WO2016210378A2 - Commande multicouche de l'expression génique dans des bactéries génétiquement modifiées - Google Patents

Commande multicouche de l'expression génique dans des bactéries génétiquement modifiées Download PDF

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WO2016210378A2
WO2016210378A2 PCT/US2016/039434 US2016039434W WO2016210378A2 WO 2016210378 A2 WO2016210378 A2 WO 2016210378A2 US 2016039434 W US2016039434 W US 2016039434W WO 2016210378 A2 WO2016210378 A2 WO 2016210378A2
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gene
payload
regulatory region
genetically engineered
encoding
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PCT/US2016/039434
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WO2016210378A3 (fr
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Dean Falb
Vincent M. ISABELLA
Jonathan W. KOTULA
Paul F. Miller
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Synlogic, Inc.
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    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01024—Phenylalanine ammonia-lyase (4.3.1.24)

Definitions

  • Methods and compositions for regulating gene expression in a cell are provided.
  • the disclosure relates to circuits for multi-layered control of gene expression in genetically engineered bacteria that can selectively express a gene of interest or payload under particular exogenous environmental conditions.
  • the genetically engineered bacteria express a therapeutic payload, particularly in low-oxygen conditions, such as in the mammalian gut.
  • Genetic engineering is a powerful tool that enables the redesign and modification of living organisms. Genetic engineering has the potential to control a wide range of biological activities from cellular function in vitro to disease therapy in vivo. Synthetic regulatory circuits in particular aim to facilitate the manipulation of complex genetic regulatory networks. However, designing circuits to control complex biological processes in living cells can be challenging (Kwok 2010), and there is significant unmet need for technologies that provide meticulous and multi-layered control of these circuits, particularly for deployment into living cells and human subjects.
  • the disclosure relates to methods and compositions that extend multi- layered control and functionality to genetic regulatory circuits in living cells.
  • the disclosure relates to genetically engineered bacteria comprising multi- layered genetic regulatory circuits, which allow the bacteria to express a payload or gene of interest under particular exogenous environmental conditions.
  • compositions described herein provide foundational technologies for genetic engineering and synthetic biology, particularly for use in advancing therapies and pharmaceutical compositions for human disease.
  • Fig. 1 illustrates an exemplary regulatory circuit comprising a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a fumarate and nitrate reductase regulator (F R)-responsive promoter; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload), wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase.
  • F R fumarate and nitrate reductase regulator
  • FIG. 1A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, and the payload is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase.
  • Fig. IB depicts the construct in the absence of oxygen (-02); FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the payload is expressed.
  • Fig. 1C depicts a construct wherein the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen (not shown), FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY
  • FIG. 2 illustrates an exemplary regulatory circuit comprising a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR- responsive promoter; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a tet regulatory region (tetO); and a third gene encoding an mf-lon degradation signal linked to a tet repressor (tetR), wherein the tetR is capable of binding to the tet regulatory region and repressing expression of the second gene or gene cassette.
  • tetR tetR
  • the mf-lon protease is capable of recognizing the mf-lon degradation signal and degrading the tetR.
  • Fig. 2A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the payload is not expressed.
  • Fig. 2B depicts the construct in the absence of oxygen (-02); FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease.
  • the mf-lon protease recognizes the mf-lon degradation signal (mf-lon deg tag) and degrades the tetR (Fig. 2C), and the payload is expressed (Fig. 2D).
  • FIG. 3 illustrates an exemplary regulatory circuit construct comprising a first gene encoding a first repressor (Repressor 1, Rl), wherein the first gene is operably linked to a FNR-responsive promoter; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a first regulatory region comprising a constitutive promoter (P-constitutive); and a third gene encoding a second repressor (Repressor 2, R2), wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette.
  • Repressor 1 first repressor
  • the third gene is operably linked to a second regulatory region comprising a constitutive promoter (P-constitutive), wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor.
  • Fig. 3A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed.
  • Fig. 3B depicts the construct in the absence of oxygen (-02); FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed.
  • Fig. 4 illustrates an exemplary regulatory circuit construct comprising a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload).
  • the second gene or gene cassette is operably linked to a constitutive promoter (P-constitutive) and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the payload.
  • the regulatory RNA is capable of eliminating the mRNA hairpin and inducing payload translation via the ribosomal binding site (RBS).
  • FIG. 4A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the payload from being translated.
  • Fig. 4B depicts the construct in the absence of oxygen (- 02); FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the payload is expressed.
  • Fig. 5 illustrates an exemplary regulatory circuit construct comprising a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload), wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter (P-constitutive); and a third gene encoding a repressor (R) operably linked to a constitutive promoter
  • Fig. 5A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed.
  • Fig. 5B depicts the construct in the absence of oxygen (-02); FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed.
  • FIG. 6 illustrates an exemplary regulatory circuit construct comprising a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR- responsive promoter (P-fnr), and a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a constitutive promoter (P-constitutive).
  • P-fnr FNR- responsive promoter
  • P-constitutive a constitutive promoter
  • the second gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites (INT), and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5' to 3').
  • Fig. 6A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR- responsive promoter, the recombinase is not expressed, the payload remains in the 3' to 5' orientation, and no functional payload is produced.
  • 6B depicts the construct in the absence of oxygen (-02); FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the payload is reverted to the 5' to 3' orientation, and functional payload is produced.
  • Fig. 7 illustrates an exemplary regulatory circuit construct comprising a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR- responsive promoter (P-fnr); a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload.
  • P-fnr FNR- responsive promoter
  • a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a T7 promoter
  • a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the payload.
  • the third gene encoding the T7 polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites (INT), and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5' to 3').
  • Fig. 7A depicts the construct in the presence of oxygen (+02); FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed.
  • FIG. 7B depicts the construct in the absence of oxygen (-02); F R dimerizes and binds the F R-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5' to 3' orientation, and the payload is expressed.
  • FIGs. 8 A-F depict schematics of component constructs of the circuits shown in Fig. 7A and 7B (SYN1331). Next, this circuitry was tested in vitro.
  • the invention includes methods of designing multi-layered circuits for control of gene expression and genetically engineered bacteria comprising those circuits.
  • the genetically engineered bacterium comprises a first gene encoding a regulatory factor, and a second gene encoding a payload or a gene cassette encoding a biosynthetic pathway for producing a payload, wherein expression of the second gene or gene cassette is directly or indirectly induced by the regulatory factor.
  • the first gene is operably linked to a first regulatory region that is directly or indirectly induced by exogenous environmental conditions or by an exogenous inducer molecule.
  • expression of the regulatory factor must first be induced by a particular setting, stimulus, or circumstance, before the payload that is regulated by said factor can be produced.
  • the regulatory circuit may optionally comprise 3 rd , 4 th , 5 th or ... n th , genes or gene cassettes that directly or indirectly regulate the activity or production of the payload.
  • the payload is a therapeutic payload and may be used to treat, prevent, and/or modulate a disease or disorder.
  • regulatory region refers to a nucleotide sequence that promotes, enhances, inhibits, represses, or otherwise controls the expression of nucleotide sequence(s) to which it is operably linked (i.e., in cis). The control may occur at the level of transcription, translation, or other forms of processing.
  • the regulatory region may be inducible, repressible, and/or controlled by exogenous environmental conditions.
  • a regulatory region may comprise one or more repressor- binding sites, promoters, constitutive promoters, enhancers, and other control regions.
  • a F R-responsive regulatory region may comprise a F R-responsive promoter, i.e., a promoter that is capable of binding the F R transcription factor and inducing expression of an operably linked gene, as well as a ribosomal binding site (RBS).
  • the regulatory region is a promoter.
  • the regulatory region comprises a promoter and a repressor-binding site, wherein repressor binding inhibits promoter activation.
  • operably linked refers a nucleic acid sequence, e.g., a gene encoding a payload, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis.
  • regulatory factor refers to a molecule that directly or indirectly upregulates, downregulates, or otherwise controls the expression of a polynucleotide sequence by its action on a regulatory region.
  • the regulatory factor directly or indirectly induces expression of a payload, as described below.
  • the regulatory factor is capable of interacting with a DNA, RNA, and/or polypeptide sequence to control, directly or indirectly, the expression of gene(s) of interest.
  • the regulatory factor is an inhibiting regulatory factor, i.e., it
  • the regulatory factor is a repressor that is capable of binding to a regulatory region comprising a repressor- binding site and downregulating expression of the gene of interest.
  • the regulatory factor is an activating regulatory factor, i.e., it upregulates expression of a gene of interest.
  • the regulatory factor is a polymerase or transcription factor that is capable of binding to a regulatory region comprising a promoter and upregulating expression of the gene of interest.
  • the regulatory factor is a first repressor that is capable of inhibiting expression of a second repressor that is capable of inhibiting the expression of the gene of interest.
  • Regulatory factors include, but are not limited to, polymerases, proteases, repressors, regulatory RNAs, guide RNAs, and recombinases. Two or more distinct regulatory factors may be used in the regulatory circuits described herein.
  • a "directly inducible" regulatory region refers to a regulatory region operably linked to a gene of interest, e.g., a therapeutic payload; in the presence of an inducer of said regulatory region, the gene of interest is expressed.
  • An "indirectly inducible" regulatory region refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a gene encoding a first molecule, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a gene encoding a second molecule, e.g., an additional transcriptional regulator or a gene encoding a therapeutic payload.
  • the second regulatory region may be activated or repressed, thereby activating or repressing expression of the gene of interest.
  • inducer of the first regulatory region Both a directly inducible and an indirectly inducible regulatory region are encompassed by the phrase "inducible regulatory region.”
  • Transcription factor or “transcriptional regulator” refers to a protein that is capable of recognizing specific DNA binding sites to control the downstream transcription of mRNA. Transcription factors may be capable of interacting with one another and/or with RNA polymerase enzymes to modulate transcription. Transcription factors may be activators that promote transcription or repressors that block
  • transcription factors include, but are not limited to, FNR, ANR, DNR, ArcA/B, ResD/E, NreA/B/C, HypR, NemR, and HypT, and corresponding DNA binding sequences are known in the art and described below (see, e.g., Table 1).
  • the term “treat” and its cognates refer to an amelioration of a disease, or at least one discernible symptom thereof. In another embodiment, “treat” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “treat” refers to inhibiting the progression of a disease, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “treat” refers to slowing the progression or reversing the progression of a disease. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease.
  • Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely
  • payload refers to one or more gene(s) of interest to be expressed by a genetically engineered bacterium comprising a regulatory circuit construct.
  • the payload is a "therapeutic payload,” which refers to a molecule, substance, or drug that is useful for treating or preventing a disease or disorder.
  • the payload is a gene encoding a therapeutic molecule.
  • the payload is a gene cassette encoding a biosynthetic pathway for producing a therapeutic molecule.
  • the payload is a gene encoding a phenylalanine- metabolizing enzyme, and the genetically engineered bacteria are capable of reducing hyperphenylalaninemia.
  • Diseases associated with hyperphenylalaninemia include, but are not limited to, phenylketonuria, classical or typical phenylketonuria, atypical phenylketonuria, permanent mild hyperphenylalaninemia, nonphenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase deficiency, and Segawa's disease.
  • the payload is a gene encoding phenylalanine ammonia lyase (PAL).
  • the payload is a gene encoding phenylalanine hydroxylase (PAH).
  • the payload is a gene encoding an anti- inflammation and/or gut barrier enhancer molecule, or a gene cassette for producing an anti-inflammation and/or gut barrier enhancer molecule, and the genetically engineered bacteria are capable of ameliorating gut inflammation and/or enhancing gut barrier function.
  • Diseases associated with gut inflammation and/or compromised gut barrier function include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases.
  • Inflammatory bowel diseases include, but are not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
  • Diarrheal diseases include, but are not limited to, acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g., dysentery, and persistent diarrhea.
  • Related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis.
  • the payload gene or gene cassette is capable of producing an anti-inflammation and/or gut barrier function enhancer molecule selected from a short-chain fatty acid, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF- ⁇ , TGF-P2, a N-acylphosphatidylethanolamines (NAPE), elafin (also called peptidase inhibitor 3 and SKALP), and trefoil factor.
  • an anti-inflammation and/or gut barrier function enhancer molecule selected from a short-chain fatty acid, butyrate, propionate, acetate, GLP-2, IL-10, IL-27, TGF- ⁇ , TGF-P2, a N-acylphosphatidylethanolamines (NAPE), elafin (also called peptidase inhibitor 3 and SKALP), and trefoil factor.
  • the payload gene or gene cassette is capable of producing a molecule that inhibits a pro-inflammatory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN- ⁇ , IL- ⁇ , IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2.
  • a pro-inflammatory molecule e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN- ⁇ , IL- ⁇ , IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2.
  • a pro-inflammatory molecule e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN
  • the payload is a gene or gene cassette for producing a molecule to treat metabolic disease.
  • Metabolic diseases include, but are not limited to, type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SFM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase
  • BDNF brain-derived neurotrophic factor
  • SFM1 Single-minded 1
  • POMC pro-opiomelanocortin
  • PCSK1 subtilisin/kexin type 1
  • SH2B1 Src homology 2B 1
  • M4R melanocortin-4-receptor
  • WAGR mental retardation
  • the payload gene or gene cassette is capable of producing a molecule selected from a n-acyl-phophatidylethanolamine (NAPE), a n-acyl-ethanolamine (NAE), a ghrelin receptor antagonist, peptide YY3-36, a cholecystokinin (CCK) family molecule, CCK58, CCK33, CCK22, CCK8, a bombesin family molecule, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, a short-chain fatty acid, butyrate, propionate, acetate, a serotonin receptor agonist, nicotinamide adenine dinucleotide (NAD), nicotinamide
  • NAPE
  • the payload gene or gene cassette is capable of producing a molecule that inhibits a metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor.
  • a metabolic disease e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor.
  • the payload is a gene or gene cassette encoding a molecule for reducing hyperammonemia.
  • disorders associated with hyperammonemia include, but are not limited to, a urea cycle disorder (UCD) such as argininosuccinic aciduria, arginase deficiency, carbamylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; hepatic encephalopathy; acute liver failure; chronic liver failure; organic acid disorders; isovaleric aciduria; 3-methylcrotonylglycinuria; methylmalonic acidemia; propionic aciduria; fatty acid oxidation defects; carnitine cycle defects; carnitine deficiency; ⁇ - oxidation deficiency; lysinuric protein intolerance; pyrroline-5-carboxy
  • UCD urea
  • the payload is an arginine feedback resistant N- acetylglutamate synthetase (argA ⁇ ), and the genetically engineered bacteria further lack functional arginine repressor (ArgR).
  • argA ⁇ arginine feedback resistant N- acetylglutamate synthetase
  • ArArgR arginine repressor
  • the payload is an arginine feedback resistant N-acetylglutamate synthetase (argA ⁇ )
  • the genetically engineered bacteria further comprise an arginine regulon, e.g., comprising genes that encode N-acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamate phosphate reductase, acetylornithine aminotransferase, N-acetylornithinase,
  • each arginine operon is mutated in at least one ArgR binding site ("ARG box") in order to reduce ArgR- mediated repression and enhance arginine production, thereby incorporating excess nitrogen and reducing ammonia.
  • ARG box ArgR binding site
  • the payload is a gene or gene cassette encoding a regulatory factor (e.g., a polymerase, a protease, a recombinase, a repressor, a regulatory RNA, a guide RNA, a transcription factor, a FNR protein, an ANR protein, a D R protein) that directly or indirectly upregulates, downregulates, or otherwise controls expression of the payload itself.
  • a regulatory factor e.g., a polymerase, a protease, a recombinase, a repressor, a regulatory RNA, a guide RNA, a transcription factor, a FNR protein, an ANR protein, a D R protein
  • polymerase and its variants refer to a protein or polypeptide that is capable of catalyzing the polymerization of a nucleotide.
  • a gene encoding a polymerase refers to a DNA sequence that encodes an
  • Polymerases include DNA
  • polymerase enzymes are known in the art.
  • the polymerase is a T7 polymerase, a T4 polymerase, T3 polymerase, SP6 polymerase, a bacteriophage polymerase, or a bacterial RNA polymerase.
  • protease and its variants refer to a protein or polypeptide that is capable of cleaving peptide or amide linkages in a polypeptide.
  • a gene encoding a protease refers to a DNA sequence that encodes an
  • proteases include, but are not limited to, prokaryotic, eukaryotic, viral, ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and 26S proteases.
  • the protease is further capable of degrading the polypeptide.
  • E. coli for example, C-terminal fusion of an ssrA tag to a protein directs endogenous ClpXP protease and/or ClpAP protease to degrade said protein.
  • the mf-Lo protease is capable of degrading a protein tagged with mf-ssrA, which is not degraded by E. coli Lon.
  • the protease is an mf-lon protease and is capable of recognizing an mf- lon degradation signal to degrade a polypeptide linked to the degradation signal.
  • Lon protease and “Lon” are used interchangeably to refer to a family of ATP-dependent serine peptidases found in archaea, bacteria, and eukaryotes. Lon proteases are capable of cleaving as well as degrading a polypeptide. Lon contains an "ATPase domain belonging to the AAA+ superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad" (Rotanova et al., 2006). Lon proteases encompass at least two known subfamilies, LonA and LonB.
  • LonA proteases comprise a large N-terminal domain
  • LonB proteases comprise a membrane- spanning domain that anchors to the cytoplasmic side of the membrane.
  • Lon protease encompasses naturally occurring as well as variant and synthetic Lon proteases (see, e.g., Cameron et al., 2014).
  • recombinase and its variants refer to a protein component of a recombination system that mediates DNA rearrangements in a specific DNA locus, including but not limited to site-specific recombinases, integrases, invertases, resolvases, and intron-encoded endonucleases.
  • a recombinase is capable of catalyzing recombination between two complementary recombination sites.
  • recombinases include, but are not limited to, FLP recombinase, Cre recombinase, Hin recombinase, Tre recombinase, recombinase A, Zygosaccharomyces rouxii R recombinase, Kluyveromyces drosophilarium R recombinase, Kluyveromyces waltii recombinase, integrases 1-34 (Intl-Int34), Bxbl, U153, PhiC31, HK022, HP1, R4, CinH, ParA, Tnl721, Tn5053, Tn21, Tn402, and Tn501 (see, e.g., WO2006026537A2; Yang et al., 2014).
  • RNA regulator and “RNA regulator” are used interchangeably to refer to a ribonucleic acid that responds to a signal to control gene expression (see, e.g., Eddy, 1999; Lease et al., 1998; US20070136827A1).
  • a RNA regulator may be capable of controlling post-transcriptional processing, translation, ribozyme activation, inhibitory RNA (RNAi), pH-dependent gene expression, and other changes to nucleic acids, e.g., a conformation change.
  • the RNA regulator is capable of eliminating an mRNA hairpin that blocks translation.
  • 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, and/or a gene.
  • non-native refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
  • the non-native nucleic acid sequence may be present on a plasmid or chromosome.
  • the genetically engineered bacteria of the invention comprise a gene encoding a payload that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature.
  • "Exogenous environmental conditions" refer to settings, stimuli, or circumstances under which the regulatory region described above is directly or indirectly induced.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal.
  • the exogenous environmental condition comprises molecules or metabolites that are specific to the mammalian gut, e.g., propionate produced by commensal bacteria.
  • the exogenous environment condition comprises an exogenous inducer molecule, i.e., an inducer molecule is co-administered with the genetically engineered bacteria in order to activate the regulatory region.
  • the exogenous environmental conditions are low-oxygen, anaerobic, or microaerobic conditions such as the environment of the mammalian gut. Bacteria have evolved transcription factors that are capable of sensing oxygen levels.
  • oxygen level-dependent promoter or “oxygen level- dependent regulatory region” refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.
  • oxygen level-dependent transcription factors include, but are not limited to, F R, ANR, and D R.
  • Corresponding promoters and/or regulatory regions, e.g., FNR-responsive promoters, A R-responsive promoters, and D R- responsive promoters are known in the art (see, e.g., Table 1).
  • Geck refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste.
  • the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine.
  • the gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas.
  • the upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine.
  • the lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal.
  • Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.
  • Non-pathogenic bacteria refer to bacteria that are not capable of causing disease or harmful responses in a host.
  • non-pathogenic bacteria include, but are not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum,
  • Bifidobacterium infantis Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus fiaecium, Escherichia coli, Lactobacillus acidophilus,
  • Lactobacillus bulgaricus Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei n 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.
  • Non-pathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut.
  • Probiotic is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism.
  • the host organism is a mammal.
  • the host organism is a human.
  • Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic.
  • Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum,
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • stable bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a regulatory circuit for expressing a payload, which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and/or propagated.
  • non-native genetic material e.g., a regulatory circuit for expressing a payload
  • the stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • the stable bacterium may be a genetically modified bacterium comprising a gene encoding a therapeutic payload, in which the plasmid or chromosome carrying the gene encoding the therapeutic payload is stably maintained in the host cell, such that the therapeutic payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo.
  • 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.
  • 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.
  • therapeutically effective dose and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition.
  • a therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disease or condition.
  • 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.
  • the genetically engineered bacteria are naturally non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Bacteroides fragilis, Bacteroides
  • Lactococcus lactis Lactococcus lactis.
  • the genetically engineered bacteria are any suitable organisms.
  • Escherichia coli strain Nissle 1917 E. coli Nissle
  • 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 a-hemolysin, P-fimbrial adhesins)" (Schultz, 2008), and E.
  • E. coli Nissle "does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic" (Sonnenborn et 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, arid pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle's "therapeutic efficacy and safety have convincingly been proven" (Ukena et al., 2007).
  • One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria.
  • the genetically engineered bacterium of the invention comprise a regulatory circuit comprising 1) a first gene encoding a regulatory factor, and 2) a second gene encoding a payload or a gene cassette encoding a biosynthetic pathway for producing a payload, wherein expression of the second gene or gene cassette is directly or indirectly induced by the regulatory factor.
  • the first gene is operably linked to a first regulatory region that is directly or indirectly induced by exogenous environmental conditions.
  • expression of the regulatory factor must first be induced by a particular setting, stimulus, or circumstance, before the payload that is regulated by said factor can be produced.
  • the first regulatory region is not associated with the regulatory factor gene in nature.
  • the second gene or gene cassette is operably linked to a second regulatory region that is not associated with the payload gene or gene cassette in nature.
  • the first regulatory region comprises a promoter.
  • the second regulatory region comprises a constitutive promoter.
  • the payload is a therapeutic payload.
  • the payload is a transcriptional regulator.
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by exogenous environmental conditions specific to the mammalian gut. In some embodiments, the regulatory region is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen, anaerobic, or microaerobic conditions. In some embodiments, the regulatory region is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal, e.g., propionate. In some embodiments, the regulatory region is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention.
  • the payload is a gene or gene cassette encoding a regulatory factor, e.g., the regulatory factor that directly or indirectly controls expression of the second gene or gene cassette.
  • the payload is a gene or gene cassette encoding a transcriptional regulator that is induced by exogenous environmental conditions, e.g., the transcriptional regulator that controls the first regulatory region.
  • the payload is a therapeutic gene or gene cassette linked in cis to a gene or gene cassette encoding a transcriptional regulator.
  • the regulatory factor gene or gene cassette is linked in cis to the gene encoding the transcriptional regulator.
  • the first regulatory region comprises a promoter selected from a fumarate and nitrate reductase regulator (F R)-responsive promoter, an anaerobic regulation of arginine deiminiase and nitrate reduction (A R)-responsive promoter, and a dissimilatory nitrate respiration regulator (D R)-responsive promoter, which are capable of being regulated by the transcriptional regulators F R, ANR, or DNR, respectively.
  • F R fumarate and nitrate reductase regulator
  • a R an anaerobic regulation of arginine deiminiase and nitrate reduction
  • D R dissimilatory nitrate respiration regulator
  • the first regulatory region comprises a FNR- responsive promoter.
  • 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.
  • multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria.
  • the first regulatory region comprises an alternate oxygen level-dependent promoter, e.g., a DNR-responsive promoter (Trunk et al., 2010) or an ANR-responsive promoter (Ray et al., 1997).
  • a DNR-responsive promoter Truenk et al., 2010
  • an ANR-responsive promoter Ray et al., 1997.
  • the ANR transcriptional regulator 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).
  • ANR activates numerous genes responsible for adapting to anaerobic growth.
  • ANR In the aerobic state, ANR is inactive. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all have functional analogs of ANR
  • 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).
  • the FNR family also includes DNR (Arai et al., 1995), a transcriptional regulator which is required in conjunction with ANR for "anaerobic nitrate respiration of Pseudomonas aeruginosa" (Hasegawa et al., 1998).
  • DNR Arai et al., 1995
  • ANR A transcriptional regulator which is required in conjunction with ANR for "anaerobic nitrate respiration of Pseudomonas aeruginosa"
  • the FNR- binding motifs "are probably recognized only by DNR" (Hasegawa et al., 1998).
  • FNR-responsive promoters are known in the art ⁇ see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991 ; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used.
  • Non-limiting examples are shown in Table 1 and known in the art ⁇ see, e.g., Fedtke et al., 2002; Gebendorfer et al., 2012; Geng et al, 2004; Morales et al., 2012; Salmon et al., 2005; Umezawa et al., 2008; Verneuil et al., 2005).
  • Table 1 Examples of transcriptional regulators and responsive genes and regulatory regions
  • the genetically engineered bacteria comprise a transcriptional regulator, for example, an oxygen level-dependent transcriptional regulator such as F R, ANR, or D R, and corresponding regulatory region from a different bacterial species.
  • a transcriptional regulator for example, an oxygen level-dependent transcriptional regulator such as F R, ANR, or D R
  • the non-native transcriptional regulator and regulatory region increase the expression of the payload under particular exogenous environmental conditions, e.g., a low-oxygen environment, as compared to the native transcriptional regulator and regulatory region in the bacteria under the same conditions.
  • the non-native transcriptional regulator is a FNR protein from N.
  • the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria.
  • the genetically engineered bacteria comprise a wild-type transcriptional regulator, for example, an oxygen level-dependent
  • the transcriptional regulator such as FNR, ANR, or DNR
  • a transcriptional regulator such as FNR, ANR, or DNR
  • the mutated regulatory region enhances binding to the wild-type transcriptional regulator and increases expression of the payload under particular exogenous environment conditions, e.g., a low-oxygen environment, as compared to the wild-type regulatory region under the same conditions.
  • the genetically engineered bacteria comprise a wild-type regulatory region, e.g., a F R-, A R-, or D R-responsive 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 regulatory region and increases expression of the payload under particular exogenous environmental conditions, e.g., a low-oxygen environment, as compared to the wild- type transcriptional regulator under the same conditions.
  • the mutant transcriptional regulator is a F R protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., 2006).
  • expression of the transcriptional regulator is controlled by a regulatory region comprising a constitutive promoter. In some embodiments, expression of the transcriptional regulator is controlled by an inducible regulatory region. In some embodiments, expression of the transcriptional regulator is controlled by a regulatory region that is directly or indirectly induced by particular exogenous environmental conditions. In some embodiments, the conditions are the same exogenous environmental conditions that induce expression of the payload. In some embodiments, the gene(s) encoding the payload are linked in cis to the gene(s) encoding the transcriptional regulator. In these embodiments, as expression of the payload increases, expression of the transcriptional regulator also increases.
  • the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the transcriptional regulator, e.g., the NR gene.
  • the transcriptional regulator gene is present on a plasmid. In some embodiments, the transcriptional regulator gene is present on a chromosome.
  • the bacteria comprise a regulatory region from a different species. In some embodiments, the bacteria comprise a gene encoding a regulatory factor from a different species. In some embodiments, the first and second genes are operably linked to and controlled by different regulatory regions. In some embodiments, the first and second genes are controlled by the same regulatory region. In some embodiments, the first and second genes are divergently transcribed from a regulatory region. In some embodiments, the genetically engineered bacteria of the invention express a payload under particular exogenous environmental conditions, such as the low-oxygen environment of the mammalian gut, to treat or prevent a disease or disorder. [059] In some embodiments, the first gene encoding the regulatory factor is present on a plasmid.
  • the first gene encoding the regulatory factor is present on a chromosome.
  • the second gene encoding the payload is present on a plasmid.
  • the second gene encoding the payload is present on a chromosome.
  • the first and second genes are present on the same plasmid.
  • the first and second genes are present on different plasmids.
  • the first and second genes are present on the same chromosome.
  • the first and second genes are present on different chromosomes.
  • the transcriptional regulator gene may be present on the same or different plasmid or chromosome as the first gene and/or the second gene.
  • gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the regulatory circuit construct, such that the construct 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.
  • expression from the plasmid may be useful for increasing expression of the gene or gene cassette.
  • expression from the chromosome may be useful for increasing stability of the gene or gene cassette.
  • the regulatory circuit is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid is useful for increasing stability of expression.
  • the regulatory circuit is expressed on a high-copy plasmid.
  • the high-copy plasmid is useful for increasing payload expression.
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a first gene encoding a polymerase regulatory factor operably linked to a first regulatory region, and a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a second regulatory region that is directly or indirectly induced by the polymerase regulatory factor.
  • any suitable polymerase may be used, and such polymerases are known in the art ⁇ see e.g. Glass et al., 1993, McAllister et al., 1993).
  • Examples of polymerases useful in these embodiments include, but are not limited to, prokaryotic polymerases, eukaryotic polymerases, bacteriophage polymerases, and bacterial polymerases.
  • the polymerase is a native bacterial polymerase of the genetically engineered bacteria of the invention.
  • the polymerase is a polymerase from a different species, strain, and/or subtype of bacteria.
  • the genetically engineered bacteria comprise a polymerase from a different species or a different bacterial species.
  • the polymerase is selected from the group consisting of a T7 polymerase, a T4 polymerase, a T3 polymerase, a SP6 polymerase and a bacterial RNA polymerase.
  • the polymerase is a T7 polymerase.
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a F R-, A R-, or D R-responsive promoter. In the presence of oxygen, F R does not dimerize and does not bind the F R-responsive promoter; the polymerase is not expressed, and the payload is not expressed (see Fig. 1). In the absence of oxygen, FNR dimerizes and binds the FNR- responsive promoter, the polymerase is expressed, and the payload is expressed (see Fig. 1).
  • the first regulatory region that controls expression of the polymerase also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the genetically engineered bacteria further comprise a third gene encoding an inhibitory factor that is capable of inhibiting the polymerase, wherein the third gene is operably linked to a third regulatory region.
  • the third regulatory region comprises a constitutive promoter.
  • the polymerase is expressed at sufficient levels when induced to overcome the inhibition by the inhibitor.
  • the third regulatory region comprises a fourth regulatory region, e.g., a FNR binding site, wherein binding of the corresponding transcriptional regulator, e.g., FNR, represses expression of the operably linked third gene encoding the inhibitory factor.
  • the third regulatory region may be upstream of the fourth regulatory region.
  • the third regulatory region may be downstream of the fourth regulatory region.
  • the third regulatory region may overlap with the fourth regulatory region.
  • F R dimerizes and binds to the FNR binding site of the third regulatory region to repress inhibitor expression;
  • FNR also dimerizes and binds the FNR binding site of the first regulatory region to induce polymerase expression.
  • the inhibitory factor is lysY (a variant of T7 lysozyme that lacks amidase activity; New England BioLabs; Zhang et al., 1997) and the polymerase is T7 polymerase (Cheng et al., 1994).
  • the inhibitory factor is an inhibitory RNA aptamer and the polymerase is SP6 polymerase (Mori et al., 2012).
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a first gene encoding a protease regulatory factor operably linked to a first regulatory region; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a second regulatory region that is directly or indirectly induced by the protease; and a third gene encoding a degradation signal linked to a repressor, wherein the repressor is capable of binding to the second regulatory region to repress expression of the second gene or gene cassette.
  • the protease regulatory factor is capable of recognizing the degradation signal and degrading the repressor.
  • any suitable repressor may be used, and such repressors are known in the art.
  • repressors useful in these embodiments include, but are not limited to, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (see, e.g., US20030166191).
  • the repressor is tetR
  • the second regulatory region is a tet regulatory region comprising a tetR- binding sequence.
  • proteases Any suitable protease may be used, and such proteases are known in the art.
  • proteases useful in these embodiments include, but are not limited to, prokaryotic, eukaryotic, viral, ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and 26S proteases.
  • the protease is a native bacterial protease of the genetically engineered bacteria of the invention.
  • the protease is a protease from a different species, strain, and/or subtype of bacteria.
  • the protease is selected from the group consisting of a Ion protease, a Clp protease, a FtsH protease, and a HslU protease (see, e.g., Langer, 2000).
  • the protease is capable of recognizing a corresponding degradation signal ("tag") and degrading a polypeptide linked to the degradation signal.
  • tags useful in these embodiments include, but are not limited to, a ssrA tag, a mecA tag, and a small molecule-responsive tag such as a HaloTag, a LID domain tag (Gottesman et al., 1998, Mei et al., 2009, Neklesa et al., 2011, Bonger et al., 2011).
  • the tag can be a wild- type degradation signal, or a variant of a wild-type degradation signal. Variants and mutants of degradation signals are also known in the art (see, e.g., McGinness et al., 2006).
  • protease-degradation signal pairs useful in these embodiments include, but are not limited to, a ssrA tag or variant and ClpXP protease, a ssrA tag or variant and ClpAP protease, and a ssrA tag or variant and Ion protease (see, e.g., Baker et al., 2006).
  • Other protease-degradation signal pairs are also known in the art. C- terminal fusion of an ssrA tag to a protein, for example, directs endogenous ClpXP protease and/or ClpAP protease to degrade said protein.
  • the protease is an mf-lon protease that is capable of recognizing an mf-lon degradation signal to degrade a polypeptide linked to the degradation signal.
  • the mf- Lon protease is capable of degrading a protein tagged with mf-ssrA, which is not degraded by E. coli Lon (Cameron et al., 2014).
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a FNR-, ANR-, or DNR-responsive promoter. In the presence of oxygen, FNR does not dimerize and does not bind the FNR-responsive promoter; the protease is not expressed, the repressor is not degraded, and the payload is not expressed (see Fig. 2). In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the protease is expressed, the repressor is degraded, and the payload is expressed (see Fig. 2).
  • the first regulatory region that controls expression of the protease also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise at least two repressors in a genetic regulatory circuit.
  • the genetically engineered bacteria comprise a first gene encoding a first repressor operably linked to a first regulatory region; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a second regulatory region; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the second regulatory region and repressing expression of the second gene or gene cassette.
  • the third gene is operably linked to a third regulatory region, wherein the first repressor is capable of binding to the third regulatory region and inhibiting expression of the second repressor.
  • any suitable repressor may be used, and such repressors are known in the art.
  • repressors useful in these embodiments include, but are not limited to, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (see, e.g., US20030166191).
  • the first repressor and the second repressor are distinct repressors, e.g., TetR and LexA.
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a F R-, ANR-, or D R-responsive promoter. In the presence of oxygen, F R does not dimerize and does not bind the FNR-responsive promoter; the first repressor is not expressed, the second repressor is expressed, and the payload is not expressed (see Fig. 3). In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the payload is expressed (see Fig. 3).
  • the first regulatory region that controls expression of the first repressor also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the two-repressor circuit described above may be adapted for other regulatory circuits comprising any suitable number of repressors, e.g., three, four, five, or more repressors, in order to modulate and control expression of the payload gene or gene cassette.
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a first regulatory region, and a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload).
  • the second gene or gene cassette is operably linked to a second regulatory region.
  • the second regulatory region comprises a constitutive promoter.
  • the second gene or gene cassette is further linked to a nucleotide sequence that is capable of inhibiting translation of the payload.
  • the further linked nucleotide sequence produces an mRNA hairpin that inhibits translation of the payload.
  • the further linked nucleotide sequence masks the ribosomal binding site (RBS) and inhibits translation of the payload.
  • a part of the further linked nucleotide sequence may recognize and bind a RBS or downstream of a RBS, and prevent the binding of the ribosome to the RBS.
  • ribosomal binding sites useful in these embodiments include, but are not limited to, a constitutive prokaryotic RBS, a non-constitutive prokaryotic RBS, a yeast RBS, a eukaryotic RBS, a Shine-Dalgarno sequence, and a synthetic RBS (see e.g., Anderson, 2006; Malys, 2011; Kosuri et al., 2013).
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a FNR-, ANR-, or DNR-responsive promoter. In the presence of oxygen, FNR does not dimerize and does not bind the FNR-responsive promoter; the regulatory RNA is not expressed, and the further linked sequence, e.g., the mRNA hairpin, inhibits production of the payload (see Fig. 4).
  • the regulatory RNA comprises a nucleotide sequence that recognizes and binds to the further linked nucleotide sequence.
  • the regulatory RNA may inhibit the mRNA hairpin produced by the further linked nucleotide sequence.
  • the first regulatory region that controls expression of the regulatory RNA also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a Cas protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a first regulatory region; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a second regulatory region; and a third gene encoding a repressor operably linked to a third regulatory region.
  • the repressor is capable of binding to the second regulatory region and repressing expression of the second gene or gene cassette.
  • the third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas protein and inhibits expression of the repressor.
  • the second regulatory region comprises a constitutive promoter.
  • the third regulatory region comprises a constitutive promoter.
  • Any suitable repressor may be used, and such repressors are known in the art. Examples of repressors useful in these embodiments include, but are not limited to, TetR, ArsR, AscG, Lad, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (see, e.g., US20030166191).
  • the repressor is tetR
  • the second regulatory region is a tet regulatory region comprising a tetR-binding sequence.
  • Any suitable Cas protein may be used, and such proteins and corresponding CRISPR systems are known in the art.
  • CRISPR-Cas systems useful in these embodiments include, but are not limited to, a type I CRISPR- associated system, a type II CRISPR-associated system, and a type III CRISPR- associated system (Sorek et al., 2013).
  • Examples of Cas proteins useful in these embodiments include, but are not limited to, Cas3, Cas6, and Cas9 (also see Haft et al., 2005).
  • Cas3 functions as a nuclease and helicase; Cas6 is an endoribonuclease that can recognize and cleave RNA at specific sites; and Cas9 is an endonuclease that can be guided to a specific genomic site by CRISPR and its guide RNA (Huo et al., 2014; Terns et al., 2013).
  • the Cas protein is Cas9.
  • the genetically engineered bacteria comprise a gene encoding a Cas protein from a different species, strain, and/or subtype of bacteria.
  • the gene encoding the Cas protein is expressed on a plasmid. In some embodiments, the gene encoding the Cas protein is expressed on a chromosome. In some embodiments, the gene encoding the Cas protein is a mutant or variant Cas protein that is capable of enhanced cleavage as compared to the unmodified protein under the same conditions.
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a FNR-, ANR-, or DNR-responsive promoter. In the presence of oxygen, FNR does not dimerize and does not bind the FNR-responsive promoter; the guide RNA is not expressed, the repressor is expressed, and the payload is not expressed (see Fig. 5). In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the payload is expressed (see Fig. 5).
  • the first regulatory region that controls expression of the CRISPR guide RNA also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator
  • transcriptional regulator e.g., FNR, that controls the first regulatory region.
  • transcription activator-like effector nucleases TALENs
  • ZFNs zinc-finger nucleases
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a first gene encoding a recombinase operably linked to a first regulatory region, and a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload).
  • the payload gene or gene cassette is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the payload gene or gene cassette by reverting its orientation (5' to 3').
  • the second gene or gene cassette is operably linked to a second regulatory region.
  • the second regulatory region comprises a constitutive promoter.
  • any suitable recombinase may be used, and such recombinases are known in the art (see, e.g., Wang et al., 2011; Yang et al., 2014; WO2006026537A2).
  • the recombinase is a bacterial recombinase or a fungal recombinase.
  • the genetically engineered bacteria comprise a recombinase from a different species or a different bacterial species.
  • the recombinase is a tyrosine recombinase or a serine recombinase. In some embodiments.
  • the recombinase is a genetic recombination enzyme capable of catalyzing DNA exchange reactions, such as excision, insertion, reversion, and/or inversion.
  • recombinases useful in these embodiments include, but are not limited to, site-specific recombinases, integrases, invertases, resolvases, and intron- encoded endonucleases.
  • the recombinase is selected from the group consisting of a FLP recombinase, a Cre recombinase, a Hin recombinase, a Tre recombinase, a recombinase A, a Zygosaccharomyces rouxii R recombinase, a
  • the recombinase is a Cre recombinase, and the recombinase binding sites are loxP sites.
  • the recombinase is a FLP recombinase, and the recombinase binding sites are FRT sites.
  • the recombinase is a Tre recombinase, and the recombinase binding sites are long terminal repeat (LTR) sites.
  • LTR long terminal repeat
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a F R-, ANR-, or D R-responsive promoter. In the presence of oxygen, F R does not dimerize and does not bind the F R-responsive promoter. The recombinase is not expressed, the payload remains in the 3 ' to 5' orientation, and no functional payload is produced (see Fig. 6).
  • the first regulatory region that controls expression of the recombinase also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the transcriptional regulator gene is also inverted in orientation (3' to 5') and flanked by recombinase binding sites, and expression of recombinase induces its 5' to 3' expression, as explained above.
  • the genetically engineered bacteria of the invention are capable of producing a payload, e.g., a therapeutic payload, and comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a first regulatory region; a second gene encoding a payload (or a gene cassette encoding a biosynthetic pathway for producing a payload) operably linked to a second regulatory region; and a third gene encoding a polymerase, wherein the polymerase is capable of binding the second regulatory region and inducing expression of the second gene or gene cassette.
  • the third gene encoding the polymerase is inverted in orientation (3' to 5') and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the polymerase gene by reverting its orientation (5' to 3').
  • the third gene is operably linked to a third regulatory region.
  • the third regulatory region comprises a constitutive promoter.
  • the first regulatory region is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the first regulatory region is directly or indirectly induced by low-oxygen conditions. In some embodiments, the first regulatory region comprises a FNR-, ANR-, or DNR-responsive promoter. In the presence of oxygen, FNR does not dimerize and does not bind the FNR-responsive promoter; the recombinase is not expressed, the polymerase gene remains in the 3' to 5' orientation, and the payload is not expressed (see Fig. 7).
  • the first regulatory region that controls expression of the recombinase also controls expression of the transcriptional regulator, e.g., FNR, that controls said regulatory region.
  • the second regulatory region that controls expression of the payload also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the third regulatory region that controls expression of the polymerase also controls expression of the transcriptional regulator, e.g., FNR, that controls the first regulatory region.
  • the transcriptional regulator gene is also inverted in orientation (3' to 5') and flanked by recombinase binding sites, and expression of recombinase induces its 5' to 3' expression, as explained above.
  • the regulatory circuits disclosed herein may be used to produce two or more distinct payloads.
  • two or more of the regulatory circuits disclosed herein may be used in combination modulate and control the expression of a single payload gene or gene cassette.
  • the genetically engineered bacteria of the invention comprise a regulatory circuit and are capable of producing at least one payload.
  • the payload is a gene encoding a therapeutic molecule, substance, or drug that is useful for treating or preventing a disease or disorder.
  • the payload is a gene cassette encoding a biosynthetic pathway for producing a therapeutic molecule, substance, or drug. Suitable therapeutic payloads and corresponding diseases and disorders are known in the art, and non-limiting examples are discussed in detail below.
  • the payload is a gene or gene cassette encoding a regulatory factor (e.g., a polymerase, a protease, a recombinase, a repressor, a regulatory RNA, a guide RNA, a transcription factor, a FNR protein, an ANR protein, a DNR protein) that directly or indirectly upregulates, downregulates, or otherwise controls expression of the payload itself.
  • a regulatory factor e.g., a polymerase, a protease, a recombinase, a repressor, a regulatory RNA, a guide RNA, a transcription factor, a FNR protein, an ANR protein, a DNR protein
  • the genetically engineered bacteria of the invention comprise a payload gene encoding a phenylalanine-metabolizing enzyme and are capable of reducing hyperphenylalaninemia.
  • Hyperphenylalaninemia a group of diseases associated with excess levels of phenylalanine, can be toxic and cause brain damage.
  • phenylketonuria classical or typical phenylketonuria, atypical phenylketonuria, permanent mild hyperphenylalaninemia, nonphenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase deficiency, and Segawa's disease.
  • Affected individuals can suffer progressive and irreversible neurological deficits, mental retardation, encephalopathy, epilepsy, eczema, reduced growth, microcephaly, tremor, limb spasticity, and/or hypopigmentation (Leonard 2006).
  • Hyperphenylalaninemia can also be secondary to other conditions, e.g., liver diseases.
  • Phenylketonuria is a severe form of hyperphenylalaninemia caused by mutations in the phenylalanine hydroxylase (PAH) gene.
  • Phenylalanine is an essential amino acid primarily found in dietary protein. Typically, a small amount is utilized for protein synthesis, and the remainder is hydroxylated to tyrosine in an enzymatic pathway that requires PAH and the cofactor tetrahydrobiopterin.
  • Primary hyperphenylalaninemia is caused by deficiencies in PAH activity that result from mutations in the PAH gene and/or a block in cofactor metabolism.
  • Current PKU therapies require substantially modified diets consisting of protein restriction.
  • PAL phenylalanine ammonia lyase
  • the payload is a gene that encodes PAH.
  • the bacteria comprise a PAH gene from a different species.
  • the bacteria comprise additional copies of a native PAH gene.
  • the payload is a gene encoding PAL.
  • PAL is encoded by a PAL gene derived from a bacterial species, including but not limited to, Achromobacter xylosoxidans, Pseudomonas aeruginosa, Photorhabdus luminescens, Anabaena variabilis, and Agrobacterium tumefaciens.
  • the bacterial species is Photorhabdus luminescens.
  • the bacterial species is Anabaena variabilis.
  • PAL is encoded by a PAL gene derived from a eukaryotic species, e.g., a yeast species, a plant species. Multiple distinct PAL proteins are known in the art.
  • the genetically engineered bacteria convert more phenylalanine when the PAL gene is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered bacteria expressing the PAL payload may be used to metabolize phenylalanine in the body into non-toxic molecules in order to treat conditions associated with hyperphenylalaninemia, including PKU.
  • the genetically engineered bacteria express Anabaena variabilis PAL ("PALI").
  • the genetically engineered bacteria express Photorhabdus luminescens PAL ("PAL3"). See Example 1.
  • a diagnostic signal of hyperphenylalaninemia in humans is a blood phenylalanine level of at least 2 mg/dL, at least 4 mg/dL, at least 6 mg/dL, at least 8 mg/dL, at least 10 mg/dL, at least 12 mg/dL, at least 14 mg/dL, at least 16 mg/dL, at least 18 mg/dL, at least 20 mg/dL, or at least 25 mg/dL.
  • the genetically engineered bacteria of the invention produce PAL under particular exogenous environmental conditions, such as the low-oxygen environment of the mammalian gut, to reduce blood phenylalanine by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold as compared to unmodified bacteria of the same subtype under the same conditions.
  • exogenous environmental conditions such as the low-oxygen environment of the mammalian gut
  • the payload gene encoding a phenylalanine-metabolizing enzyme may be used in one or more of the regulatory circuits described above, e.g., the polymerase- dependent genetic regulatory circuit, the protease-dependent genetic regulatory circuit, the multi-repressor genetic regulatory circuit, the regulatory RNA genetic regulatory circuit, the CRISPR genetic regulatory circuit, the recombinase-dependent genetic regulatory circuit, or the recombinase- and polymerase-dependent genetic regulatory circuit.
  • the genetically engineered bacteria comprise a regulatory circuit comprising a first gene encoding a polymerase operably linked to a first regulatory region comprising a F R-responsive promoter, and a second gene encoding a PAL payload gene operably linked to a second regulatory region that is directly or indirectly induced by the polymerase; and a third gene encoding an inhibitory factor, e.g., lysY, that is capable of inhibiting the polymerase.
  • F R does not bind the first regulatory region
  • the polymerase is not expressed
  • the PAL payload gene is not expressed.
  • FNR dimerizes and binds to the first regulatory region, the polymerase is expressed at a level sufficient to overcome the action of the inhibitory factor, and the PAL payload gene is expressed to reduce hyperphenylalaninemia.
  • the genetically engineered bacteria are capable of metabolizing phenylalanine in the diet in order to treat a disease associated with hyperphenylalaninemia, e.g., PKU.
  • the genetically engineered bacteria are delivered simultaneously with dietary protein.
  • the genetically engineered bacteria and dietary protein are delivered after a period of fasting or phenylalanine-restricted dieting.
  • a patient suffering from hyperphenylalaninemia may be able to resume a substantially normal diet, or a diet that is less restrictive than a phenylalanine-free diet.
  • the genetically engineered bacteria may be capable of metabolizing phenylalanine from additional sources, e.g., the blood, in order to treat a disease associated with
  • the genetically engineered bacteria need not be delivered simultaneously with dietary protein, and a phenylalanine gradient is generated, e.g., from blood to gut, and the genetically engineered bacteria metabolize phenylalanine and reduce hyperphenylalaninemia.
  • the genetically engineered bacteria of the invention may be evaluated by methods known in the art, e.g., in vivo in an animal model. Any suitable animal model of a disease or condition may be used.
  • the animal model may be a mouse model of PKU ⁇ see, e.g., Sarkissian et al., 1999), e.g., an Enu2 PAH mutant BTBR mouse (BTBR- a/f ⁇ 2 , Jackson Laboratories).
  • the genetically engineered bacteria of the invention is administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by measuring blood phenylalanine and/or cinnamate before and after treatment.
  • the animal may be sacrificed, and tissue samples are collected and analyzed.
  • the genetically engineered bacteria of the invention comprise a payload gene encoding an anti-inflammation and/or gut barrier enhancer molecule, or a gene cassette for producing an anti-inflammation and/or gut barrier enhancer molecule, and are capable of ameliorating gut inflammation and/or enhancing gut barrier function.
  • Diseases associated with gut inflammation and/or compromised gut barrier function include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases.
  • Inflammatory bowel diseases include, but are not limited to, Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease, and indeterminate colitis.
  • Diarrheal diseases include, but are not limited to, acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g., dysentery, and persistent diarrhea.
  • Related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left- sided colitis, pancolitis, and fulminant colitis.
  • IBD Inflammatory bowel disease
  • T cells and activated macrophages are characterized by significant local inflammation in the gastrointestinal tract driven by T cells and activated macrophages, and by compromised function of the epithelial barrier that separates the luminal contents of the gut from the host circulatory system.
  • IBD pathogenesis is linked to both genetic and environmental factors and may be caused by altered interactions between gut microbes and the intestinal immune system.
  • Current approaches to treat IBD are focused on therapeutics that modulate the immune system and suppress inflammation (Cohen et al., 2014).
  • Drawbacks from this approach are associated with systemic immunosuppression, which includes greater susceptibility to infectious disease and cancer.
  • Short-chain fatty acids produced by commensal bacteria are capable of regulating the immune system in the gut (Smith et al., 2013).
  • butyrate plays a direct role in inducing the differentiation of regulatory T cells and suppressing immune responses associated with inflammation in IBD (Atarashi et al., 2011;
  • Butyrate is normally produced by microbial fermentation of dietary fiber and plays a central role in maintaining colonic epithelial cell homeostasis and barrier function (Hamer et al., 2008).
  • One main reason why these engineered microbes have not been successful in treating patients is that their viability and stability are compromised, because they constitutively produce large amounts of foreign proteins.
  • additional therapies to treat diseases and conditions associated with gut inflammation and/or compromised gut barrier function and that avoid undesirable side effects.
  • the payload produced by the genetically engineered bacteria of the invention is an anti-inflammation molecule and/or a gut barrier function enhancer molecule.
  • Anti -inflammation molecules and/or 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- ⁇ , TGF-p2, N- acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), and trefoil factor.
  • Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-a, IFN- ⁇ , IL- ⁇ , IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2.
  • a molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2.
  • a molecule may be both anti-inflammatory and gut barrier function enhancing.
  • An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene.
  • an anti-inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway comprising multiple genes, e.g., butyrate.
  • the genetically engineered bacteria are capable of producing each of the enzymes in said biosynthetic pathway.
  • the payload produced by the genetically engineered bacteria of the invention is a butyrogenic gene cassette.
  • the butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from Peptoclostridium difficile (also called Clostridium difficile): bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransf erase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013).
  • Peptoclostridium difficile strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiAl, hbd, crt2, pbt, and buk.
  • a butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiAl from Peptoclostridium difficile strain 630, and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain 1296.
  • a single gene from Treponema denticola (ter, encoding Zram ⁇ -enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile.
  • a butyrogenic gene cassette may comprise thiAl, hbd, crt2, pbt, and buk irom 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.
  • the payload gene or gene cassette for producing the anti-inflammation and/or gut barrier enhancer molecule may be used in one or more of the regulatory circuits described above, e.g., the polymerase-dependent genetic regulatory circuit, the protease-dependent genetic regulatory circuit, the multi-repressor genetic regulatory circuit, the regulatory RNA genetic regulatory circuit, the CRISPR genetic regulatory circuit, the recombinase-dependent genetic regulatory circuit, or the recombinase- and polymerase-dependent genetic regulatory circuit.
  • the genetically engineered bacteria comprise a regulatory circuit comprising a first gene encoding a polymerase operably linked to a first regulatory region comprising a F R-responsive promoter, and a butyrate gene cassette payload operably linked to a second regulatory region that is directly or indirectly induced by the polymerase; and a third gene encoding an inhibitory factor, e.g., lysY, that is capable of inhibiting the polymerase.
  • F R does not bind the first regulatory region
  • the polymerase is not expressed, and the butyrate gene cassette payload is not expressed.
  • FNR dimerizes and binds to the first regulatory region, the polymerase is expressed at a level sufficient to overcome the action of the inhibitory factor, and the butyrate gene cassette payload is expressed to ameliorate inflammatory bowel disease.
  • the genetically engineered bacteria of the invention may be evaluated by methods known in the art, e.g., in vivo in an animal model. Any suitable animal model of a disease or condition associated with gut inflammation and/or compromised gut barrier function may be used ⁇ see, e.g., Mizoguchi 2012), e.g., a mouse model of IBD. IBD may be induced by treatment with dextran sodium sulfate in the mouse model.
  • the genetically engineered bacteria of the invention may be administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by endoscopy, colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics.
  • the animal is sacrificed, and tissue samples are collected and analyzed.
  • tissue samples are collected and analyzed.
  • colonic sections from the animal are fixed and scored for inflammation and ulceration, and/or homogenized and analyzed for myeloperoxidase activity and cytokine levels (e.g., IL- ⁇ , TNF-a, IL-6, IFN- ⁇ and IL- 10).
  • myeloperoxidase activity and cytokine levels e.g., IL- ⁇ , TNF-a, IL-6, IFN- ⁇ and IL- 10).
  • the genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a therapeutic molecule for treating metabolic disease.
  • Metabolic diseases include, but are not limited to, type 1 diabetes; type 2 diabetes; obesity; metabolic syndrome; Bardet-Biedel syndrome;
  • Prader-Willi syndrome non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency;
  • BDNF brain-derived neurotrophic factor
  • SIM1 Single-minded 1
  • POMC proopiomelanocortin
  • POMC proprotein convertase subtilisin/kexin type 1
  • SH2B1 Src homology 2B 1
  • M4R melanocortin-4-receptor
  • Obesity is caused by an imbalance between energy intake and
  • Type 2 diabetes Patients suffering from obesity are at increased risk of developing adverse physiological conditions, e.g., non-alcoholic fatty liver, cardiovascular diseases, type 2 diabetes.
  • adverse physiological conditions e.g., non-alcoholic fatty liver, cardiovascular diseases, type 2 diabetes.
  • the incidence of type 2 diabetes has increased 300% in the last three decades in the United States.
  • Type 2 diabetics are resistant to the effects of insulin, a hormone that regulates blood glucose levels, and frequently experience hyperglycemia, a condition in which blood glucose is above physiologically tolerable levels.
  • hyperglycemia can result in severe complications such as hypertension, cardiovascular disease, inflammatory disease, blood vessel damage, nerve damage, cancer, and diabetes-induced coma.
  • gut bacteria have demonstrated the close relationship between gut bacteria and metabolic disease (Harley et al., 2012). In obese mice, the ratio of firmicutes to bacteroidetes bacteria is increased (Harley et al., 2012; Mathur et al., 2015). These bacteria extract different amounts of energy from food, which may contribute to changes in energy balance. Similar changes have been also been observed in human studies (Harley et al., 2012; Mathur et al., 2015). Several molecules that are produced by gut bacteria have been shown to be metabolic regulators. For example, gut bacteria digest and break down dietary fiber into molecules such as acetate, butyrate, and propionate.
  • NAPEs N- acylphosphatidylethanolamines
  • the payload gene or gene cassette is capable of producing a molecule selected from a n-acyl-phophatidylethanolamine (NAPE), a n- acyl-ethanolamine (NAE), a ghrelin receptor antagonist, peptide YY3-36, a
  • cholecystokinin (CCK) family molecule CCK58, CCK33, CCK22, CCK8, a bombesin family molecule, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, a short-chain fatty acid, butyrate (described above), propionate (described above), acetate, a serotonin receptor agonist, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA).
  • GRP gastrin releasing peptide
  • P neuromedin B
  • glucagon GLP-1, G
  • the payload gene or gene cassette is capable of producing a molecule that inhibits a metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor.
  • a metabolic disease e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor.
  • the payload expressed by the genetically engineered bacteria of the invention is a propionate gene cassette.
  • the local production of propionate reduces food intake and ameliorates metabolic disease (Lin et al., 2012).
  • 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 propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002).
  • acrylate pathway propionate biosynthesis genes e.g., pet, IcdA, IcdB, IcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA
  • the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrA ⁇ " " , thrB, thrC, ilvA ⁇ ” " , aceE, aceF, and Ipd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively.
  • pyruvate pathway propionate biosynthesis genes see, e.g., Tseng et al., 2012
  • thrA ⁇ " " , thrB, thrC, ilvA ⁇ " " , aceE, aceF, and Ipd which
  • the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase.
  • 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 butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.
  • the propionate biosynthesis genes may be from a different species, strain, or substrain of bacteria.
  • the payload gene or gene cassette for producing the molecule for treating metabolic disease may be used in one or more of the regulatory circuits described above, e.g., the polymerase-dependent genetic regulatory circuit, the protease- dependent genetic regulatory circuit, the multi-repressor genetic regulatory circuit, the regulatory RNA genetic regulatory circuit, the CRISPR genetic regulatory circuit, the recombinase-dependent genetic regulatory circuit, or the recombinase- and polymerase- dependent genetic regulatory circuit.
  • the genetically engineered bacteria comprise a regulatory circuit comprising a first gene encoding a polymerase operably linked to a first regulatory region comprising a F R-responsive promoter, and a propionate gene cassette payload operably linked to a second regulatory region that is directly or indirectly induced by the polymerase; and a third gene encoding an inhibitory factor, e.g., lysY, that is capable of inhibiting the polymerase.
  • F R does not bind the first regulatory region
  • the polymerase is not expressed
  • the propionate gene cassette payload is not expressed.
  • FNR dimerizes and binds to the first regulatory region, the polymerase is expressed at a level sufficient to overcome the action of the inhibitory factor, and the propionate gene cassette payload is expressed to ameliorate metabolic disease.
  • the genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model.
  • Any suitable animal model of a metabolic disease may be used ⁇ see, e.g., Mizoguchi 2012).
  • the animal is a C57BL/6J mouse that is fed a high fat diet in order to induce obesity and type 2 diabetes-related symptoms such as hyperinsulinemia and hyperglycemia.
  • an animal harboring a genetic deficiency that causes a metabolic disease e.g., a
  • the genetically engineered bacteria of the invention comprise a gene or gene cassette encoding molecule for reducing hyperammonemia.
  • Disorders associated with hyperammonemia include, but are not limited to, a urea cycle disorder (UCD) such as argininosuccinic aciduria, arginase deficiency, carbamylphosphate synthetase deficiency, citrullinemia, N-acetylglutamate synthetase deficiency, and ornithine transcarbamylase deficiency; hepatic
  • UCD urea cycle disorder
  • the mitochondrial disorders 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.
  • Prolonged low protein diets can cause muscle breakdown due to the low level of amino acids in blood, and adversely increase ammonia production.
  • Patients taking scavenging drugs may experience severe side effects including nausea, vomiting, irritability, anorexia, and menstrual disturbance in females. When these treatment options fail, a liver transplant may be required (National Urea Cycle Disorders Foundation).
  • National Urea Cycle Disorders Foundation National Urea Cycle Disorders Foundation
  • arginine biosynthesis converts glutamate, which comprises one nitrogen atom, to arginine, which comprises four nitrogen atoms. Enhancement of arginine biosynthesis may be used to incorporate excess nitrogen in the body into non-toxic molecules, i.e., arginine rather than ammonia. Since more than 70% of excess ammonia in hyperammonemic patients accumulates in the gastrointestinal tract, enhancement of arginine biosynthesis in gut bacteria can be a useful method to treat conditions associated with hyperammonemia.
  • arginine biosynthesis converts 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.
  • the additional three steps involve carbamoylphosphate utilization to generate arginine.
  • the first and fifth steps in arginine biosynthesis may be catalyzed by the bifunctional enzyme ornithine acetyltransferase. All of the genes encoding these enzymes are subject to repression by arginine via its interaction with the arginine repressor (ArgR) to form a complex that binds to the regulatory region of each gene and inhibits transcription.
  • ArgR arginine repressor
  • 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).
  • 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 (ArgR), which Maas and Clark (1964) termed a "regulon.” Within each operon, the repressor protein can bind to one or more regulatory regions, called ARG boxes, and repress the expression of the operably linked genes. Any combination of the genes encoding the enzymes responsible for arginine biosynthesis may be organized, naturally or synthetically, into an operon.
  • the payload is a gene encoding an arginine feedback resistant N-acetylglutamate synthetase (argA fbr ), which is less sensitive to arginine-mediated feedback inhibition as compared to wild-type ArgA (see, e.g., Eckhardt et al., 1975; Rajagopal et al., 1998).
  • the genetically engineered bacteria further comprise an arginine biosynthesis regulon comprising one or more nucleic acid mutations in at least one ARG box for each of the operons.
  • the mutation in the one or more ARG boxes results in the reduction or the elimination of ArgR binding to the ARG boxes.
  • the nucleotide sequences of the ARG boxes may vary for each operon, and the consensus ARG box sequence is ⁇ / ⁇ nTGAAT A / T A / T T /A T /A ATTCAn t /A (Maas, 1994).
  • the payload is a gene encoding argA ⁇ r
  • the genetically engineered bacteria further lack functional ArgR.
  • the genetically engineered bacteria comprising the argA fbr gene are capable of producing more arginine and incorporating more excess nitrogen compared to unmodified bacteria under the same condition in order to treat hyperammonemia.
  • the argA ⁇ r payload may be used in one or more of the regulatory circuits described above, e.g., the polymerase-dependent genetic regulatory circuit, the protease-dependent genetic regulatory circuit, the multi-repressor genetic regulatory circuit, the regulatory RNA genetic regulatory circuit, the CRISPR genetic regulatory circuit, the recombinase-dependent genetic regulatory circuit, or the recombinase- and polymerase-dependent genetic regulatory circuit.
  • the regulatory circuits described above e.g., the polymerase-dependent genetic regulatory circuit, the protease-dependent genetic regulatory circuit, the multi-repressor genetic regulatory circuit, the regulatory RNA genetic regulatory circuit, the CRISPR genetic regulatory circuit, the recombinase-dependent genetic regulatory circuit, or the recombinase- and polymerase-dependent genetic regulatory circuit.
  • the genetically engineered bacteria comprise a regulatory circuit comprising a first gene encoding a polymerase operably linked to a first regulatory region comprising a FNR-responsive promoter, and a argA fbr payload operably linked to a second regulatory region that is directly or indirectly induced by the polymerase; and a third gene encoding an inhibitory factor, e.g., lysY, that is capable of inhibiting the polymerase.
  • an inhibitory factor e.g., lysY
  • the genetically engineered bacteria of the invention may be evaluated by methods known in the art, e.g., in vivo in an animal model. Any suitable animal model of a disease or condition associated with hyperammonemia may be used ⁇ see e.g., Nicaise et al., 2008), e.g., a mouse model of acute liver failure and hyperammonemia. Hyperammonemia may be induced by treatment with thiol acetamide in the mouse model.
  • the genetically engineered bacteria of the invention may be administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by measuring blood ammonia levels in mice. Blood ammonia level in mice can be measured by mandibular bleed, and ammonia level can be determined by ammonia analyzer. 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.
  • the genetically engineered bacteria further comprise a deletion or mutation in a gene required for cell survival and/or growth. Any gene required for cell survival and/or growth may be targeted, including but not limited to, dapD, leuB, metB, metC, proAB, thi-1, thr, and thyA, as long as the corresponding wild-type gene product is not produced in the bacteria.
  • thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death.
  • the thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003).
  • the genetically engineered bacterium is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene.
  • a thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo.
  • the genetically engineered bacterium 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.
  • the auxotrophic modification is used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
  • Diaminopimelic acid is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971).
  • the genetically engineered bacterium is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene.
  • a dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies.
  • the auxotrophic modification is used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product. Kill switch
  • the genetically engineered bacteria of the invention further comprise a kill switch.
  • the kill switch is intended to kill the genetically engineered bacteria of the invention in response to external stimuli.
  • the kill switch is triggered by the presence of a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.
  • toxins 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), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive nitrogen 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.
  • transcriptional activation toggle switches
  • riboregulators translation
  • DNA recombination recombinase-based switches
  • 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.
  • One example of an AND riboregulator switch is activated by tetracycline, isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and
  • 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.
  • 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 expression a payload gene or gene cassette.
  • the regulatory regions described herein are operably linked to a detectable product, e.g., GFP, which can be used to screen for mutants.
  • the regulatory region is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces.
  • FACS fluorescence-activated cell sorting
  • one or more transcription factor binding sites or regulatory factor binding sites are mutagenized to increase or decrease binding.
  • the wild-type binding sites are left intact and the remainder of the regulatory region is subjected to mutagenesis.
  • the mutant regulatory region is inserted into the genetically engineered bacteria of the invention to increase production of the payload under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions.
  • the regulatory region and/or transcription factor gene is a synthetic, non- naturally occurring sequence.
  • the payload gene or gene cassette is mutated to increase its expression and/or stability under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions.
  • compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent diseases.
  • Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers are provided.
  • the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to express at least one therapeutic payload. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to express at least one therapeutic payload.
  • compositions of the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use.
  • physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use.
  • 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.
  • the pharmaceutically acceptable carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, or suspensions.
  • 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
  • polyethylene glycol PEG
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.
  • the composition is formulated for oral
  • the invention provides 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.
  • a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc.
  • a single dosage form may be administered over a period of time, e.g., by infusion.
  • Single dosage forms of the pharmaceutical composition of the invention 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.
  • Dosage regimens may be adjusted to provide a therapeutic response. 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.
  • Another aspect of the invention provides methods of treating a disease or disorder.
  • the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders.
  • 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.
  • the genetically engineered bacteria of the invention are administered orally, e.g., in a liquid suspension.
  • the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally.
  • the genetically engineered bacteria of the invention are administered via a feeding tube or gastric shunt.
  • the genetically engineered bacteria of the invention are administered rectally, e.g., by enema.
  • the pharmaceutical composition described herein is administered to treat a subject.
  • the subject is a human subject.
  • the genetically engineered bacteria are 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.
  • the pharmaceutical composition comprising the genetically engineered bacteria may be re-administered at a therapeutically effective dose and frequency.
  • the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.
  • the methods of the invention may comprise administration of a composition of the invention alone or in combination with one or more additional molecules, e.g., a therapeutic molecule, or an inducer that is capable of activating the genetically engineered bacteria of the invention.
  • additional molecules e.g., a therapeutic molecule, or an inducer that is capable of activating the genetically engineered bacteria of the invention.
  • 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 interfere with or kill the bacteria.
  • 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 disease. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.
  • aeruginosa requires a novel CRP/F R-r elated transcriptional regulator, DNR, in addition to A R. FEBS Lett. 1995 Aug 28;371(l):73-6. PubMed PMID: 7664887. Atarashi et al. Induction of colonic regulatory T cells by indigenous Clostridium
  • Staphylococcus carnosus are positively controlled by the novel two-component system NreBC. J Bacterid. 2002 Dec; 184(23):6624-34. PubMed PMID: 12426351. Furusawa et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013; 504:446-450.
  • Mei et al. Molecular determinants of MecA as a degradation tag for the ClpCP protease.
  • Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA.
  • CRISPR-based technologies prokaryotic defense weapons repurposed.
  • N-acyl phosphatidylethanolamines affect the lateral distribution of
  • aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli. Mol Microbiol. 1991 Jun;5(6): 1483-90. PubMed PMID: 1787798.
  • PAL Anabaena variabilis
  • PAL3 Photorhabdus luminescens
  • transcriptional and translational elements are synthesized (Gen9, Cambridge, MA) and cloned into vector pBR322.
  • the PAL payload is placed under control of a T7 polymerase promoter comprising the binding sequence of TAATACGACTCACTATAGGGAGA.
  • a FNR-responsive promoter drives expression of bacteriophage T7 polymerase
  • a lac promoter drives expression of lysY (New England Biolabs C3010I).
  • a FNR binding site is further placed downstream of the lac promoter, wherein FNR binding inhibits lysY expression. Exemplary nucleotide sequences are shown in Table 2.
  • Table 2 Exemplary nucleotide sequences for T7 polymerase-regulated PAL construct
  • RR exemplary regulatory regions
  • Table 3 The nucleotide sequences of exemplary regulatory regions (“RR") comprising a FNR-responsive promoter sequence are shown in Table 3. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning.
  • RR2 is the promoter sequence of nirB
  • RR3 is the promoter sequence oiydfZ.
  • RR4 is the promoter sequence of nirB fused to a strong ribosome binding site
  • RR5 is the promoter sequence oiydfZ fused to a strong ribosome binding site.
  • RR6 is the anaerobically induced small RNA gene, fir S.
  • Table 3 Exemplary nucleotide sequences for FNR-responsive regulatory regions
  • the plasmids of Example 1 are transformed into . coli Nissle.
  • the genetically engineered Nissle comprises the first plasmid (comprising a gene encoding PAL) and the second plasmid (comprising a gene encoding T7 polymerase and a gene encoding its inhibitor, lysY). All tubes, solutions, and cuvettes are pre-chilled to 4° C.
  • An overnight culture of E. coli Nissle is diluted 1 : 100 in 5 mL of lysogeny broth (LB) containing a suitable selection marker, e.g., ampicillin, and grown until it reaches an OD 60 o of 0.4-0.6.
  • LB lysogeny broth
  • the 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 ⁇ g of the plasmid 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 and incubated overnight.
  • the regulatory circuit comprising PALI or PAL3 can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, MA).
  • homologous recombination To create a vector capable of integrating the synthesized construct into the chromosome, Gibson assembly is first used to add lOOObp 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 is used to clone the fragment between these arms.
  • PCR is used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the regulatory circuit construct between them.
  • This PCR fragment is used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells are 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-).
  • Cultures of transformed E. coli Nissle are grown overnight and then diluted 1 : 100 in LB.
  • the cells are grown with shaking at 250 rpm either aerobically or anaerobically in a Coy anaerobic chamber supplied with 90% N 2 , 5% C0 2 , and 5% H 2 .
  • bacteria are pelleted, washed, and resuspended in minimal media, and supplemented with 4 mM phenylalanine. Aliquots are removed at Oh, 2h, and 4h for phenylalanine and cinnimate quantification by mass spectrometry.
  • mice For in vivo studies, the BTBR-Pah enu2 mice are obtained from Jackson Laboratory and bred to homozygosity for use as a model of PKU. Bacteria harboring the PALI or PAL3 payload are grown as described above. Control Nissle bacteria or low oxygen level-induced bacteria are resuspended in phosphate buffered saline and administered by oral gavage.
  • mice are fasted by removing chow overnight (10 hours) and then blood samples are collected by mandibular bleeding the next morning to determine baseline phenylalanine levels. Blood samples are collected in heparinized tubes and spun at 2G for 20 minutes to produce plasma that is removed and stored at -80° C. Mice are given chow again and after 1 hour gavaged with 100 microliters (10 9 CFU) of bacteria that had previously been induced. Mice are put back on chow for 2 hours; plasma samples are prepared as described above. [0149] As noted above, in alternative embodiments, other regulatory regions and corresponding transcriptional regulators may be used, and the PALI and PAL3 payloads may be used with any of the regulatory circuits described herein. In addition, other suitable payloads may be produced by these circuits and methods, as described above.

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Abstract

Des bactéries génétiquement modifiées comprenant des circuits pour la commande multi-couche de l'expression génique sont divulguées. Des méthodes de régulation et d'expression d'un gène de charge utile particulier ou d'une cassette de gènes dans des conditions environnementales exogènes particulières sont également divulguées.
PCT/US2016/039434 2015-06-25 2016-06-24 Commande multicouche de l'expression génique dans des bactéries génétiquement modifiées WO2016210378A2 (fr)

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