1x109/animal) received 5 mL bicarbonate flush instead of 14 mL followed by test article dose administration.
B. Analysis
Overall Condition and Weight [1422] Overall condition: Clinical observations were performed twice daily, beginning on Day -6 through 44. The first observation was in the AM. The second observation was no sooner than 4 hours after the AM observation. During the dosing phase, the second observation was performed 4 hour (±10 minutes) post dose administration. Additional clinical observations were performed, as necessary.
[1423] No test article-related clinical observations were identified in this study.
[1424] Incidental and/or procedural findings, commonly noted in similarly housed animals, occurred sporadically among individuals, were noted in control animals, were also present during the acclimation phase, and did not increase in severity and incidence among dose groups. These included findings related to skin (scab/crust, wounds, abrasions, abnormal skin discoloration, bruising), hair loss, kinked tail, sunken eyes, inappetence, emesis, and urogenital discharge.
[1425] Weight: Body weights were measured on Days -6, 1, 8, 11, 15, 22, 29, 36, and 43 prior to the first feeding and dose administration, as applicable.
[1426] No test article-related effects on body weights were identified in this study. Decreases of 5 to 10% body weight, relative to baseline (Day 1), was apparent across all groups, including control, by Day 36 and determined to be study
procedure-related. Compared to Day 36, an upward trend was noted in the available body weight data on Day 43, albeit values did not completely return to baseline.
Clinical Pathology [1427] Blood Collection: Animals were fasted overnight prior to daily dose administration and at least 4 hours prior to each series of collections that included specimens for serum chemistry and plasma bioanalysis. In these instances, associated clinical pathology evaluations were from fasted animals. Blood was collected by venipuncture from a peripheral vein of restrained, conscious animals via a single draw (if possible) and divided into appropriate tubes for analysis, as follows: Hematology: approximately 1.3 mL, K2EDTA tube; Coagulation: approximately 1.8 mL, 3.2% sodium citrate tube; Serum chemistry: approximately 1.0 mL, serum separator tube (SST); Plasma sample: approximately 1.0 mL, Lithium Heparin; The blood for clinical pathology assessment was processed to serum or plasma, or used intact, according to SNBL USA SOPs.
[1428] Whenever possible, blood was collected via a single draw and then divided appropriately. Specimen collection frequency is summarized in Table 29.
Table 29. Specimen collection frequency
Figure imgf000627_0001
– = Not applicable
x = Number of times procedure performed within the week [1429] Table 30 Summarizes the Clinical Pathology Assay Information. Table 30. Clinical pathology assay information
Figure imgf000628_0001
a Residual samples were discarded prior to study finalization [1430] No test article-related effects in hematology, coagulation, and serum chemistry parameters were identified in this study.
[1431] Plasma Samples: Animals were fasted for 4 hours prior to removal of the sample. Approximately 1 mL of blood were collected from the femoral vein and transferred into 2 mL lithium heparin tubes on Days -1 and 30, and prior to dose administration on Days 2, 7, 14, and 28. After collection of the target volume of blood in the tube, approximately 0.05 (on Days -1 and 2) or 0.1 mL (on Days 7, 14, 28, and 30) of mineral oil was added to cover the surface of blood in the tubes. Tubes were not inverted and were placed on wet ice.
[1432] The samples were centrifuged within 15 minutes of collection at 2 to 8 °C to obtain plasma and the plasma was maintained on dry ice prior to storage at -60 to -86°C [1433] Analysis of specimens is conducted using a blood ammonia analyzer instrument.
[1434] Fecal Sample Collection: Two fecal samples per animal were collected at the target time points listed in Table 29. Sample collection dates and times were recorded. 50 mL falcon tube with approximately 5mL PBS were used as the container (If feces is liquid, no PBS is added). To get the fecal sample weight, pre- and post- sampling weight of container was taken. Samples were collected from the bottom of the cage from each animal. To get fresh and un-contaminated samples, remaining food was removed and the cage pan was cleaned and squeegeed to remove debris and/or water before the collection. On Days -5, 2, 4, 7, and 14, food removals and pan cleanings were performed after the first feeding and/ or dose administration. On Days 18, 20, 24, 28, 30, 35, 40, 46, and 50, the food removals and pan cleanings were performed the night before each collection. Visually fresh fecal samples were also collected in the morning before any procedures, except clinical observations, occurred.
[1435] Fecal Swab Collection: When fecal samples were not collected by the end of the scheduled day, fecal swab samples were collected with a cotton tip applicator from the rectum of animals restrained in a procedure cage.
[1436] Samples were put on wet ice immediately after the collection. Samples were stored at -20 to -15 °C until analysis. Analysis of specimens was conducted using a PCR analytical method as described in Example 31.
[1437] Two fecal samples per animal were collected on Day -5 and after dose administration on Days 2, 4, 7, and 14, and three fecal samples per animal were collected on Days 29, 30, 35, 40, 46, and 50, and prior to dose administration on Days 18 through 28. Fifty milliliter falcon tubes with approximately 5 mL PBS were used for the collections on Days -5 through 14, and 50 mL falcon tubes with
approximately 5 mL PBS for Tube 1, 20 mL of 50% glycerol and 10 mM thymidine for Tube 2, and 20 mL of 50% glycerol/PBS for Tube 3, were used for the collections on Days 18 through 50. Specimens were put on wet ice immediately after the collection and the contents of each tube collected on Days 18 through 50 were broken-up and mixed using a sterile tongue depressor.
[1438] Since no fecal samples were collected from the animals listed in Table 29 by the end of the collection day, two fecal swab samples were collected per animal. After sample collection, the cotton part of the swab was transferred to a 5 mL cryovial with 1 mL of PBS and immediately put on wet ice.
[1439] Results are shown in FIG. 37, and show that the amount of bacteria quantified from fecal samples follows a similar pattern for Kanamycin resistant control Nissle (SYN-UCD107) and SYN-UCD303. Bacteria in the fecal samples reach a level of less than 1000 bacteria/per ml of feces by day 35. Results indicate that under these dosing conditions, similar amounts of bacteria were recovered with the auxotroph SYN- UCD303 as control kanamycin resistant Nissle in the feces. Similar results have been observed in mice. In conclusion, SYN-UCD303 appeared to be present in the NHP feces at nearly the same concentration as Nissle (SYN-UCD107).
[1440] In overall conclusion, the test article, SYN-UCD303, 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 test article-related mortality occurred and no test article-related effects were identified upon clinical observation, body weight, and clinical pathology assessment.
Example 28. Repeat-Dose Pharmacokinetic and Pharmacodynamic Study of SYN- UCD-303 Following Daily Nasogastric Gavage Dose Administration for 28-days in
Cynomolgus Monkeys (non-GLP) [1441] Pharmacokinetics and pharmacodynamics of SYN-UCD303, SYN- UCD304, SYN-UCD305, and SYN-UCD306 and are studied following daily nasogastric gavage (NG) dose administration for 28-days to female cynomolgus monkeys essentially as described in Example 27. Cynomolgus monkeys are selected because this species is closely related, both phylogenetically and physiologically, to humans and is a species commonly used for nonclinical toxicity evaluations. The genetically engineered bacteria are administered by nasal gastric gavage, consistent with the proposed route of administration in humans. Animals overall well-being (clinical observations), weight clinical pathology (serum chemistry, hematology, and
coagulation) are tracked. Plasma is analyzed for ammonia levels, and fecal samples are examined for bacterial load.
Example 29. 4-Week Toxicity Study in Cynomolgus Monkeys with a 4-Week
Recovery (GLP) [1442] To evaluate any potential toxicities arising from administration of the genetically engineered bacteria, the pharmacokinetics and pharmacodynamics of SYN- UCD303 is studied following daily nasogastric gavage (NG) dose administration for 28- days to female cynomolgus monkeys under GLP conditions.
[1443] In other embodiments, the study is conducted SYN-UCD304, SYN- UCD305, and/or SYN-UCD306.
[1444] The study is conducted in compliance with nonclinical Laboratory Studies Good Laboratory Practice Regulations issued by the U.S. Food and Drug Administration (Title 21 of the Code of Federal Regulations, Part 58; effective June 20, 1979) and the OECD Principles on Good Laboratory Practice (C[97]186/Final; effective 1997). The animals are individually housed based on the recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).
[1445] Animals are administered SYN-UCD303 or control vehicle essentially as described in the Example 27, except that all materials are manufactured under GMP standards. Dosing is tabulated in Table 31. Additionally, animals are acclimated for 14 days and the dosing period is daily for 28 days followed by a recovery period of 28 days. Additionally, animals are euthanized at the end of the study to conduct histological analysis.
Table 31. Dosing Period and Regimen
Figure imgf000631_0001
Figure imgf000632_0002
[1446] Study Analysis is conducted as described in Table 32. Hematology, Coagulation, Serum Chemistry and Plasma Samples parameters are essentially as described in Example 27, and are analyzed using the methods described in Example 27. Collection and analysis of fecal samples is essentially conducted as described in Example 27.
Table 32. Study Analysis
Figure imgf000632_0001
Figure imgf000633_0001
[1447] To evaluate any potential toxicities arising from administration of the genetically engineered bacteria, the pharmacokinetics and pharmacodynamics of SYN- UCD303 is studied following daily gavage dose administration for 4 weeks followed by a two week recovery under GLP conditions.
[1448] All materials are manufactured under GLP conditions. CD-1 Mice, 6-8 weeks of age at initiation of study are acclimated for 7 days. Groups of males and female mice are studied separately. SYN-UCD303 or vehicle control are administered as described in Table 33.
[1449] In certain embodiments, the study is conducted SYN-UCD304, SYN- UCD305, and/or SYN-UCD306. Table 33. Study Design
Figure imgf000634_0001
a Toxicity (Tox) Animals, Terminal Necropsy Day 29; b Toxicity (Tox) Animals, Recovery Necropsy Day 42
[1450] The study analysis is described in Table 34. Hematology, Coagulation, Serum Chemistry and Plasma Samples parameters are essentially as described in Example 27, and are analyzed using the methods described in Example 27. Collection and analysis of fecal samples is essentially conducted as described in Example 27. Histology is conducted as in Example 29.
Table 34. Study Analysis
Figure imgf000634_0002
Figure imgf000635_0001
[1451] To analyze fecal samples from non-human primates (NHPs) for the presence of Nissle, the number of bacteria from NHP samples was quantified based on the quantity of DNA in the sample using qPCR. In some embodiments, this protocol is used for the analysis of fecal samples from other mammals, including, but not limited to, mice and humans.
[1452] Sample Homogenization: Fecal samples (stored at -20°C) were thawed at room temperature for 90 minutes. For solid fecal samples, the approximate solid volume was estimated, and phosphate buffered saline (PBS) was added to double the volume. For liquid fecal samples, no additional PBS was added. Samples were then vortexed for 30 seconds per tube. Fecal samples were homogenized using a disposable Pestle (Fisher 12-141-363) in PBS in Eppendorf tubes and kept on ice for subsequent procedures.
[1453] DNA purification: The homogenized samples (250 µL) were removed using sterilized filter tips (Racked Gilson Expert Sterilized Filter Tips) cut at the first gradation line and transferred to Eppendorf tubes. DNA was purified from the homogenized samples (250 µl) using the MoBio PowerLyzer PowerSoil DNA Isolation Kit (12855-100) following manufacturer’s protocol. The amount of purified DNA recovered from each sample was quantified by measuring the OD260 of the sample on a Eppendorf BioSpectrometer Basic. [1454] PCR reaction: Two reactions (Reaction 1 and Reaction 2) were assembled and run in triplicate. The first reaction served to quantify the amount of Nissle, and the second to quantify the total amount of bacteria present in the fecal samples. For the first qPCR reaction, purified DNA (5ng), 0.4 uL of Primer 1 (10 uM), 0.4 uL of Primer 2 (10 uM), and 10 uL of SYBR Green PCR Master Mix (Thermo Fisher Scientific: 4368577) were brought up to 20 uL with water. For the second qPCR reaction, purified DNA (5ng), 0.4 uL of Primer 3 (10 uM), 0.4 uL of Primer 4 (10 uM), and 10 uL of SYBR Green PCR Master Mix (Thermo Fisher Scientific: 4368577) were brought up to 20 uL with water.
[1455] The sequences of primers used in the reactions at a concentration of 10 ^M are found in Table 35. Table 35. Primer Sequences
Figure imgf000636_0001
[1456] For quantification of the amount of bacterial DNA amplified, a standard curve was run for each reaction. To generate the standard curve, Fragment #1
(synthesized at a set concentration) (Table 36) was diluted in 10-fold serial dilutions, resulting in the following amounts: no DNA, 1 copy of Fragment #1, 10 copies of Fragment #1, 100 copies of Fragment #1, and so on, until the eighth well has 106 copies of fragment #1. Standard DNAs were then added to the qPCR reaction mix for Reaction #1.
Table 36. Standard DNA Sequence (Fragment #1)
Figure imgf000636_0002
[1457] PCR reaction conditions for standard curve, and Reactions 1 and 2 are shown in Table 37.
Table 37. PCR Reaction Conditions
Figure imgf000637_0001
[1458] As a quality control measure, the melt-curves of the test qPCR reactions were compared to the positive control for each primer set, to ensure that the melt-curve of the positive controls matched the melt-curve of the test samples. The presence and quantity of Nissle and total bacteria was determined by analyzing CT values (Cycle threshold values) against the standard curve.
Example 32. Construction of vectors for overproducing butyrate [1459] In addition to the ammonia conversion circuit, GABA transport circuit, GABA metabolic circuit, and/or manganese transport circuit described above, the E. coli Nissle bacteria further comprise one or more circuits for producing a gut barrier enhancer molecule.
[1460] To facilitate inducible production of butyrate in E. coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322. The butyrate gene cassette is placed under the control of a FNR regulatory region selected from SEQ ID NOs: 18-29 (Table III). In certain constructs, the FNR- responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. [1461] In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Tables 38 and 39). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Tables 14-17). In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.
Table 38. pLogic031-nsrR-norB-butyrate construct (SEQ ID NO: 79)
Figure imgf000638_0001
Figure imgf000639_0001
Figure imgf000640_0001
Figure imgf000641_0001
Figure imgf000642_0002
Table 39. Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct
Figure imgf000642_0001
Figure imgf000643_0001
Figure imgf000644_0001
Figure imgf000645_0001
[1462] The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA and may exhibit dependence on oxygen as a co- oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from Treponema denticola, trans-2-enoynl-CoA reductase (ter), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, a second butyrate gene cassette in which the ter gene replaces the bcd2-etfA3-etfB3 genes of the first butyrate cassette is synthesized (Genewiz,
Cambridge, MA). The ter gene is codon-optimized for E. coli codon usage using Integrated DNA Technologies online codon optimization tool
(https://www.idtdna.com/CodonOpt). The second butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322. The second butyrate gene cassette is placed under control of a FNR regulatory region as described above. In certain constructs, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 38 and Table 39). In certain constructs, the butyrate gene cassette is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 40).
Table 40. ROS regulated constructs, OxyR construct, Tet-regulated constructs
Figure imgf000646_0001
Figure imgf000647_0001
Figure imgf000648_0001
Figure imgf000649_0001
Figure imgf000650_0001
Figure imgf000651_0001
Figure imgf000652_0001
Figure imgf000653_0001
Figure imgf000654_0001
Figure imgf000655_0001
Figure imgf000656_0001
[1463] In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter.
[1464] In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB. TesB is a thioesterase found in E. Coli that cleaves off the butyrate from butyryl- coA, thus obviating the need for pbt-buk.
[1465] In one embodiment, tesB is placed under the control of a FNR regulatory region selected from any of the sequences in Table III. In an alternate embodiment, tesB is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS- responsive transcription factor, e.g., nsrR. In yet another embodiment, tesB is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR. In certain constructs, the different described butyrate gene cassettes are each placed under the control of a tetracycline-inducible or constitutive promoter. For example, genetically engineered Nissle are generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB expressed under the control of a nitric oxide-responsive regulatory element. SEQ ID NO: 86 comprises a reverse complement of the nsrR repressor gene from Neisseria gonorrhoeae (underlined), intergenic region containing divergent promoters controlling nsrR and the butyrogenic gene cassette and their respective RBS (bold), and the butyrate genes (ter- thiA-hbd-crt-tesB) separated by RBS.
Table 41. SEQ ID NO: 86
Figure imgf000657_0001
Figure imgf000658_0001
[1466] To facilitate inducible production of butyrate in Escherichia coli Nissle, the eight genes of the butyrate production pathway from Peptoclostridium difficile (bcd, etfB, etfA, thiA, hbd, crt, bpt, and buk; NCBI), as well as transcriptional and
translational elements, were synthesized (Gen, Cambridge, MA) and cloned into vector pBR to create pLogic. As synthesized, the genes were placed under control of a tetracycline-inducible promoter, with the tet repressor (tetR) expressed constitutively, divergent from the tet-inducible synthetic butyrate operon. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a base pair ribosome binding site derived from the T promoter.
[1467] The gene products of bcd-etfA-etfB form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co- oxidant. Because an effective probiotic should be able to function in an oxygen-limited environment (e.g. the mammalian gut), and because it has been shown that a single gene from Treponema denticola can functionally replace this three gene complex in an oxygen-independent manner (trans--enoynl-CoA reductase; ter), we created a second plasmid capable of butyrate production in E. coli. Inverse PCR was used to amplify the entire sequence of pLogic outside of the bcd-etfA-etfB region. The ter gene was codon optimized for E. coli codon usage using Integrated DNA technologies online codon optimization tool (https://www.idtdna.com/CodonOpt), synthesized (Genewiz,
Cambridge, MA), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic.
Example 34. Transforming E. coli [1468] Each plasmid is transformed into E. coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of E. coli Nissle or E. coli DH5a is diluted 1:100 in 5 mL of lysogeny broth (LB) and grown until it reached an OD600 of 0.4-0.6. The cell culture medium contains a selection marker, e.g., ampicillin, that is suitable for the plasmid. The E. coli cells are then centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 1 mL of 4° C water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C water. The E. coli are again centrifuged at 2,000 rpm for 5 min. at 4° C, the supernatant is removed, and the cells are finally resuspended in 0.1 mL of 4° C water. The
electroporator is set to 2.5 kV. 0.5 µg of one of the above plasmids is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. One mL of room- temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C for 1 hr. The cells are spread out on an LB plate containing ampicillin and incubated overnight.
[1469] In alternate embodiments, the butyrate cassette can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, MA).
Organization of the constructs and nucleotide sequences are provided herein. To create a vector capable of integrating the synthesized butyrate cassette construct into the chromosome, Gibson assembly was first used to add 1000bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the butyrate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20ug/mL at 37 degrees C. Growth at 37 degrees C also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac-).
Example 35. Production of butyrate in recombinant E. coli [1470] Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.
Example 36. Production of butyrate in recombinant E. coli [1471] Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hrs) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. Example 37. Production of Butyrate in Recombinant E. coli using tet-inducible promoter [1472] FIG. 48 shows butyrate cassettes described above under the control of a tet-inducible promoter. Production of butyrate is assessed using the methods described below in Example 40. The tet-inducible cassettes tested include (1) tet-butyrate cassette comprising all eight genes (pLOGIC031); (2) tet-butyrate cassette in which the ter is substituted (pLOGIC046) and (3) tet-butyarte cassette in which tesB is substituted in place of pbt and buk genes. FIG. 51A 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 CoA).
[1473] Overnight cultures of cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of 100ng/ml to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at OD600=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS. FIG. 51B 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.
[1474] FIG. 52 shows the BW25113 strain of E. Coli, which is a common cloning strain and the background of the KEIO collection of E. Coli mutants. NuoB mutants having NuoB deletion were obtained. NuoB is a protein complex involved in the oxidation of NADH during respiratory growth (form of growth requiring electron transport). Preventing the coupling of NADH oxidation to electron transport allows an increase in the amount of NADH being used to support butyrate production. FIG. 52 shows that compared with wild-type Nissle, deletion of NuoB results in grater production of butyrate.
Figure imgf000662_0001
Figure imgf000663_0001
Figure imgf000664_0001
Example 38. Production of Butyrate in Recombinant E. coli [1475] Production of butyrate is assessed in E. coli Nissle strains containing the butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of E. coli strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5%H2). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment.
[1476] In an alternate embodiment, overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Genetically engineered Nissle comprising pLogic031-nsrR- norB-butyrate operon construct; SYN-UCD507) or (pLogic046-nsrR-norB-butyrate operon construct; SYN-UCD508) produce significantly more butyrate as compared to wild-type Nissle.
[1477] Genetically engineered Nissle were generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB (SEQ ID NO: 48) expressed under the control of a tetracycline promoter (pLOGIC046-tesB-butyrate; SEQ ID NO: 88). SEQ ID NO: 88 comprises a reverse complement of the tetR repressor (underlined), an intergenic region containing divergent promoters controlling tetR and the butyrate operon and their respective RBS (bold), and the butyrate genes (ter-thiA1- hbd-crt2-tesB) separated by RBS.
Table 43. pLOGIC046-tesB-butyrate sequence SEQ ID NO: 88 gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttcta atccgcatatgatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca aataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccct ttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcc ccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaag gctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtac ttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgta aaaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaac atctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgt aggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattccga cctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatc attaattcctaatttttgttgacactctatcattgatagagttattttaccactcccta tcagtgatagagaaaagtgaactctagaaataattttgtttaactttaagaaggagata tacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcag ggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcaga agtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggtt acggcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcggcgtg tcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaataattt ggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatg cgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaa tttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtat catgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatc cgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagcc gccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaa ggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaag ctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggcc acagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataa aggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccagct tgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacgcgt ctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcg cattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgttga tggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccat gatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttga acgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgcca gtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggta gagttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagatat gatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaa gacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatata gtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtga tgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtac caagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaa gatggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagc
Figure imgf000666_0001
Figure imgf000667_0001
[1478] Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, anhydrous tetracycline (ATC) was added to cultures at a final concentration of 100 ng/mL to induce expression of butyrate genes from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. Replacement of pbt and buk with tesB leads to greater levels of butyrate production.
[1479] FIG. 53C shows butyrate production in strains comprising an FNR- butyrate cassette syn 363 (having the ter substitution) in the presence/absence of glucose and oxygen. FIG. 53C shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containing no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicated time pints and supernatant fractions were assessed for butyrate concentration using LC-MS. These data show that SYN 363 requires glucose for butyrate production and that in the presence of glucose butyrate production can be enhanced under anaerobic conditions when under the control of the anaerobic FNR-regulated ydfZ promoter. Example 39. In vitro Activity of Bacterial Strain Comprising Ammonia- Metabolizing and Butyrate Producing Circuits [1480] To determine whether ammonia uptake and conversion to arginine and production of butyrate could be accomplished in one strain, a plasmid (Logic156) comprising a butyrate production cassette construct, was used for initial proof-of- concept experiments. The following strains were generated using the plasmid: SYN- UCD501 (comprising Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance); and SYN-UCD601, which is SYN-UCD-305, additionally comprising Logic156 (i.e., SYN-UCD601 comprises∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)). Arginine and butyrate production was the compared between the butyrate only producer SYN-UCD501, the arginine only producer SYN-UCD305, and the combined butyrate/arginine producer SYN-UCD-601. Sequences for the butyrate cassette used are shown in Table 38.
[1481] Briefly, 3ml LB (containing selective antibiotics (Amp for SYN- UCD501 and SYN-UCD601) and 3mM thymidine for SYN-UCD-305 and SYN- UCD601) with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 dilution into 10ml LB
(containing antibiotics and thymidine where necessary as above) in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (1, 2, 24h), 120 ul cells were removed and pelleted at 14,000rpm for 1min, and 100ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for arginine and butyrate concentrations (as described in Example 13 and Example 40).
[1482] Results are depicted in FIG. 53A and FIG. 53B, and 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.
Figure imgf000668_0001
Figure imgf000669_0001
Figure imgf000670_0001
Figure imgf000671_0001
[1483] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 104 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 SEQ ID NO: 104 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 104 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 104 or a functional fragment thereof.
[1484] Non-limiting FNR promoter sequences are provided herein. In some embodiments, the genetically engineered bacteria of the invention comprise a butyrate cassette under the control of one or more of promoter sequences found in Table III, e.g., nirB promoter, ydfZ promoter, nirB promoter fused to a strong ribosome binding site, ydfZ promoter fused to a strong ribosome binding site, fnrS, an anaerobically induced small RNA gene (fnrS promoter), nirB promoter fused to a crp binding site, and fnrS fused to a crp binding site.
[1485] In some embodiments, the butyrate cassette is under the control of a promoter which is inducible by metabolites present in the gut. In some embodiments the butyrate cassette is induced by HE-specific molecules or metabolites indicative of liver damage, e.g., bilirubin. In some embodiments, the butyrate cassette is placed under the control of promoter, which is inducible by inflammation or an inflammatory response (e.g., RNS or ROS promoter).
[1486] In some embodiments, the genetically engineered bacteria comprise a butyrate cassette driven by a promoter induced by a molecule or metabolite, 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, and manganese in their blood and intestines. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein.
[1487] In some embodiments, the butyrate cassette is inducible by arabinose and is driven by the AraBAD promoter.
Example 40. Quantification of Butyrate by LC-MS/MS [1488] To obtain the butyrate measurements in Example 37 a LC-MS/MS protocol for butyrate quantification was used.
Sample preparation
[1489] First, fresh sodium butyrate stock solution (10mg/m), and 1000, 500, 250, 100, 20, 4 and 0.8µg/mL of sodium butyrate standards were prepared in water. Then, 10µL of sample (bacterial supernatants and standards) were pipetted into a V- bottom polypropylene 96-well plate, and 90µL of 67% ACN (60uL ACN+30uL water per reaction) with 4ug/mL of butyrate-d7 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 20µL of diluted samples were added to 180µL of a buffer containing 10mM MES pH4.5, 20mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour.
LC-MS/MS method
[1490] Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 45 and Table 46A. Tandem Mass Spectrometry details are found in Table 47. Table 45. HPLC Details
Figure imgf000673_0001
Table 46A. HPLC Method
Figure imgf000673_0002
Table 47. Tandem Mass Spectrometry Details
Figure imgf000673_0003
Example 41. Efficacy of genetically engineered bacteria producing arginine and/or butyrate in a bile duct ligation model [1491] Ligation of the common bile duct in rodents has used as an experimental procedure in research for many years to induce liver cholestasis and fibrosis (see e.g., Tag et a., Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis, Journal of Visualized Experiments, February 2015; 96; e52438, and references therein).
[1492] To determine the efficacy of a strain comprising an arginine and butyrate producing circuit in reducing symptoms of liver inflammation and fibrosis, a bile duct ligation model is used. A Nissle control (SYN-UCD107, kanamycin resistant Nissle), an arginine producing strain (SYN-UCD305), a butyrate producing strain (SYN-UCD502), and a strain producing both butyrate and arginine (SYN-UCD605) are compared in the study. ALT/AST levels, fibrosis (portal, perisinusoidal and total) and hepatic inflammation are the primary endpoints of the study; overall animal health is the secondary endpoint of the study.
[1493] Animals (C57BL6, 8 weeks) are treated by oral gavage with either H2O control (n=12), or SYN-UCD107 (n=12; kanamycin resistant Nissle), or arginine producing SYN-UCD305 (n=12; comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and no antibiotic resistance), or butyrate producing SYN-UCD502 (n=12; comprising a PydfZ-ter butyrate cassette integrated on the chromosome) or SYN-UCD605 (n=12; comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and PydfZ-ter butyrate cassette integrated on the chromosome, and no antibiotic resistance). Bacteria are administered at a dose of >10e10 cells/ml.
[1494] In some embodiments, SYN-UCD501 (comprising wild type ArgR, no FNR-ArgAfbr, wild type ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance) and SYN-UCD602 (comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)) are used instead of SYN-UCD502 and SYN- UCD605.
[1495] On Day 0, bile duct ligation surgery is performed as described in
Example 40. On day 1, mice are weighed and randomized. Mice are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. On day two, mice are gavaged with 100ul H2O, SYN798 and SYN993-in the AM and PM. On day three, mice are gavaged with 100ul H2O, SYN-UCD107, SYN- UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. At 4h post AM dose, blood is collected for ALT/AST analysis. On days 4-6, mice are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. On day 7, mice are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. At 4h post AM dose, blood is collected for ALT/AST analysis. On days 8-9, mice are gavaged with 100ul H2O, SYN- UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. On day 10, mice are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. At 4h post AM dose, blood is collected for ALT/AST analysis. On days 11-13, mice are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM and PM. On day 14, animals are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502 or SYN-UCD605 in the AM. Then, 4h post dose animals are euthanized, and blood is collected by cardiac bleed for ALT/AST analysis. The liver tissue is harvested for fibrosis analysis by histological assessment.
Example 42. Bile Duct Ligation Procedure Bile Duct Ligation
Pre-surgical Preparation:
[1496] During the complete experimentation, the animal is kept on a warming plate at a temperature of 37 °C, permanently connected to an anesthesia system, and the operational area is covered overall with fluid-impermeable, self-adhesive drapes.
[1497] The mouse is anaesthetized with inhalation of 4 vol% isoflurane in 100% oxygen at a flow rate of 4 L/min for the induction of the anesthesia. The depth of anesthetization is sufficient when the following vital criteria are reached: regular spontaneous breathing, no reflex after setting of pain stimuli between toes, and no response to pain. The abdominal fur of the mouse is shaved with an electric fur shaver and the eyes are protected from drying out by usage of eye and nose ointment. The mouse is placed on a 37 °C heated hot plate, the mouse snout is inserted in the Fluovac mask of a Fluovac anesthesia system, and the legs of the animal are fixed with stripes of silk tape. Anesthesia of the mouse is maintained by inhalation of 1.5-3 vol% isoflurane in 100% oxygen at a flow rate of 1 L/min and induct perioperative analgesia via intraperitoneal injection of buprenorphine solution (0.1 mg/kg BW dissolved in 0.9% NaCl solution). [1498] The shaved abdominal skin is sterilized with a gauze swab that is moistened with a standard antiseptic, ready to use alcoholic solution for preoperative treatment of the skin.
[1499] Surgical Procedures:
[1500] The abdomen is opened with a midline laparotomy of a length of approximately 2 cm by cutting the cutis plus fascia at the same time with an 11.5cm surgical scissor. The connective tissue on top of the peritoneum is dissected by using the scissor as a spreader. The peritoneum is cut along the linea alba to open the peritoneal cavity. The cavity is enlarged by inserting a holding suture in the sternum, raising the filament of the suture, and fixing it on top of the Fluovac mask. The operation area is spread by inserting a Colibri retractor in the peritoneal cavity. The liver is lifted with a moisturized (0.9% NaCl solution) cotton swab so that the ventral side of it sticks to the diaphragm and the hilum is clearly visible. The bile duct is exposed by caudal movement of the gut. The bile duct is separated carefully from the flanking portal vein and hepatic artery using a micro-serrations forceps. The 5-0 suture is placed around the bile duct and secured with two surgical knots. When tying the knots the tractive force is increased continuously to ensure effective obstruction without severing the bile duct. Asecond cranial ligation is added in the same manner without dissecting the bile duct in between. The ends of the sutures are cut, the sternum lowered, and the retractor removed. The peritoneal cavity is rinsed with 0.9% NaCl solution and the abdominal organs replaced to the physiological positions. Both abdominal layers (peritoneum and cutis plus facia) are closed with separate running sutures with 6-0 Mersilk. The ends of the sutures are cut and the operation area is sterilized with a gauze swab moistened with antiseptic solution.
Postoperative Treatment and Follow-up:
[1501] The mouse is allowed to recover in a cage warmed up by an infrared lamp until the mouse is fully awake and active. Afterwards, the mouse is moved to a normal cage and provided ad libitum access to water and food. After the surgery, the animals are monitored at regular intervals and follow-up postoperative treatments are carried out with suitable analgesia (e.g., buprenorphine solution) following the local recommendation of the internal animal care and use committees. Animals are kept with free access to food and water ad libitum until the end of the experiment. Example 43. TAA model of Hepatic Encephalopathy [1502] TAA treatment of mice has previously been employed in the literature to model increased blood ammonia levels associated with UCDs, acute and chronic liver disease and HE (Wallace MC, et al., Lab Anim. 2015 Apr;49(1 Suppl):21-9. Standard operating procedures in experimental liver research: thioacetamide model in mice and rat)s. In some embodiments, a TAA-induced mouse model of hyperammonemia is employed to investigate the duration of activity of the genetically engineered bacteria and to generate additional data to support this approach to the treatment of hepatic encephalopathy.
[1503] To determine the efficacy of a strain comprising an arginine and butyrate producing circuit in alleviating symptoms of liver inflammation and fibrosis, Nissle control (SYN-UCD107, kanamycin resistant Nissle), an arginine producing strain (SYN-UCD305), a butyrate producing strain (SYN-UCD502), and a strain producing both butyrate and arginine (SYN-UCD605) are compared in a TAA model study.
ALT/AST levels, fibrosis (portal, perisinusoidal and total) and hepatic inflammation are the primary endpoints of the study. Overall animal health is the secondary endpoint of the study.
[1504] To investigate the effects of engineered bacteria on prolonged elevations of blood ammonia, the bacteria are administered to C57BL6 mice that are also administered a dose of 300mpk thioacetamide (TAA).
[1505] C57BL6 (10 weeks old) are administered one daily dose of SYN- UCD107, or SYN-UCD305 (n=12), SYN-UCD502 (n=12), or SYN-UCD605 (n=12) (100 ul of > 1 X1010 cells/ml) or vehicle control. Alternatively, mice are administered 2 daily doses of bacteria (100 ul of > 1 X1010 cells/ml) (n=5 for each treatment group), once in the AM and once in the PM. After three days of pre-dosing with the bacteria, the mice are treated intraperitoneally with thioacetamide (TAA) at 300mpk or with H2O as control. Alternatively, the mice are treated twice daily, once in the AM and once in the PM with 250mpk. The duration of the study is five days. Ammonium levels are measured and overall health survival, body weight change is monitored.
[1506] In brief, animals are acclimated for 7 days. On day 1 of the time course, animals are weighed, bled to measure baseline ALT/AST and collect fecal pellets (per cage), and are randomized based on ALT/AST levels. Animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD107, SYN-UCD305, SYN- UCD502, or SYN-UCD605 (100ul/dose/animal). Water is changed to H2O(+)20mg/ml ATC. On day 2, animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605
(100ul/dose/animal). On day 3, animals are weighed and dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605 (100ul/dose/animal). Additionally, animals are dosed intraperitoneally with 300mpk TAA (or saline control). Alternatively, animals are dosed with 250mpk TAA (or saline control) once in the AM and once in the PM. On day 4, animals are weighed and blood is collected for ALT/AST analysis. Fecal pellets are collected per cage. Animals are dosed per oral gavage either once or twice (AM and PM) with H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605 (100ul/dose/animal). Animals may also be dosed with 250mpk TAA (or saline control). On day 5, animals are weighed and blood is collected for ALT/AST analysis. Fecal pellets are collected (per cage). Animals are dosed by oral gavage either once or twice (AM and PM) with H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605
(100ul/dose/animal). ALT/AST levels, bacterial load in the fecal pellets, and overall health survival, and body weight changes are monitored. The liver tissue is harvested for fibrosis analysis by histological assessment.
Example 44. Carbontetrachloride (CCl4) Model of Hepatic Encephalopathy [1507] CCl4 is often used to induce hepaticfibrosis and cirrhosis in animals because the underlying iochemical mechanisms and histological characteristics are similar to those observed in human liver cirrhosis (Nhung et al., Establishment of a standardized mouse model of hepatic fibrosis for biomedical research; Biomedical Research and Therapy 2014, 1(2):43-49).
[1508] CYP2E1 is an enzyme which is expressed in perivenular hepatocytes, and which converts CCl4 into a CCl3+ radical. The accumulation of the CCl3+ radical causes centrilobular necrosis and changes the permeability of the hepatocyte plasma and mitochondrial membranes. As a result, an increase in inflammation and fibrogenesis, and extracellular matrix deposition is observed. Chronic CCl4 exposure causes the formation of nodules and fibrosis, products of the wound healing process. CCl4 treatment has been shown to cause fibrosis after 2–4 weeks, significant bridging fibrosis after 5–7 weeks, cirrhosis after 9–11 weeks, and micronodular cirrhosis after 10–20 weeks (Nhung et al., Establishment of a standardized mouse model of hepatic fibrosis for biomedical research; Biomedical Research and Therapy 2014, 1(2):43-49, and references therein).
[1509] To determine the efficacy of a strain comprising an arginine and butyrate producing circuit in alleviating symptoms of liver inflammation and fibrosis, a CCl4 mouse model of liver cirrhosis is used. A Nissle control (SYN-UCD107, kanamycin resistant Nissle), an arginine producing strain (SYN-UCD305), a butyrate producing strain (SYN-UCD502), and a strain producing both butyrate and arginine (SYN- UCD605) are compared in the study. ALT/AST levels, fibrosis (portal, perisinusoidal and total) and hepatic inflammation are the primary endpoints of the study. Overall animal health is the secondary endpoint of the study. Study duration is 8 weeks.
[1510] Animals (C57BL6, 8 weeks) are treated by oral gavage with either H2O control (n=12), or SYN-UCD107 (n=12; kanamycin resistant Nissle), or arginine producing SYN-UCD305 (n=12; comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and no antibiotic resistance), or butyrate producing SYN-UCD502 (n=12; comprising wild type ArgR, no FNR-ArgAfbr, wild type ThyA , and a PydfZ-ter butyrate cassette integrated on the chromosome) or SYN- UCD605 (n=12; comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, PydfZ-ter butyrate cassette integrated on the chromosome, and no antibiotic resistance. Bacteria are administered at a dose of >10e10 cells/ml in 100 ul.
[1511] In some embodiments, SYN-UCD501 (comprising Wild type ArgR, no FNR-ArgAfbr, wild type ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance) and SYN-UCD602 (comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)) are used instead of SYN-UCD502 and SYN- UCD605.
[1512] On Day 1, mice are weighed and randomized. Animals are gavaged with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605 in the AM. Additionally animals are gavaged with olive oil (sham control) or 1ml/kg CCL4 in olive oil (all other treatment groups). Animals are gavaged with 100ul H2O, SYN- UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605 in the PM. [1513] Through Week 1-8, animals are gavaged BID with 100ul H2O, SYN- UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605. Additionally, animals are gavaged 3x/week with olive oil (sham control) or 1ml/kg CCL4 in olive oil (all other treatment groups). Animals are weighed 3x/week, and blood for ALT/AST analysis is collected 1x/week.
[1514] At the end of Week 8 animals are gavaged with 100ul with 100ul H2O, SYN-UCD107, SYN-UCD305, SYN-UCD502, or SYN-UCD605 in the AM. At 4h post dose, animals are weighed, euthanized and blood is collected by cardiac bleed for ALT/AST analysis. The liver is harvested for fibrosis analysis by histological assessment.
Example 45. Efficacy of Butyrate-Expressing Bacteria in a DSS Mouse Model [1515] Bacteria harboring the butyrate cassettes described above are grown overnight in LB. Bacteria are then diluted 1:100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters is administered by oral gavage to mice. Damage to the gut is induced in mice by supplementing drinking water with 3% dextran sodium sulfate for 7 days prior to bacterial gavage. Mice are treated daily for 1 week and bacteria in stool samples are detected by plating stool homogenate on agar plates supplemented with a suitable selection marker, e.g., ampicillin. After 5 days of bacterial treatment, gut damage is scored in live mice using endoscopy. Endoscopic damage score is determined by assessing colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. Mice are sacrificed and colonic tissues are isolated. Distal colonic sections are fixed and scored for inflammation and ulceration. Colonic tissue is homogenized and measurements are made for myeloperoxidase activity using an enzymatic assay kit and for cytokine levels (IL-1β, TNF-α, IL-6, IFN-γ and IL-10). Example 46. Generating a DSS-Induced Mouse Model of HE [1516] The genetically engineered bacteria described can be tested in the dextran sodium sulfate (DSS)-induced mouse model. The administration of DSS to animals results in chemical injury to the intestinal epithelium, allowing
proinflammatory intestinal contents (e.g., luminal antigens, enteric bacteria, bacterial products) to disseminate and trigger inflammation (Low et al., 2013). To prepare mice for DSS treatment, mice are labeled using ear punch, or any other suitable labeling method. Labeling individual mice allows the investigator to track disease progression in each mouse, since mice show differential susceptibilities and responsiveness to DSS induction. Mice are then weighed, and if required, the average group weight is equilibrated to eliminate any significant weight differences between groups. Stool is also collected prior to DSS administration, as a control for subsequent assays.
Exemplary assays for fecal markers of inflammation (e.g., cytokine levels or myeloperoxidase activity) are described below.
[1517] For DSS administration, a 3% solution of DSS (MP Biomedicals, Santa Ana, CA; Cat. No. 160110) in autoclaved water is prepared. Cage water bottles are then filled with 100 mL of DSS water, and control mice are given the same amount of water without DSS supplementation. This amount is generally sufficient for 5 mice for 2-3 days. Although DSS is stable at room temperature, both types of water are changed every 2 days, or when turbidity in the bottles is observed.
[1518] Acute, chronic, and resolving models of intestinal inflammation are achieved by modifying the dosage of DSS (usually 1-5%) and the duration of DSS administration (Chassaing et al., 2014). For example, acute and resolving gut damage may be achieved after a single continuous exposure to DSS over one week or less, whereas chronic gut damage is typically induced by cyclical administration of DSS punctuated with recovery periods (e.g., four cycles of DSS treatment for 7 days, followed by 7-10 days of water).
[1519] FIG. 53D shows that butyrate produced in vivo in DSS mouse models under the control of an FNR promoter can be gut protective. LCN2 and calprotectin are both a measure of gut barrier disruption (measure by ELISA in this assay). FIG. 53D shows that Syn 363 (ter substitution) reduces inflammation and/or protects gut barrier as conmpared to Syn 94 (wildtype Nissle).
Example 47. Monitoring Disease Progression in Vivo [1520] Following initial administration of DSS, stool is collected from each animal daily, by placing a single mouse in an empty cage (without bedding material) for 15-30 min. However, as DSS administration progresses and inflammation becomes more robust, the time period required for collection increases. Stool samples are collected using sterile forceps, and placed in a microfuge tube. A single pellet is used to monitor occult blood according to the following scoring system: 0, normal stool consistency with negative hemoccult; 1, soft stools with positive hemoccult; 2, very soft stools with traces of blood; and 3, watery stools with visible rectal bleeding. This scale is used for comparative analysis of intestinal bleeding. All remaining stool is reserved for the measurement of inflammatory markers, and frozen at -20◦C.
[1521] The body weight of each animal is also measured daily. Body weights may increase slightly during the first three days following initial DSS administration, and then begin to decrease gradually upon initiation of bleeding. For mouse models of acute colitis, DSS is typically administered for 7 days. However, this length of time may be modified at the discretion of the investigator.
Example 48. In Vivo Efficacy of Genetically Engineered Bacteria Following DSS
Induction [1522] The genetically engineered bacteria described herein can be tested in DSS-induced animal models of HE. Bacteria are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by
centrifugation. Bacteria are then resuspended in phosphate buffered saline (PBS). Gut damage is induced in mice by supplementing drinking water with 3% DSS for 7 days prior to bacterial gavage. On day 7 of DSS treatment, 100 µL of bacteria (or vehicle) is administered to mice by oral gavage. Bacterial treatment is repeated once daily for 1 week, and bacteria in stool samples are detected by plating stool homogenate on selective agar plates.
[1523] After 5 days of bacterial treatment, gut damage is scored in live mice using the Coloview system (Karl Storz Veterinary Endoscopy, Goleta, CA). In mice under 1.5-2.0% isoflurane anesthesia, colons are inflated with air and approximately 3 cm of the proximal colon can be visualized (Chassaing et al., 2014). Endoscopic damage is scored by assessing colon translucency (score 0-3), fibrin attachment to the bowel wall (score 0-3), mucosal granularity (score 0-3), vascular pathology (score 0-3), stool characteristics (normal to diarrhea; score 0-3), and the presence of blood in the lumen (score 0-3), to generate a maximum score of 18. Mice are sacrificed and colonic tissues are isolated using protocols described in Examples 8 and 9. Distal colonic sections are fixed and scored for inflammation and ulceration. Remaining colonic tissue is homogenized and cytokine levels (e.g., IL-1β, TNF-α, IL-6, IFN-γ, and IL-10), as well as myeloperoxidase activity, are measured using methods described below.
Example 49. Euthanasia Procedures for Rodent Models of HE [1524] Four and 24 hours prior to sacrifice, 5-bromo-2’-deooxyuridine (BrdU) (Invitrogen, Waltham, MA; Cat. No. B23151) may be intraperitoneally administered to mice, as recommended by the supplier. BrdU is used to monitor intestinal epithelial cell proliferation and/or migration via immunohistochemistry with standard anti-BrdU antibodies (Abcam, Cambridge, MA).
[1525] On the day of sacrifice, mice are deprived of food for 4 hours, and then gavaged with FITC-dextran tracer (4 kDa, 0.6 mg/g body weight). Fecal pellets are collected, and mice are euthanized 3 hours following FITC-dextran administration. Animals are then cardiac bled to collect hemolysis-free serum. Intestinal permeability correlates with fluorescence intensity of appropriately diluted serum (excitation, 488 nm; emission, 520 nm), and is measured using spectrophotometry. Serial dilutions of a known amount of FITC-dextran in mouse serum are used to prepare a standard curve.
[1526] Alternatively, intestinal inflammation is quantified according to levels of serum keratinocyte-derived chemokine (KC), lipocalin 2, calprotectin, and/or CRP-1. These proteins are reliable biomarkers of inflammatory disease activity, and are measured using DuoSet ELISA kits (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions. For these assays, control serum samples are diluted 1:2 or 1:4 for KC, and 1:200 for lipocalin 2. Samples from DSS-treated mice require a significantly higher dilution.
Example 42. Non-obese Diabetes (NOD) Model of Hepatic Encephalopathy [1527] NOD mice can be used as an in vivo model for testing the efficacy of bacteria comprising a gut-barrier enhancing circuit, e.g., a butyrate biosynthetic cassette, as these mice exhibit a pronounced“leaky gut” phenotype, which is evidenced by a decrease in the level of tight junction proteins (e.g., occludin, zonula occludin, mucin, E-cadherin).
[1528] To determine the efficacy of a strain comprising an arginine and butyrate producing circuit in alleviating symptoms associated with“leaky gut”, a NOD mouse model is used. NOD mice result from the autoimmune destruction of pancreatic islet β cells. A Nissle control (SYN-UCD107, kanamycin resistant Nissle), an arginine producing strain (SYN-UCD305), a butyrate producing strain (SYN-UCD502), and a strain producing both butyrate and arginine (SYN-UCD605) are compared in the study. Epithelial gut integrity (occludin and other tight junction markers) and gut inflammation (inflammatory biomarkers, e.g., IL-1A, IL-6, TNFa, IL-21) are the primary endpoints of the study. Overall animal health is the secondary endpoint of the study. Study duration is 8 weeks.
[1529] Animals (C57BL6, 8 weeks) are treated by oral gavage with either H2O control (n=12), or 100mM butyrate (n=12), or arginine producing SYN-UCD305 (n=12; comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and no antibiotic resistance), or butyrate producing SYN-UCD502 (n=12; comprising wild type ArgR, no FNR-ArgAfbr, wild type ThyA , and a PydfZ-ter butyrate cassette integrated on the chromosome) or SYN-UCD605 (n=12; comprising ∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, PydfZ-ter butyrate cassette integrated on the chromosome, and no antibiotic resistance. Bacteria are administered at a dose of >10e10 cells/ml in 100 ul.
[1530] In some embodiments, SYN-UCD501 (comprising Wild type ArgR, no FNR-ArgAfbr, wild type ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance) and SYN-UCD602 (comprising∆ArgR, PfnrS- ArgAfbr integrated into the chromosome at the malEK locus,∆ThyA, and Logic156 (pSC101 PydfZ-ter butyrate plasmid; amp resistance)) are used instead of SYN-UCD502 and SYN- UCD605.
[1531] On Day 1, mice are weighed and randomized. Animals are gavaged with 100ul H2O, 100 mM butyrate, SYN-UCD305, SYN-UCD502, or SYN-UCD605 in the AM. Animals are gavaged with 100ul H2O, 100mM butyrate, SYN-UCD305, SYN- UCD502, or SYN-UCD605 in the PM. Animals are gavaged BID with 100ul H2O, 100mM butyrate, SYN-UCD305, SYN-UCD502, or SYN-UCD605. Animals are weighed daily.
[1532] On day 5, mice are fasted for 4h and then gavaged with 0.6mg/g FITC- dextran (40kD). 3h post FITC-dex administration mice are weighed, and blood is collected by cardiac bleed and colons and fecal pellets are harvested.
[1533] Haptoglobin/zonulin and Lcn2 are measured. RNA levels of TJP1, OCLN, CLDN25, and EPCAM are measured in colon samples (increased levels of these markers indicate therapeutic effect). RNA levels of inflammatory biomarkers are measured in the blood samples (decreased levels of these biomarkers indicate therapeutic effect). Example 51. GABA transport and metabolic circuits [1534] In addition to the ammonia conversion circuit described above, the E. coli Nissle bacteria further comprise one or more GABA transport and/or one or more GABA metabolic circuits. Genetically engineered strains comprising at least one GABA transport circuit comprise a gene encoding an exemplary GABA transport protein, such as GabP (SEQ ID NO: 105, Table 48; SEQ ID NO: 106, Table 49), and are constructed using methods described above. Genetically engineered strains comprising at least one GABA metabolic circuit comprise genes encoding enzymes required for GABA catabolism, including, but not limited to GSST and SSDH, and are constructed using methods described above.
[1535] The genes encoding GabP, GSST, and SSDH are expressed under the control of a tetracycline-inducible promoter, a FNR promoter selected from SEQ ID NOs: 18-29, or a promoter induced by HE-related molecules or metabolites. The genes encoding GabP, GSST, and SSDH may be expressed under the control of the same or different promoters. As discussed herein, other promoters may be used. The genes encoding GabP, GSST, and SSDH are expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. The genes encoding GabP, GSST, and SSDH are 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. 18.
Table 48
Figure imgf000685_0001
Table 49
Figure imgf000686_0001
[1536] In some embodiments, the genetically engineered bacteria comprise the nucleic acid sequence of SEQ ID NO: 106 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 SEQ ID NO: 106 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 106 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 106 or a functional fragment thereof. Example 52. Manganese transport circuit [1537] In addition to the circuits described above, the E. coli Nissle bacteria further comprise one or more manganese transport circuits. Genetically engineered strains comprising at least one manganese transport circuit comprise a gene encoding an exemplary manganese transport protein, such as MntH (SEQ ID NO: 107, Table 50, SEQ ID NO: 108, Table 51), and are constructed using methods described above.
[1538] The gene encoding MntH is expressed under the control of a tetracycline-inducible promoter, a FNR promoter selected from SEQ ID NOs: 18-29, or a promoter induced by HE-related molecules or metabolites. As discussed herein, other promoters may be used. The gene encoding MntH is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. The gene encoding MntH 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. . FIG. 18.
Figure imgf000687_0001
Figure imgf000688_0001
Example 54. Assessment of intestinal butyrate levels in response to SYN501 administration in mice [1539] To determine efficacy of butyrate production by the genetically engineered bacteria in vivo, the levels of butyrate upon administration of SYN501 (Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid)) to C57BL6 mice was first assessed in the feces. Water containing 100 mM butyrate was used as a control.
[1540] On day 1, C57BL6 mice (24 total animals) were weighed and randomized into 4 groups; Group 1: H20 control (n=6); Group 2-100 mM butyrate (n=6); Group 3-streptomycin resistant Nissle (n=6); Group 4-SYN501 (n=6). Mice were either gavaged with 100 ul streptomycin resistant Nissle or SYN501, and group 2 was changed to H20(+)100 mM butyrate at a dose of 10e10 cells/100ul. On days 2-4, mice were weighted and Groups 3 and 4 were gavaged in the AM and the PM with streptomycin resistant Nissle or SYN501. On day 5, mice were weighed and Groups 3 and 4 were gavaged in the am with streptomycin resistant Nissle or SYN501, and feces was collected and butyrate concentrations determined as described in Example 55. Results are depicted in FIG. 55. 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.
[1541] Next the effects of SYN501 on levels of butyrate in the cecum, cecal effluent, large intestine, and large intestine effluent are assessed. Because baseline concentrations of butyrate are high in these compartments, an antibiotic treatment is administered in advance to clear out the bacteria responsible for butyrate production in the intestine. As a result, smaller differences in butyrate levels can be more accurately observed and measured. Water containing 100 mM butyrate is used as a control.
[1542] During week 1 of the study, animals are treated with an antibiotic cocktail in the drinking water to reduce the baseline levels of resident microflora. The antibiotic cocktail is composed of ABX-ampicillin, vancomycin, neomycin, and metronidazole. During week 2 animals are orally administered 100 ul of streptomycin resistant Nissle or engineered strain SYN501 twice a day for five days (at a dose of 10e10 cells/100ul).
[1543] On day 1, C57BL6 (Female, 8 weeks) are separated into four groups as follows: Group 1: H20 control (n=10); Group 2: 100 mM butyrate (n=10); Group 3: streptomycin resistant Nissle (n=10); Group 4: SYN501 (n=10). Animals are weighed and feces is collected from the animals (T=0-time point). Animals are changed to H2O (+) antibiotic cocktail. On day 5, animals are weighed and feces is collected (time point T=5d). The H2O (+) antibiotic cocktail bottles are changed. On day 8, the mice are weighed and feces is collected. Mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. The water in all cages is changed to water without antibiotic. Group 2 is provided with 100 mM butyrate in H2O. On days 9-11, mice are weighed, and mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. On day 12, mice are gavaged with streptomycin resistant Nissle or SYN501 in the AM, and 4 hours post dose, blood is harvested, and cecal and large intestinal contents, and tissue, and feces are collected and processed for analysis. Example 55. Quantification of Butyrate in feces by LC-MS/MS Sample preparation [1544] Fresh 1000, 500, 250, 100, 20, 4 and 0.8µg/mL sodium butyrate standards were prepared in water. Single fecal pellets were ground in 100uL water and centrifuged at 15,000 rpm for 5min at 4°C. 10µL of the sample (fecal supernatant and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90µL of the derivatizing solution containing 50mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5ug/mL of butyrate-d7 were added to each sample. The plate was heat-sealed and incubated at 60°C for 1hr. The plate was then centrifuged at 4,000rpm for 5min and 20µL of the derivatized samples mixed to 180µL of 22% acetonitrile with 0.1% formic acid.
LC-MS/MS method
[1545] Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 53, 54, and 55. Tandem Mass Spectrometry details are found in Table 54.
Table 53. HPLC Details
Figure imgf000690_0001
[01]
Table 54. HPLC Method
Figure imgf000690_0002
Table 55. Tandem Mass Spectrometry Details
Figure imgf000690_0003
Figure imgf000691_0001
Example 56. Comparison of Butyrate production levels between the genetically engineered bacteria encoding a butyrate cassette and selected Clostridia strains [1546] The efficacy of butyrate production in SYN501 (pSC101 PydfZ-ter - >pbt-buk butyrate plasmid) was compared to CBM588 (Clostridia butyricum
MIYARISAN, a Japanese probiotic strain), Clostridium tyrobutyricum VPI 5392 (Type Strain), and Clostridium butyricum NCTC 7423 (Type Strain).
[1547] Briefly, overnight cultures of SYN501 were diluted 1:100 were grown in RCM (Reinforced Clostridial Media, which is similar to LB but contains 0.5% glucose) at 37 C with shaking for 2 hours, then either moved into the anaerobic chamber or left aerobically shaking. Clostridial strains were only grown anaerobically. At indicated times (2, 8, 24, and 48h), 120 ul cells were removed and pelleted at 14,000rpm for 1min, and 100ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate concentrations (as described in Example 40). Results are depicted in FIG. 56, and show that SYN501 produces butyrate levels comparable to Clostridium spp. in RCM media Example 57. Comparison of in vitro butyrate production efficacy of chromosomal insertion and plasmid-bearing engineered bacterial strains [1548] The in vitro butyrate production efficacy of engineered bacterial strains harboring a chromosomal insertion of a butyrate cassette was compared to a strain bearing a butyrate cassette on a plasmid. SYN1001 and SYN1002 harbor a
chromosomal insertion between the agaI/rsmI locus of a butyrate cassette (either ter^tesB or ter^pbt-buk, respectively) driven by an fnr inducible promoter. These strains were compared side by side with the low copy plasmid strain SYN501
(Logic156 (pSC101 PydfZ-ter ->pbt-buk butyrate plasmid) also driven by an fnr inducible promoter. Butyrate levels in the media were measured at 4 and 24 hours post anaerobic induction.
[1549] Briefly, 3ml LB was inoculated with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 dilution into 10ml LB (containing antibiotics) in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (4 and 24h), 120 ul cells were removed and pelleted at 14,000rpm for 1min, and 100ul of the supernatant was transferred to a 96- well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate concentrations (as described in Example 40). Results are depicted in FIG. 57A, and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501.
[1550] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 110, 111, 112, or 113, or a functional fragment thereof.
Table 56. FRNRs Butyrate Cassette Sequences
Figure imgf000692_0001
Figure imgf000693_0001
Figure imgf000694_0001
Figure imgf000695_0001
Figure imgf000696_0001
Figure imgf000697_0001
Figure imgf000698_0001
Figure imgf000699_0001
Figure imgf000700_0002
Example 58. Biochemical Analysis of Butyrate Production in SYN1001 [1551] SYN1001 was assessed for its ability to produce butyrate in vitro. An overnight culture of LB-grown SYN1001 was diluted 1:100 into fresh LB (10mL in a 125mL baffled flask). The culture was grown aerobically with shaking at 250 rpm, 37oC, for 1.5h. The culture was then moved into an anaerobic chamber (Coy Lab Products, MI) supplying an atmosphere of 85% N2, 10% CO2, and 5% H2. Anaerobic incubation commenced at 37oC for 4 hours in order to induce the expression of the butyrate operon from the PfnrS promoter.
[1552] After the 4 hour anaerobic induction of the butyrate operon, the culture was removed from the anaerobic chamber and approximately 2x108 activated cells were used to inoculate 1 mL of M9 minimal medium containing 0.5% glucose. Assay cultures were incubated statically at 37oC for 18 hours in the presence of O2. For sample collection, 200uL aliquots were removed from assay cultures and spun down at maximum speed for 1 min in a microcentrifuge. The culture supernatant was retained, and LC-MS-MS was used to determine the concentration of butyrate in the supernatant fraction (Table 57-data are average of assay performed in triplicate for three different manufacturing runs).
Table 57: Butyrate production in SYN1001 from three different experiments
Figure imgf000700_0001
[1553] Equivalent concentrations of butyrate were obtained from 3 independent production runs of SYN1001. In production run 3, SYN94 and SYN2001 control strains were included and supernatants from these strains contained negligible amounts of butyrate (0.47 and 0.38mM respectively) compared to SYN1001, which contained significantly higher levels (6.98mM; n=3). SYN94 is a streptomycin-resistant version of the parental E.coli Nissle strain. SYN2001 is an engineered E.coli strain that has been modified to over-produce acetate and does not contain a synthetic butyrate operon, described elsewhere herein. Run 3 culture supernatants were used to generate bioactivity in cell based assays described below. Example 62. Cell-based Assay Development and In-vitro Butyrate Strain
Assessment Methods
[1554] Mammalian Cell Culture: HT-29 colon adenocarcinoma cells were obtained from ATCC (Cat#: HTB38). Cells were cultured at 370C, 5% CO2 in RPMI media supplemented with 10% FBS, 1% pen-strep (complete media). Cells were allowed to grow to ~80% confluency before passaging for activity assays.
[1555] Alkaline Phosphatase (AP) Activity Assay: HT-29 colon
adenocarcinoma cells were plated in complete media at either 1x105 cells/well (24 well plates) or 1x104 cells/well (96 well plates) and allowed to recover overnight at 370C, 5% CO2. The following day media was replaced with fresh complete media containing either PBS, synthetic acetate (SIGMA-Cat#S8750) or butyrate (SIGMA-Cat#B5887), or bacterial supernatants of interest. Cells were incubated for 4 days under these conditions and then media was removed and cellular lysates were prepared (10 min on ice with vendor-supplied lysis buffer (BioVision-see below) followed by clarification for 10 min @14K rpm, 40C). Lysates from each condition were then assessed for AP activity using an alkaline phosphatase activity kit (BioVision, Cat#K412-500) according to manufacturer’s recommendations.
[1556] Cell Viability Assay:HT-29 colon adenocarcinoma cells were plated in 2 separate plates in complete media at either 1x105 cells/well (24-well plates) or 1x104 cells/well (96-well plates) and allowed to recover overnight at 370C, 5% CO2. The following day, one plate of cells, which served as the day 1 time point read out (input), was treated with trypsin (5 min at 370C, 5% CO2) and cells were counted using a Cellometer K2 instrument (Nexcelom). Live and dead cells were distinguished by trypan blue exclusion. For the remaining plate, media was replaced with fresh complete media containing either PBS, synthetic acetate or butyrate, or bacterial supernatants of interest. Cells were incubated for 4 days under these conditions and then media was removed. Cells were detached from plates with trypsin and counted using the
Cellometer K2 as described above.
In vitro Assessment of Engineered Butyrate-producing Strain SYN1001
[1557] To assess the activity of the butyrate-producing strain SYN1001 in vitro, we employed the AP cell-based assay. HT-29 cells were plated in triplicate at 1x104 cells/well 96-well plates in complete media and allowed to recover overnight. The following day, media was removed and fresh media containing a dilution series of exogenous synthetic butyrate (5mM-0.3mM), or culture supernatants from the SYN94 control (0.26mM-0.016mM), SYN1001 butyrate-producing strain (3.5mM-0.11mM) or SYN2001 acetate-producing strain (0.22mM-0.01mM) were added, and the cells were incubated for 4 days. After the incubation period, media was removed and the plates were processed for assessment of AP activity. FIG. 57B shows that incubation of HT-29 cells with the supernatants from the butyrate-producing SYN1001 strain demonstrated a similar AP activity profile to cells incubated with synthetic butyrate. In contrast, the unengineered strain SYN94 or the acetate-producing strain SYN2001 had little to no effect on AP activity at any concentration tested. To better visualize the similarity in AP activity induction between synthetic butyrate and SYN1001-produced butyrate, the values from the AP activity assay were fit to a non-linear equation algorithm and graphed. As shown in FIG. 57C, the activity profile for butyrate produced by SYN1001 is comparable to synthetic butyrate. Incubation with synthetic butyrate, SYN94, SYN1001 or SYN2001 did not have any appreciable effect on cell viability (Data not shown).
Summary
[1558] The results describe the design and evaluation of an engineered, butyrate-producing strain, SYN1001, which contains a modified butyrate module comprised of the
Figure imgf000702_0001
(ter) gene from Treponema denticola, the thiolase (thiA1), 3-hydroxyybutyryl-CoA dehydrogenase (hbd), and crotonase (crt2) genes from Clostridium difficile, and the thioesterase B gene (tesB), which is endogenous to E. coli. SYN1001 is capable of producing ~7mM butyrate in vitro under the conditions described here. This in vitro butyrate production translates to activity in a cell-based assay that is comparable on an equimolar basis to that observed with pure, synthetic butyrate. Table 58 summarizes the final pharmacological characteristics of the SYN1001. Table 58. Final characterization of the pharmacological characteristics of
SYN1001
Figure imgf000703_0001
Example 63. Production of Propionate through the Sleeping Beauty Mutase Pathway in genetically engineered E. coli BW25113 and Nissle [1559] In E. coli, a four gene operon, sbm-ygfD-ygfG-ygfH (sleeping beauty mutase pathway) has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro. While the sleeping beauty mutase pathway is present in E. coli, it is not under the control of a strong promoter and has shown low activity in vivo.
[1560] The utility of this operon for the production of propionate was assessed. Because E. coli Nissle does not have the complete operon, initial experiments were conducted in E. coli K12 (BW25113).
[1561] First, the native promoter for the sleeping beauty mutase operon on the chromosome in the BW25113 strain was replaced with a fnr promoter (BW25113 ldhA::frt; PfnrS-SBM-cam). The sequence for this construct is provided in Table 59. Mutation of the lactate dehydrogenase gene (ldhA) reportedly increases propionate production, and this mutation is therefore also added in certain embodiments.
[1562] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 114, or 115, or a functional fragment thereof. Table 59. SBM Construct Sequences
Figure imgf000703_0002
Figure imgf000704_0001
Figure imgf000705_0001
Figure imgf000706_0001
Figure imgf000707_0001
Figure imgf000708_0001
Figure imgf000709_0001
Figure imgf000710_0001
[1563] Next, this strain was tested for propionate production.
[1564] Briefly, 3ml LB (containing selective antibiotics (cam) where necessary was inoculated from frozen glycerol stocks with either wild type E. coli K12 or the genetically engineered bacteria comprising the chromosomal sleeping beauty mutase operon under the control of a FNR promoter. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10ml LB in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, and 24 hours, 120 ul of cells were removed and pelleted at 14,000rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for propionate concentrations, as described in
[1565] Results are depicted in FIG. 59D and show that the genetically engineered strain produces ~2.5mM after 24h, while very little or no propionate production was detected from the E. coli K12 wild type strain. Propionate was measured as described in Example 65. Example 63. Evaluation of the Sleeping Beauty Mutase Pathway for the
Production of Propionate in E coli Nissle [1566] Next, the SBM pathway is evaluated for propionate production in E. coli Nissle. Nissle does not have the full 4-gene sleeping beauty mutase operon; it only has the first gene and a partial gene of the second, and genes 3 and 4 are missing. Therefore, recombineering is used to introduce this pathway into Nissle. The frt-cam-frt-PfnrS- sbm, ygfD, ygfG, ygfH construct is inserted at the location of the endogenous, truncated Nissle SBM. Next, the construct is transformed into E coli Nissle and tested for propionate production essentially as described above. Example 64. Evaluation of the Acrylate Pathway from Clostridium propionicum for Propionate Production [1567] The acrylate pathway from Clostridium propionicum is evaluated for adaptation to propionate production in E. coli. A construct (Ptet-pct-lcdABC-acrABC), codon optimized for E. coli, is synthesized by Genewiz and placed in a high copy plasmid (Logic051). Additionally, another construct is generated for side by side testing, in which the acrABC genes (which may be the rate limiting step of the pathway) are replaced with the acuI gene from Rhodobacter sphaeroides (Ptet- acuI-pct-lcdABC). Subsequently these constructs are transformed into BW25113 and are assessed for their ability to produce propionate, as compared to the type BW5113 strain as described above in Example 63. Propionate was measured as described in Example 65.
Table 60. of Exemplary Propionate Cassette Sequences
Figure imgf000712_0001
Figure imgf000713_0001
Figure imgf000714_0001
Figure imgf000715_0001
Figure imgf000716_0001
Figure imgf000717_0001
Figure imgf000718_0001
Figure imgf000719_0001
Figure imgf000720_0001
Figure imgf000721_0001
Figure imgf000722_0001
Figure imgf000723_0001
Figure imgf000724_0001
Figure imgf000725_0001
Figure imgf000726_0001
Figure imgf000727_0001
[1568] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 116, 117, 118, or 119, or a functional fragment thereof.
Example 65. Quantification of Propionate by LC-MS/MS Sample preparation
[1569] First, fresh 1000, 500, 250, 100, 20, 4 and 0.8µg/mL sodium propionate standards were prepared in water. Then, 25µL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 75µL of 60% ACN (45uL ACN+30uL water per reaction) with 10ug/mL of butyrate-d5 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 5µL of diluted samples were added to 95µL of a buffer containing 10mM MES pH4.5, 20mM EDC (N-(3-Dimethylaminopropyl)-N′- ethylcarbodiimide), and 20mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour.
LC-MS/MS method
[1570] Propionate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 61 and Table 62. Tandem Mass Spectrometry details are found in Table 63.
Table 61. HPLC Details
Figure imgf000728_0001
Table 62. HPLC Method
Figure imgf000728_0002
Table 63. Tandem Mass Spectrometry Details
Example 66. Tet-driven and RNS driven in vitro Butyrate Production in
Recombinant E. coli [1571] All incubations were performed at 37oC. Lysogeny broth (LB)-grown overnight cultures of E. coli Nissle transformed with pLogic031 or pLogic046 were subcultured 1:100 into 10mL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2h, at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression the butyrate operon from pLogic031 or pLogic046. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points ((0 up to 24 hours, as shown in FIG. 84A) to assess levels of butyrate production by LC-MS. As seen in FIG. 84B butyrate production is greater in the strain comprising the pLogic046 construct than the strain comprising the pLogic031 construct.
[1572] Production of butyrate was also assessed in E. coli Nissle strains containing the butyrate cassettes driven by an RNS promoter described above
(pLogic031-nsrR-norB-butyrate operon construct and pLogic046-nsrR-norB-butyrate operon construct) in order to determine the effect of nitrogen on butyrate production. Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA- NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final
concentration of 0.3mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points (0 up to 24 hours, as shown in FIG. 84B) to assess levels of butyrate production. As seen in FIG. 84B, genetically engineered Nissle comprising pLogic031-nsrR-norB-butyrate operon construct) or (pLogic046-nsrR-norB-butyrate operon construct) produced significantly more butyrate as compared to wild-type Nissle.
Example 67. Assessment of in vitro and in vivo activity of Biosafety System
Containing Strain
[1573] The activity of the following strains is tested:
[1574] SYN-21002 comprises a construct shown in FIG. 67D knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67B, except that the bla gene is replaced with a FNRS-fbrArgA construct. In other embodiments, other inducible or constitutive promoters are used.
[1575] SYN-21003 comprises a construct shown in FIG. 67E knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67B, except that the bla gene is replaced a FNRS- fbrArgA construct. In other embodiments, other inducible or constitutive promoters are used.
[1576] SYN-21005 comprises a construct shown in FIG. 67D knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67C, except that the bla gene is replaced with a FNRS-fbrArgA construct. In other embodiments, other inducible or constitutive promoters are used.
[1577] SYN-21007 comprises a construct shown in FIG. 67E knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67C, except that the bla gene is replaced with a FNRS-fbrArgA construct. In other embodiments, other inducible or constitutive promoters are used.
[1578] SYN-21009 a construct shown in FIG. 67D knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67B, except that the bla gene is replaced with the construct FIG. 50G/50H (FNR-ter-thiA1-hbd-crt2-tesB butyrate cassette). In other embodiments, other inducible or constitutive promoters are used.
[1579] SYN-21011 comprises a construct shown in FIG. 67E knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67B, except that the bla gene is replaced with the construct of FIG. 50G/50H (FNR-ter-thiA1-hbd-crt2-tesB butyrate cassette). In other embodiments, other inducible or constitutive promoters are used.
[1580] SYN-21013 comprises a construct shown in FIG. 67D knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67C, except that the bla gene is replaced with the construct FIG. 50G/50H (FNR-ter-thiA1-hbd-crt2-tesB butyrate cassette). In other embodiments, other inducible or constitutive promoters are used.
[1581] SYN-21014 comprises a construct shown in FIG. 67E knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in FIG. 67C, except that the bla gene is replaced with the construct of FIG. 50G/50H (FNR-ter-thiA1-hbd-crt2-tesB butyrate cassette). In other embodiments, other inducible or constitutive promoters are used. Table 64. Biosafety System Constructs and Sequence Components
Figure imgf000731_0001
Figure imgf000732_0001
Figure imgf000733_0001
Figure imgf000734_0001
Figure imgf000735_0001
Figure imgf000736_0001
Figure imgf000737_0001
Figure imgf000738_0001
Example 68. Generation of Butyrate and Acetate Producing Strains
A. Generation of an Acetate Overproducing Strain
[1582] E. coli generates high levels of acetate as an end product of fermentation. In order generate enhanced acetate production, strain SYN2001 was generated, which harbors a deletion in the endogenous ldh (lactate dehydrogenase) gene, with the intention to prevent or reduce flux through the metabolic arm generating lactate, and thereby enhancing the flux through the metabolic arm generating acetate (see, e.g., FIG. 85).
[1583] Briefly, We deleted the gene encoding L-lactate dehydrogenase A (ldhA) to block carbon flux from pyruvate to lactate and improve acetate biosynthetic yield in E. coli Nissle. Knockout primers were synthesized (IDT) and a chloramphenicol- resistance antibiotic marker was inserted in place of the ldhA coding region to ensure the removal of the targeted gene. The ldhA gene on the E. coli Nissle genome was knocked out and replaced with the chloramphenicol resistance gene through allelic exchange, which was facilitated by the lambda red recombinase system. Proper knockout of the target gene in the Nissle genome was validated by the ability of the resulting Nissle strain to grow on chloramphenicol-containing LB plates or medium and further confirmed by PCR. This strain was designated SYN2001.
[1584] For this study, media M9 media containing 50mM MOPS with 0.5% glucose was compared to media containing 0.5/% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut.
[1585] SYN2001 and streptomycin resistant E coli Nissle (SYN094) were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, 3, 4, 5, and 6 hours, cells were removed and pelleted at 14,000rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for acetate concentrations as described herein, e.g., in Example 69.
Table 66. Acetate production by SYN2001 from three different manufacturing experiments
Figure imgf000739_0001
[1586] Culture supernatants of SYN2001 produced between 21.2 and 31.5 mM acetate and an undetectable amount of butyrate (data not shown) under the above conditions in 3 independent production runs. Culture supernatant from run 3 was then used to generate the bioactivity results from cell based assays presented below in
Example 63.
[1587] As seen in FIG. 86A and FIG. 86B, the ldhA knockout E. coli Nissle strain SYN2001 has improved acetate productivity during over a 6 hour time course using either glucose or glucuronic acid as the main carbon source.
B. Generation of strains which produces butyrate and acetate a. Knock out of the endogenous adhE and ldhA genes
[1588] In order to improve acetate production while also producing high levels butyrate production, deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were generated to prevent or reduce metabolic flux through pathways which do not result in acetate or butyrate production (see, e.g., FIG. 85). Aldehyde-alcohol dehydrogenase converts acetylCoA into acetaldehyde, which is then converted to ethanol. As a result, a mutation or deletion of adhE is expected to prevent the metabolic flux towards ethanol production and consequently allow for additional acetylCoA to be used for butyrate production. For this study, Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes were used. Additionally, media M9 media containing 50mM MOPS with 0.5% glucose was compared to media containing 0.5/% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut.
[1589] Briefly, bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10ml LB (containing antibiotics) in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96- well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 69.
[1590] As seen in FIG. 86C and FIG. 86D, both integrated strains made similar amounts of acetate, and FNRS-ter-pbt-buk butyrate cassettes produced slightly more butyrate. Deletions in adhE and ldhA have similar effects on butyrate and acetate production. Acetate production was much greater in media containing 0.5% glucuronic acid.
b. Knock out of the endogenous frdA gene
[1591] FrdA is one of two catalytic subunits in the four subunit fumarate reductase complex. Fumarate reductase converts fumarate (derived from
phosphoenolpyruvate) to succinate along one arm of anaerobic metabolism. In a second study, the effect of a deletion in the endogenous frdA gene, which prevents metabolic flux through the phosphoenolpyruvate -> succinate pathway, on acetate and butyrate production was assessed. For this study, SYN2005 (comprising FNRS-ter-tesB butyrate cassette integrated at the HA1/2 site and a deletion in the endogenous frd gene) was compared to SYN1004 (comprising the FNRS-ter-tesB butyrate cassette integrated at the HA1/2 site).
[1592] Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10ml LB (containing antibiotics) in a 125ml baffled flask.
Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 69.
[1593] Results are depicted in FIG. 86E and indicate that the frdA mutation in SYN2005 allowed increased acetate production relative to SYN1173. SYN1173 produces greater levels of butyrate than acetate, while SYN2005 produces similar levels of both acetate and butyrate.
[1594] In other studies, strains are generated with combinations of deletions in two or more of the aldE, ldhA, and frd genes and the effect of the deletions on acetate and butyrate production are assessed.
C. Butyrate only producing strains
[1595] In order to generate a strain which can produce butyrate, but has a reduced ability to produce acetate, a deletion in the pta gene was introduced into a strain that contains an integrated butyrate cassette (Ter/TesB cassette) under the control of an FNR promoter (SYN2002). Phosphate acetyltransferase (Pta) catalyzes the conversion between acetyl-CoA and acetylphosphate, the first step in the metabolic arm leading to the generation of acetate (see., e.g., FIG.85). As such inhibition of this step was assumed to help prevent accumulation of acetate. Additionally, a mutation in the adhE (aldehyde-alcohol dehydrogenase) gene was introduced.
[1596] Acetate and butyrate production in both strains was compared to a third strain which contains both the FNR-driven ter-pbt-buk butyrate cassette and the deletion in the endogenous ldhA gene (e.g., as described above).
[1597] For this study, bacteria from all three strains were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10ml LB (containing antibiotics) in a 125ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5h, and then transferred to the anaerobic chamber at 37 C for 4h. Bacteria (2X108 CFU) were added to 1ml M9 media containing 50mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at -80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 69.
[1598] Results are depicted in FIG. 86F, and show that the strain comprising the deletion in the endogenous ldhA gene produced acetate but no butyrate, the strain comprising the FNR-ter-tesB butyrate cassette and the aldhE deletion produced butyrate, but very low levels of acetate. The third strain, comprising the FNRter-tesB butyrate cassette and the deletions in the adhE and pta genes, made equal amounts of acetate and butyrate. Example 69. Acetate and Butyrate quantification in bacterial supernatant by LC- MS/MS Sample Preparation
[1599] Ammonium acetate and Sodium butyrate stock (10 mg/mL) was prepared in water and aliquoted in 1.5 mL microcentrifuge tubes (100 µL) and stored at -20°C. Standards (1000, 500, 250, 100, 20, 4, 0.8 µg/mL) were prepared in water.
Sample and standards (10µL) were pipetted in a V-bottom polypropylene 96-well plate on ice. Derivatizing solution (90µL) containing 50mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphosphine in acetonitrile with 2 ug/mL of Sodium butyrate-d7 was added into the final solution. The plate was then heat-sealed with a ThermASeal foil and mixed well, and the samples were incubated at 60°C for 1hr for derivatization and centrifuged at 4000rpm for 5min. The derivatized samples (20µL) were added to 180µL of 0.1% formic acid in water/ACN (140:40) in a round-bottom 96-well plate. The plate was then heat-sealed with a ClearASeal sheet and mixed well. LC-MS/MS method
[1600] Derivatized metabolites were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 67, Table 68, Table 69 provide the summary of the LC-MS/MS method.
Table 67
Figure imgf000742_0001
Figure imgf000743_0001
Example 20. Synthesis of Constructs for Tryptophan Biosynthesis and Indole
Metabolite Synthesis. [1601] Various constructs are synthesized, and cloned into vector pBR322 for transformation of E. coli. In some embodiments, the constructs encoding the effector molecules are integrated into the genome. Table 46B. Exemplary Sequences and Construct Sequences for Tryptophan and
Indole Metabolite Synthesis
Figure imgf000743_0002
Figure imgf000744_0001
Figure imgf000745_0001
Figure imgf000746_0001
Figure imgf000747_0001
Figure imgf000748_0001
Figure imgf000749_0001
Figure imgf000750_0001
Figure imgf000751_0001
Figure imgf000752_0001
Figure imgf000753_0001
Figure imgf000754_0001
Figure imgf000755_0001
Figure imgf000756_0001
Figure imgf000757_0001
Figure imgf000758_0001
Figure imgf000759_0001
Figure imgf000760_0001
[1602] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 46B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 46B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 46B. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 46B. In another embodiment, the gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 46B. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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 one or more sequences of Table 46B. In another
embodiment, the genetically engineered bacteria comprise one or more sequence of Table 46B. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 46B.
[1603] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 103. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 103. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 103. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 103. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 103. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 103. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 103. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 103.
[1604] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 120. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 120. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 120. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 120. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 120. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 120. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 120. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 120.
[1605] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 121. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 121. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 121. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 121. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 121. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 121. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 121. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 121.
[1606] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 122. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 122. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 122. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 122. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 122. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 122. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 122. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 122.
[1607] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 123. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 123. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 123. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 123. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 123. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 123. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 123. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 123.
[1608] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 124. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 124. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 124. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 124. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 124. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 124. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 124. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 124.
[1609] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 125. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 125. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 125. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 125. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 125. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 125. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 125. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 125.
[1610] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 126. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 126. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 126. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 126. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 126. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 126. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 126. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 126.
[1611] In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 127. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 127. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 127. In some embodiments, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 127. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 127. Accordingly, In some embodiments, the genetically engineered bacteria comprise a sequence which 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: 127. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 127. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 127. Example 52. Tryptophan Production in an Engineered Strain of E. coli Nissle [1612] A number of tryptophan metabolites, either host-derived (such as tryptamine or kynurenine) or intestinal bacteria-derived (such as indole acetate or indole), have been shown to downregulate inflammation and promote gut barrier health, via the activation of the AhR receptor. Other tryptophan metabolites, such as the bacteria-derived indole propionate, have been shown to help restore intestinal barrier integrity, in experimental models of colitis. In this example, the E. coli strain Nissle was engineered to produce tryptophan, the precursor to all those beneficial metabolites.
[1613] First, in order to remove the negative regulation of tryptophan biosynthetic genes mediated by the transcription factor TrpR, the trpR gene was deleted form the E. coli Nissle genome. The tryptophan operon trpEDCBA was amplified by PCR from the E. coli Nissle genomic DNA and cloned in the low-copy plasmid pSC101 under the control of the tet promoter, downstream of the tetR repressor gene. This tet- trpEDCBA plasmid was then transformed into the∆trpR mutant to obtain the∆trpR, tet- trpEDCBA strain. Subsequently, a feedback resistant version of the aroG gene (aroGfbr) from E. coli Nissle, coding for the enzyme catalyzing the first committing step towards aromatic amino acid production, was synthetized and cloned into the medium copy plasmid p15A, under the control of the tet promoter, downstream of the tetR repressor. This plasmid was transformed into the∆trpR, tet-trpEDCBA strain to obtain the∆trpR, tet-trpEDCBA, tet-aroGfbr strain. Finally, a feedback resistant version of the tet- trpEBCDA construct (tet-trpEfbrBCDA) was generated from the tet-trpEBCDA. Both the tet-aroGfbr and the tet-trpEfbrBCDA constructs were transformed into the∆trpR mutant to obtain the∆trpR, tet-trpEfbrDCBA, tet-aroGfbr strain.
[1614] All generated strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, 100ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 0.5% glucose and placed at 37C at 250rpm. 200uL were collected at 2h, 4h and 16h and tryptophan was quantified by LC- MS/MS in the bacterial supernatant. FIG. 95A shows that tryptophan is being produced and secreted by the∆trpR, tet-trpEDCBA, tet-aroGfbr strain. The production of tryptophan is significantly enhanced by expressing the feedback resistant version of trpE.
Example 53. Improved Tryptophan by Using a non-PTS Carbon Source and by
Deleting the tnaA Gene Encoding Tryptophanase [1615] One of the precursor molecule to tryptophan in E. coli is
phosphoenolpyruvate (PEP). Only 3% of available PEP is normally used to produce aromatic acids (that include tryptophan, phenylalanine and tyrosine). When E. coli is grown using glucose as a sole carbon source, 50% of PEP is used to import glucose into the cell using the phosphotransferase system (PTS). In order to increase tryptophan production, a non-PTS oxidized sugar, glucuronate, was used to test tryptophan secretion by the engineered E. coli Nissle strain∆trpR, tet-trpEfbrDCBA, tet-aroGfbr. In addition, the tnaA gene, encoding the tryptophanase enzyme, was deleted in the∆trpR, tet-trpEfbrDCBA, tet-aroGfbr strain in order to block the conversion of tryptophan into indole to obtain the∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbr strain.
[1616] The∆trpR, tet-trpEfbrDCBA, tet-aroGfbr and∆trpR∆tnaA, tet- trpEfbrDCBA, tet-aroGfbr strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, 100ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 1% glucose or 1% glucuronate and placed at 37C at 250rpm or at 37C in an anaerobic chamber. 200uL were collected at 3h and 16h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant. FIG. 95B shows that tryptophan production is doubled in aerobic condition when the non- PTS oxidized sugar glucoronate was used. In addition, the deletion of tnaA had a positive effect on tryptophan production at the 3h time point in both aerobic and anaerobic conditions and at the 16h time point, only in anaerobic condition. Example 54. Improved Tryptophan Production by Increasing the Rate of Serine
Biosynthesis in E. coli Nissle [1617] The last step in the tryptophan biosynthesis in E. coli consumes one molecule of serine. In this example, we demonstrate that serine availability is a limiting factor for tryptophan production and describe the construction of the tryptophan producing E. coli Nissle strains∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbrserA and ∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbrserAfbr strains.
[1618] The∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbr strain was grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3mL LB with antibiotics in culture tubes. After two hours of growth at 37C at 250rpm, 100ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000rpm for 5min and resuspended in 3mL M9 minimal media. Cells were spun down again at 4,000rpm for 5min, resuspended in 3mL M9 minimal media with 1% glucuronate or 1% glucuronate and 10mM serine and placed at 37C an anaerobic chamber. 200uL were collected at 3h and 16h and tryptophan was quantified by LC- MS/MS in the bacterial supernatant. FIG. 95C shows that tryptophan production is improved three-fold by serine addition.
[1619] In order to increase the rate of serine biosynthesis in the∆trpR∆tnaA, tet- trpEfbrDCBA, tet-aroGfbr strain, the serA gene from E. coli Nissle encoding the enzyme catalyzing the first step in the serine biosynthetic pathway was amplified by PCR and cloned into the tet-aroGfbr plasmid by Gibson assembly. The newly generated tet- aroGfbr-serA construct was then transformed into a∆trpR∆tnaA, tet-trpEfbrDCBA strain to generate the∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbr-serA strain. The tet-aroGfbr- serA construct was further modified to encode a feedback resistant version of serA (serAfbr). The newly generated tet-aroGfbr-serAfbr construct was used to produce the ∆trpR∆tnaA, tet-trpEfbrDCBA, tet-aroGfbr-serAfbr strain, optimized to improve the rate of serine biosynthesis and maximize tryptophan production. Example 55. Comparison of Various Tryptophan Producing Strains [1620] Compare the rates of tryptophan production in the different strains generated, the following constructs and strains were generated according to methods and sequences described herein (e.g. Example 43), and assayed for tryptophan production in the presence of glucuronate as a carbon source under aerobic conditions. SYN2126 comprises∆trpR∆tnaA (∆trpR∆tnaA). SYN2323 comprises∆trpR∆tnaA and a tetracycline inducible construct for the expression of feedback resistant aroG on a plasmid (∆trpR∆tnaA, tet-aroGfbr). SYN2339 comprises∆trpR∆tnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (∆trpR∆tnaA, tet-aroGfbr, tet-trpEfbrDCBA). SYN2473 comprises∆trpR∆tnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG and SerA on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (∆trpR∆tnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA). SYN2476 comprises∆trpR∆tnaA and a tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a plasmid (∆trpR∆tnaA, tet-trpEfbrDCBA).
[1621] Overnight cultures were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Next, cells were induced with 100ng/mL ATC for 2 hours (37C, 250rpm), spun down, washed with cmL M9, spun down again and resuspended in 3mL M9+1% glucuronate. Cells were plated for CFU counting. For the assay, the cells were placed af 37C with shaking at 250rpm. Supernatants were collected at 1h, 2h, 3h, 4h 16h for HPLC analysis for tryptophan. As seen in FIG. 96, 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. Example 56. Bacterial Production of Indole Acetic Acid (IAA) [1622] The ability of a strain comprising tryptophan production circuits and additionally Indole-3-pyruvate decarboxylase from Enterobacter cloacae (IpdC) and Indole-3-acetaldehyde dehydrogenase from Ustilago maydis (Iad1) to produce indole acetic acid (IAA) was tested. The following strains were generated according to methods described herein and tested.
[1623] SYN2126: comprises∆trpR and∆tnaA (∆trpR∆tnaA). SYN2339 comprises circuitry for the production of tryptophan;∆trpR and∆tnaA, a first tetracline inducible trpEfbrDCBA construct on a first plasmid(pSC101), and a second tetracycline inducible aroGfbr construct on a second plasmid (∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)) (FIG. 90B). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises trpDH-ipdC-iad1 incorporated at the end of the second construct
(∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A))(FIG. 93B).
[1624] Overnight cultures of the strains were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Strains were then induced with 100ng/mL ATC for 2 hours (37C, 250rpm). Cells were spun down, and resuspended in 1mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 1h, 2.5h and 18h for LCMS analysis of tryptophan and indole acetic acid as described herein.
[1625] As seen in FIG. 99, SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 containing the additional IAA producing circuitry produces amounts of IAA that are comparable to the amounts of tryptophan produced in its parent SYN2339. No tryptophan is measured, indicating that all tryptophan produced in SYN2342 is efficiently converted into IAA. Example 57. Tryptamine Production Comparing Two Tryptophan Decarboxylases [1626] The efficacy of two tryptophan decarboxylases (tdc), one from
Catharanthus roseus (tdcCr)and a second from Clostridium sporogenes (tdcCs) in producing tryptamine from tryptophan was tested. The following strains were generated according to methods described herein and tested.
[1627] SYN2339 comprises∆trpR and∆tnaA and a tetracycline inducible trpEfbrDCBA construct on a plasmid and another tetracycline inducible construct expressing aroGfbr on a second plasmid (∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)). SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine. SYN2340 comprises∆trpR and∆tnaA and a tetracycline inducible trpEfbrDCBA construct on a plasmid and another tetracycline inducible construct expressing aroGfbr tdcCr on a second plasmid (∆trpR∆tnaA, tetR-Ptet- trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-tdcCr (p15A)). SYN2794 comprises∆trpR and ∆tnaA and a tetracycline inducible trpEfbrDCBA construct on a plasmid and another tetracycline inducible construct expressing aroGfbr tdcCs on a second plasmid
(∆trpR∆tnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-tdcCs (p15A)).
[1628] Overnight cultures of the strains were diluted 1/100 in 3mL LB plus antibiotics and grown for 2 hours (37C, 250rpm). Strains were then induced with 100ng/mL ATC for 2 hours (37C, 250rpm). Cells were spun down, and resuspended in 1mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 3h and 18h for LCMS analysis of tryptophan and tryptamine, as described herein.
[1629] As seen in FIG. 100, TdcCs from Clostridium sporogenes is more efficient than TdcCr from Catharanthus roseus in tryptamine production and converts all the tryptophan produced into tryptamine Example 58. Tryptophan and Anthranilic Acid Quantification in Bacterial Supernatant by LC-MS/MS [1630] Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 µL), and stored at -20°C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 µg/mL) were prepared in water. Samples (10 µL) and standards were mixed with 90 µL of ACN/H2O (60:30, v/v) containing 1µg/mL of Tryptophan-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for 5min. The solution (10µL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.
[1631] LC-MS/MS method
[1632] Tryptophan and Anthranilic acid were measured by liquid
chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 82., Table 83, and Table 84 provide the summary of the LC-MS/MS method.
Figure imgf000771_0001
Example 59. Quantification of Tryptamine in Bacterial Supernatant by Liquid Chromatography-Mass Spectrometry (LC-MS) [1633] Tryptamine acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 µL), and stored at -20°C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 µg/mL) were prepared. Samples (10 µL) and standards were mixed with 90 µL of ACN/H2O (60:30, v/v) containing 1µg/mL of tryptamine-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000rpm for 5min. The solution (10µL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well.
LC-MS/MS method [1634] Tryptamine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 85., Table 86, and Table 87 provide the summary of the LC- MS/MS method.
Figure imgf000772_0001
Example 60. Quantification of Tryptophan, Indole-3-acetate, Indole-3-lactate, Indole-3-propionate in Bacterial Supernatant by High-pressure Liquid
Chromatography (HPLC) [1635] Samples were thawed on ice and centrifuged at 3,220 x g for 5min at 4°C. 80µL of the supernatant was pipetted, mixed with 20µL 0.5% formic acid in water, and analyzed by HPLC using a Shimadzu Prominence-I. HPLC conditions used for the quantification of tryptophan, indole-3-acetate, indole-3-lactate and indole-3-propionate are described in Table 88.
Figure imgf000773_0001
Example 61. Generation of constructs for overproducing therapeutic molecules for secretion [1636] To produce strain capable of secreting anti-inflammatory or gut barrier enhancer polypeptides, e.g., GLP2, IL-22, several constructs are designed employing different secretion strategies. Various GLP2, IL-22, constructs are synthesized, and cloned into vector pBR322 for transformation of E. coli. In some embodiments, the constructs encoding the effector molecules are integrated into the genome. In some embodiments, the constructs encoding the effector molecules are on a plasmid, e.g., a medium copy plasmid. Table 89. lists exemplary polypeptide coding sequences used in h n r
Figure imgf000774_0001
Figure imgf000775_0002
[1637] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 or a functional fragment thereof.
[1638] Table 90 lists exemplary secretion tags, which can be added at the N- terminus when the diffusible outer membrane (DOM) method or the fliC method is used.
Table 90. Secretion Tags and FliC components
Figure imgf000775_0001
Figure imgf000776_0001
Figure imgf000777_0002
[1639] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and SEQ ID NO: 151.
Table 91. Non-limiting Examples of Secretion Constructs
Figure imgf000777_0001
Figure imgf000778_0001
Figure imgf000779_0001
Figure imgf000780_0001
Figure imgf000781_0001
[1640] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, and SEQ ID NO: 159. Table 92 lists exemplary secretion constructs.
Table 92. Non-limiting Examples of Secretion Constructs
Figure imgf000782_0001
Figure imgf000783_0001
[1641] In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 160, SEQ ID NO: 161.
Example 62. Bacterial Secretion of GLP-2 [1642] To determine whether the human GLP-2 expressed by engineered bacteria is secreted, the concentration of GLP-2 in the bacterial supernatant from two engineered strains comprising GLP-2 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing GLP-2 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing GLP-2, further comprising a deletion in degP (see Table 93).
[1643] E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBusterTM (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4◦C then placed on ice until further processing.
[1644] The concentration of GLP-2 in the cell-free medium and in the bacterial cell extract was measured by Human GLP2 ELISA Kit (Competitive EIA) (LSBio), according to manufacturer’s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of GLP-2. Standard curves were generated using recombinant GLP-2. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. As seen in Table 93, deletion of degP, a periplasmic protease, improved secretion levels over 3-fold.
Table 93. GLP-2 Secretion
Figure imgf000784_0001
Co-culture studies
[1645] To determine whether the hGLP-2 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant (from both strains shown above) containing secreted human GLP-2 is added to the growth medium of Caco-2 cells and CCD-18Co cells. The Caco-2 cell line is a continuous cell of heterogeneous human epithelial colorectal adenocarcinoma cells. As described e.g., in Jasleen et al. (Dig Dis Sci. 2002
May;47(5):1135-40) GLP-2 stimulates proliferation and [3H]thymidine incorporation in Caco-2 and T84 cells. Additionally, GLP-2 stimulates VEGFA secretion in these cells (see., e.g., Bulut et al, Eur J Pharmacol. 2008 Jan 14;578(2-3):279-85.
[1646] Functional activity of bacterially secreted GLP-2 is therefore assessed by its ability to induce proliferation and VEGF secretion.
[1647] Caco-2 are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Caco-2 cells (1e6/24 well) are serum starved overnight. Titrations of either recombinant human GLP-2 (50 and 250 nM) diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for a defined time.
[1648] For cell proliferation assays, cells are harvested and resuspended in lysis buffer. The cells are assayed after 12, 24, 48, and 72 hours of incubation. Cell proliferation is measured using a Cell proliferation assay kit according to
manufacturer’s instruction (e.g., a Cell viability was assessed by a 3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay).
[1649] For the measurements of VEFA secretion, cells are harvested and resuspended in lysis buffer, and concentrations of GLP-2 in the medium are determined ELISA
[1650] PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity.
Competition studies
[1651] As an additional control for specificity, a competition assay is performed. Titrations of anti-GLP-2 antibody are pre-incubated with constant concentrations of either recombinant GLP-2 or supernatants from the engineered bacteria for 15min. Next, the supernatants/ rhIL2 solutions are added to serum-starved Cac-2 cells
(1e6/well) and cells are incubated for 30 min followed by VEGFA ELISA as described above.
Example 63. Bacterial Secretion of IL-22 [1652] To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant from a two engineered strains comprising IL-22 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing IL-22 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing IL-22, further comprising a deletion in degP (Table 93).
[1653] E. coli Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22 micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBusterTM (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes unsoluble debris is spun down at 20 min at 12,000 rcf at 4◦C then placed on ice until further processing.
[1654] The concentration of IL-22 in the cell-free medium and in the bacterial cell extract was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL- 22), according to manufacturer’s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Standard curves were generated using recombinant IL-22. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 94 summarizes levels of IL-22 measured in the supernatant. The data show that both hIL-22 are secreted at various levels from the different bacterial strains.
Table 94. IL-22 Secretion
Figure imgf000786_0001
Figure imgf000787_0001
Example 64. Bacterial Secretion of IL-22 and Functional Assays Generation of Bacterial Supernatant and Measurement of IL-22 concentration [1655] To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant was measured.
[1656] E. coli Nissle comprising a tet-inducible integrated construct (delta pal::CmR expressing PhoA-IL22 from Ptet) was grown overnight in LB medium.
Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100ng/mL to induce expression of hIL- 22. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the supernatant was centrifuged, and filtered through a 0.22 micron filter to remove any remaining bacteria.
[1657] The concentration of hIL-22 in the cell-free medium was measured by hIL-22 ELISA (R&D Systems (DY782) ELISA for hIL-22), according to
manufacturer’s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Additionally, samples were diluted to ensure absence of matrix effects and to demonstrate linearity. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. The engineered bacteria comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag were determined to be secreting at 199 ng/ml supernatant.
Co-culture studies [1658] To determine whether the hIL-22 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation was conducted, in which the bacterial supernatant containing secreted human IL-22 was added to the growth medium of a mammalian colonic epithelial cell line. IL-22 is known to induce the phosphorylation of STAT1 and STAT3 in Colo205 cells (see, e.g., Nagalakshmi et al., Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells. Int Immunopharmacol. 2004 May;4(5):679-91). Functional activity of bacterially secreted IL-22 was therefore assessed by its ability to phosphorylate STAT3 in Colo205 cells.
[1659] Colo205 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Colo205 (1e6/24 well) were serum starved overnight. Titrations of either recombinant human IL-22 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria were added to cells for 30 minutes. Cells were harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) was run in triplicate for all samples, according to manufacturer’s instructions. PBS-treated cells and PBS were added as negative controls. Dilutions of samples were included to demonstrate linearity. No signal was observed for wild type Nissle. Results for the engineered strain comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag are shown in FIG. 101A, and demonstrate that hIL-22 secreted from the engineered bacteria is functionally active.
Competition studies [1660] As an additional control for specificity, a competition assay was performed. Titrations of anti-IL22 antibody (MAB7821, R&D Systems) were pre- incubated with constant concentrations of either rhIL22 (data not shown) or
supernatants from the engineered bacteria for 15min. Next, the supernatants/ rhIL2 solutions were added to serum-starved Colo205 cells (1e6/well) and cells were incubated for 30 min followed by pSTAT3 ELISA as described above. As shown in FIG. 101B, the phospho-Stat3 signal induced by the secreted hIL-22 is competed by the hIL-22 antibody MAB7821. Example 64. Generation and Analysis of an engineered IL-22-producing E. coli
Nissle strain Engineering and Production of IL-22
[1661] A synthetic construct was generated in which expression of IL-22 is controlled by the tetracycline-inducible promoter (Ptet), which is derepressed via the addition of the tetracycline analog anhydrotetracycline (aTc), and translation is driven by a strong ribosome binding site (RBS) located immediately upstream from the IL-22 coding sequence. To promote translocation to the periplasm, a 21-amino acid PhoA- secretion tag was added to the N-terminus of IL-22.
[1662] The corresponding engineered element was constructed using a synthetic DNA cassette encoding the IL-22 protein coding sequence (IDT Technologies,
Coralville, Iowa) which was cloned into an initial plasmid vector, creating the plasmid Logic435. The IL-22 sequence was later amplified and cloned using Gibson assembly technology and the NEBuilder Hifi Mastermix (NEB). The final pBR322-based plasmid was sequence-verified by Sanger sequencing (Genewiz) and designated Logic522.
[1663] To create a Gram-negative bacterium capable of secreting bioactive proteins, a diffusible outer membrane (DOM) phenotype was engineered in the E. coli Nissle background. A series of DOM mutants were created by deleting different periplasmic proteins leading to a‘leaky’ phenotype. Deletions of several different genes were tested including lpp, pal, tolA and nlpI. For example, the pal mutant (SYN3000) showed a good secretion phenotype with little-to-no deleterious effect on growth rate while supporting strong production of effectors in the extracellular medium. Logic522 was inserted into SYN3000 to create the IL-22 secretion strain, SYN3001.
[1664] To assay for production of IL-22, cultures were grown and induced, then supernatants were harvested and quantified using ELISA. Overnight cultures were harvested by centrifugation at 12.5K x g for 5 minutes. The supernatants of the cultures were removed from the cell pellet and filtered through a 0.22 µm filter to separate any remaining bacteria from the supernatant. This supernatant was run immediately in the ELISA, stored short-term at 4oC, or aliquoted and stored at -20oC.
[1665] To evaluate the production of IL-22 in the filtered supernatants, samples of SYN3000 and SYN3001 were diluted in triplicate and run on an R&D Systems IL-22 Quantikine® ELISA Kit (Minneapolis, MN). The results from 3 independent production runs are shown in Table 95. The results demonstrated that the SYN3001 supernatants contained an average of 312 ng/ml (+/- 11.38) of material that reacted positively in the IL-22 ELISA assay. In contrast, the SYN3000 supernatants had undetectable levels (not shown). Culture supernatant from run 3 was then used to generate the bioactivity results from the cell based assays described below. Table 95 SYN3001 supernatant results from three different production runs.
Figure imgf000790_0001
In Vitro Assessment of IL-22 Produced by the Engineered Strain SYN3001
[1666] To assess the biological activity of IL-22 produced by SYN3001 (IL-22 secreting strain), titrations of SYN3001 and SYN3000 (DOM mutant, non IL-22 secreting negative control strain) supernatants (starting at 150ng/mL and titrated in 1:3 dilutions) were added to Colo205 cells and the activation of STAT3 was assessed. FIG. 101C shows the results from 5 independent experiments (each performed in triplicate). Supernatants from SYN3001 induced activation of STAT3 with an average EC50 of 4.8 ng/mL (+/- 1.74 ng/mL). In contrast, SYN3000 had no effect on STAT3 activity.
[1667] To verify that the STAT3 activation elicited by supernatants from SYN3001 was indeed due to IL-22 signaling, Colo205 cells were stimulated with IL-22 supernatants derived from SYN3001 at 3 ng/mL in the presence of increasing concentrations of an anti-IL-22 neutralizing antibody. rLI-22 in the absence of the neutralizing antibody served as a positive control. FIG. 101D shows the results from 3 independent experiments (performed in triplicate), demonstrating that the anti-IL-22 antibody inhibited SYN3001-induced activation of STAT3 in a dose-dependent manner. The average IC50 for the anti-IL-22 antibody mediated inhibition of SYN3001-derived IL-22 was 3.45 ng/mL for SYN3001, in line with the value observed using rIL-22, 3.70 ng/mL.
Summary
[1668] The results describe the design and evaluation of an engineered IL- 22 producing strain, SYN3001, which contains a tetracycline-inducible promoter driving the expression of IL-22 fused to a cleavable PhoA-secretion tag to mediate Sec- dependent secretion into the periplasm and a pal mutation to create a diffusible outer membrane phenotype (DOM) that facilitates extracellular secretion. This strain is capable of producing >300 ng/mL IL-22 in vitro under the conditions described here. This in vitro IL-22 production translates to biological activity in a cell-based assay that is comparable to that observed with recombinant IL-22. In addition, the specific activity of the bacterially-produced IL-22 was verified by demonstrating that this signal could be inhibited by a neutralizing antibody against IL-22. Table 96 summarizes the final pharmacological characteristics of SYN3001. Table 96. Final characterization of the pharmacological characteristics of SYN300
Figure imgf000791_0001
Example 67. Table 98. Other Sequences of interest
Figure imgf000791_0002
Figure imgf000792_0001

Claims

Claims 1. A bacterium comprising at least one gene or gene cassette for the
consumption of ammonia and at least one genes or gene cassette for producing butyrate, wherein the bacterium comprises an endogenous pta gene which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
2. The bacterium of claim 1, wherein the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, pbt, and buk genes.
3. The bacterium of claim 1, wherein the at least one gene cassette for producing butyrate comprises ter, thiA1, hbd, crt2, and tesb genes.
4. The bacterium of any one of claims 1-3, wherein the bacterium comprises an endogenous adhE gene which is knocked down via mutation or deletion.
5. The bacterium of any one of claims 1-4, wherein the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion.
6. The bacterium of any one of claims 1-3, wherein the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.
7. The bacterium of any one of claims 1-6, wherein the promoter operably
linked to the at least one gene or gene cassette is induced by exogenous environmental conditions.
8. The bacterium of claim 7, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by low-oxygen or anaerobic conditions.
9. The bacterium of claim 8, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is selected from a FNR- inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.
10. The bacterium of claim 7, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by one or more molecules or metabolites indicative of liver damage.
11. The bacterium of claim 7, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive nitrogen species.
12. The bacterium of claim 7, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive oxygen species.
13. The bacterium of claim 7, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an
environmental factor that is not naturally present in a mammalian gut.
14. A bacterium comprising at least one gene or gene cassette for the
consumption of ammonia and at least one genes or gene cassette for producing butyrate, wherein the bacterium comprises at least one endogenous gene selected from frd, ldhA, and adhE, which is knocked down via mutation or deletion, and wherein the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature.
15. The bacterium of claim 14, wherein the at least one gene cassette for
producing butyrate comprises ter, thiA1, hbd, crt2, pbt, and buk genes.
16. The bacterium of claim 14, wherein the at least one gene cassette for
producing butyrate comprises ter, thiA1, hbd, crt2, and tesb genes.
17. The bacterium of any one of claims 14-16, wherein the bacterium comprises an endogenous adhE gene which is knocked down via mutation or deletion.
18. The bacterium of any one of claims 14-17, wherein the bacterium comprises an endogenous frd gene which is knocked down via mutation or deletion.
19. The bacterium of any one of claims 14-18, wherein the bacterium comprises an endogenous ldhA gene which is knocked down via mutation or deletion.
20. The bacterium of any one of claims 14-19, wherein the promoter operably linked to the at least one gene or gene cassette is induced by exogenous environmental conditions.
21. The bacterium of claim 20, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by low- oxygen or anaerobic conditions.
22. The bacterium of claim 21, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is selected from a FNR- inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.
23. The bacterium of claim 14, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by one or more molecules or metabolites indicative of liver damage.
24. The bacterium of claim 14, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive nitrogen species.
25. The bacterium of claim 14, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by the presence of reactive oxygen species.
26. The bacterium of claim 14, wherein the promoter operably linked to the at least one gene or gene cassette for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
27. The bacterium of claim 1 or claim 14, wherein the ammonia conversion
circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter that is induced by exogenous environmental conditions; and wherein the bacterium lacks a functional ArgR.
28. The bacterium of claim 27, wherein each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been
independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions.
29. The bacterium of claim 28, wherein each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
30. The bacterium of any one of claims 27-29, wherein under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
31. The bacterium of claim 1 or 14, wherein the ammonia conversion circuit comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous
environmental conditions; wherein the arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase; and wherein each operon except the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine- mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the genes in the operon.
32. The bacterium of claim 31, wherein the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine- mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the argininosuccinate synthase gene.
33. The bacterium of claim 32, wherein the operon comprising the gene encoding argininosuccinate synthase comprises a constitutively active promoter that regulates transcription of the argininosuccinate synthase gene.
34. The bacterium of any one of claims 28-33, wherein arginine feedback
resistant N-acetylglutamate synthetase is controlled by endogenous environmental conditions.
35. The bacterium of claim 34, wherein arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced under low oxygen conditions.
36. The bacterium of claim 35, wherein arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter selected from a FNR- inducible promoter, an ANR-inducible promoter, and a DNR-inducible promoter.
37. The bacterium of claim 34, wherein arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage.
38. The bacterium of claim 34, wherein arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species.
39. The bacterium of claim 34, wherein arginine feedback resistant N- acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species.
40. The bacterium of claim 34, wherein the promoter operably linked to the at least one gene or gene cassettes for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
41. The bacterium of any one of claims 1-40, wherein the bacterium is a non- pathogenic bacterium.
42. The bacterium of any one of claims 1-41, wherein the bacterium is a probiotic bacterium.
43. The bacterium of any one of claims 1-42, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
44. The bacterium of any one of claims 1-43, wherein the bacterium is
Escherichia coli strain Nissle.
45. The bacterium of any one of claims 1-44, wherein the at least one gene or gene cassette for producing a butyrate is present on a plasmid in the bacterium and operably linked on the plasmid to the inducible promoter.
46. The bacterium of any one of claims 1-44, wherein the at least one gene or gene cassette for producing butyrate is present on a bacterial chromosome and operably linked on chromosome to the inducible promoter.
47. The bacterium of claim 1, comprising at least one gene or gene cassette
selected from (1) a GABA metabolic gene or gene cassette (2) a GABA transport gene or gene cassette, (3) a manganese transport gene or gene cassette.
48. The bacterium of claim 47, wherein the at least one gene for the consumption of GABA is capable of producing a GABA catabolism enzyme.
49. The bacterium of claim 48, wherein the GABA catabolism enzyme is selected from GABA α-ketoglutarate transaminase (GSST) and succinate- semialdehyde dehydrogenase (SSDH).
50. The bacterium of claim 47, wherein the GABA transport circuit is capable of producing a GABA membrane transport protein.
51. The bacterium of claim 50, wherein the GABA membrane transport protein is GabP.
52. The bacterium of claim 47, wherein the manganese transport circuit is capable of producing a manganese membrane transport protein.
53. The bacterium of claim 52, wherein the manganese membrane transport protein is MntH.
54. The bacterium of any one of claims 47-52, wherein the at least one genes or gene cassettes is controlled by a promoter induced by exogenous
environmental conditions.
55. The bacterium of claim 54, wherein the at least one genes or gene cassettes is controlled by a promoter induced under low oxygen conditions.
56. The bacterium of claim 54, wherein the at least one genes or gene cassettes is controlled by a promoter selected from a FNR-inducible promoter, an ANR- inducible promoter, and a DNR-inducible promoter.
57. The bacterium of claim 54, wherein the at least one genes or gene cassettes is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage.
58. The bacterium of claim 54, wherein the at least one genes or gene cassettes is controlled by a promoter induced by the presence of reactive nitrogen species.
59. The bacterium of claim 54, wherein the at least one genes or gene cassettes is controlled by a promoter induced by the presence of reactive oxygen species.
60. The bacterium of claim 54, wherein the promoter operably linked to the at least one gene or gene cassettes for producing butyrate is induced by an environmental factor that is not naturally present in a mammalian gut.
61. The bacterium of claim 47, wherein the ammonia conversion circuit
comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous
environmental conditions; and wherein the bacterium has been engineered to lack a functional ArgR.
62. The bacterium of claim 61, wherein each copy of a functional argR gene normally present in a corresponding wild-type bacterium has been
independently deleted or rendered inactive by one or more nucleotide deletions, insertions or substitutions.
63. The bacterium of claim 62, wherein each copy of the functional argR gene normally present in a corresponding wild-type bacterium has been deleted.
64. The bacterium of any one of claims 47-63, wherein arginine feedback
resistant N-acetylglutamate synthetase is controlled by a promoter selected from a FNR-inducible promoter, an ANR-inducible promoter, and a DNR- inducible promoter.
65. The bacterium of any one of claims 47-64, wherein arginine feedback
resistant N-acetylglutamate synthetase is controlled by a promoter induced by one or more molecules or metabolites indicative of liver damage.
66. The bacterium of any one of claims 47-65, wherein arginine feedback
resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive nitrogen species.
67. The bacterium of any one of claims 47-66, wherein arginine feedback
resistant N-acetylglutamate synthetase is controlled by a promoter induced by the presence of reactive oxygen species.
68. The bacterium of any one of claims 47-67, wherein under conditions that induce the promoter that controls expression of the arginine feedback resistant N-acetylglutamate synthetase, the transcription of each gene that is present in an operon comprising a functional ARG box and which encodes an arginine biosynthesis enzyme is increased as compared to a corresponding gene in a wild-type bacterium under the same conditions.
69. The bacterium of claim 47, wherein the ammonia conversion circuit
comprises an arginine regulon comprising a gene encoding a functional N-acetylglutamate synthetase with reduced arginine feedback inhibition as compared to a wild-type N-acetylglutamate synthetase from the same bacterial subtype under the same conditions, wherein expression of the gene encoding arginine feedback resistant N-acetylglutamate synthetase is controlled by a promoter that is induced by exogenous
environmental conditions; wherein the arginine regulon comprises one or more operons comprising genes that encode arginine biosynthesis enzymes N-acetylglutamate kinase, N- acetylglutamate phosphate reductase, acetylornithine aminotransferase, N- acetylornithinase, carbamoylphosphate synthase, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase; and wherein each operon except the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine- mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the genes in the operon.
70. The bacterium of claim 69, wherein the operon comprising the gene encoding argininosuccinate synthase comprises one or more mutated ARG box(es) characterized by one or more nucleic acid mutations that reduces arginine- mediated repression of the operon via ArgR binding, and retains RNA polymerase binding with sufficient affinity to promote transcription of the argininosuccinate synthase gene.
71. The bacterium of claim 70, wherein the operon comprising the gene encoding argininosuccinate synthase comprises a constitutively active promoter that regulates transcription of the argininosuccinate synthase gene.
72. The bacterium of any one of claims 47-71, wherein the bacterium is a non- pathogenic bacterium.
73. The bacterium of any one of claims 47-72, wherein the bacterium is a probiotic bacterium.
74. The bacterium of any one of claims 47-73, wherein the bacterium is selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus.
75. The bacterium of any one of claims 47-74, wherein the bacterium is Escherichia coli strain Nissle.
76. The bacterium of any one of claims 47-75, wherein the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit, is present on a plasmid in the bacterium and operably linked on the plasmid to the inducible promoter.
77. The bacterium of any one of claims 47-75, wherein the ammonia conversion circuit, GABA metabolic circuit, GABA transport circuit, or the manganese transport circuit, is present on a bacterial chromosome and operably linked on chromosome to the inducible promoter.
78. The bacterium of any one of claims 47-77, wherein the bacterium is an
auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut.
79. The bacterium of claim 78, wherein mammalian gut is a human gut.
80. A pharmaceutically acceptable composition comprising the bacterium of any one of claims 1-79.
81. The pharmaceutically acceptable composition of claim 80, wherein the
composition is formulated for oral or rectal administration.
82. A method of treating a disease, disorder or condition associated with
hyperammonemia, or symptom(s) thereof in a subject in need thereof comprising the step of administering to the subject the composition of claim 80 for a period of time sufficient to lessen the severity of the disease or
symptom(s).
83. The method of claim 82, wherein the disease, disorder, condition is selected from hepatic encephalopathy, Huntington’s disease, or symptom(s) thereof.
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CN109172613A (en) * 2018-08-14 2019-01-11 景岳生物科技(中国)有限公司 The topical compositions of the dead bacterium culture containing lactobacillus, medical composition and its in promoting wound healing and reduce the purposes of scar
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