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WO2024096123A1 - Micro-organisme génétiquement modifié et son procédé de production - Google Patents

Micro-organisme génétiquement modifié et son procédé de production Download PDF

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WO2024096123A1
WO2024096123A1 PCT/JP2023/039717 JP2023039717W WO2024096123A1 WO 2024096123 A1 WO2024096123 A1 WO 2024096123A1 JP 2023039717 W JP2023039717 W JP 2023039717W WO 2024096123 A1 WO2024096123 A1 WO 2024096123A1
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gene
genes
microorganism
modification
group
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Japanese (ja)
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南部 由美子 西田
大雅 田宮
亮 奥村
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Bio Palette Co Ltd
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Bio Palette Co Ltd
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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

Definitions

  • the present disclosure relates to microorganisms having modifications that do not substantially restore the normal function of the proteins encoded by the genes and/or nucleic acids, and methods for producing such microorganisms.
  • the present disclosure provides: (Item X1) A microorganism, in which at least one gene has been modified such that the modification does not substantially restore a function normally possessed by the protein encoded by said gene.
  • (Item X2) The microorganism described in the above item, wherein the gene is selected from the group consisting of an essential gene, a pharmacological effect-related gene, a morphology-related gene, and a colonization-related gene.
  • the gene is an essential gene and the modification is such that, when an activity normally possessed by a protein encoded by the essential gene is reduced or deleted, the microorganism does not substantially spread in an environment or a subject.
  • the amino acid synthesis enzyme is selected from the group consisting of genes encoding lysine biosynthetic enzymes: dapA, cysK, cysM, pabA, pabB, pabC, glyA, proB, proA, leuB, alr, dadX, serC, argH, hisD, ilvA, leuB, lysA, metB, pheA, proC, thrC, trpC, and tyrA.
  • nucleic acid synthetase is selected from the group consisting of a gene encoding thymidylate synthase (thyA), purK, purN, purT, pyrB, pyrC, pyrD, and pyrE.
  • thyA thymidylate synthase
  • the vitamin synthesis enzyme is selected from the group consisting of thiC, thiE, thiF, thiS, thiG, thiH, ribB, ribC, ribD, nadA, nadB, panC, pdxB, bioH, and metE.
  • the microorganism according to any one of the preceding items, wherein the modification comprises a point mutation in the gene.
  • the modification includes a mutation that generates a stop codon.
  • the modification comprises at least two mutations in the at least one gene.
  • the modification includes at least one mutation in each of at least two types of genes.
  • a method for producing a microorganism having at least one genetic modification comprising the steps of: modifying said at least one gene in said microorganism by converting one or more nucleotides of a target nucleic acid sequence of said gene into another nucleotide or deleting said nucleotide or by inserting one or more nucleotides into said target nucleic acid sequence of said gene; wherein the modification does not substantially restore a function normally possessed by a protein encoded by the gene.
  • the gene is selected from the group consisting of an essential gene, a pharmacological effect-related gene, a morphology-related gene, and a fixation-related gene.
  • amino acid synthesis enzyme is selected from the group consisting of genes encoding lysine biosynthetic enzymes: dapA, cysK, cysM, pabA, pabB, pabC, glyA, proB, proA, leuB, alr, dadX, serC, argH, hisD, ilvA, leuB, lysA, metB, pheA, proC, thrC, trpC, and tyrA.
  • nucleic acid synthetase is selected from the group consisting of a gene encoding thymidylate synthase (thyA), purK, purN, purT, pyrB, pyrC, pyrD, and pyrE.
  • the vitamin synthesis enzyme is selected from the group consisting of thiC, thiE, thiF, thiS, thiG, thiH, ribB, ribC, ribD, nadA, nadB, panC, pdxB, bioH, and metE.
  • the present disclosure makes it possible to modify microorganisms in such a way that their normal functions are not substantially restored, and the modified microorganisms cannot grow unless an external factor that restores their normal functions (which could be, for example, nutrients (essential nutrients) or certain drugs) is provided, making it possible to control and manage the survival and growth of the microorganisms depending on the presence or absence of the external factor.
  • an external factor that restores their normal functions which could be, for example, nutrients (essential nutrients) or certain drugs
  • FIG. 1 is a graph showing the results of non-growth tests of a thyA gene knockout strain, a dapA gene knockout strain, and a double knockout strain of thyA and dapA genes in one embodiment of the present disclosure.
  • microorganisms refers to minute living organisms, including prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, fungi, viruses, and even individual, separate cells of multicellular organisms such as animals and plants. Microorganisms also include natural microorganisms, as well as those cultured and artificially propagated, mutated microorganisms, and microorganisms artificially modified by transformation or other techniques.
  • modification of a gene means that a nucleotide (e.g., dC) on a DNA strand is converted to another nucleotide (e.g., dT, dA, or dG) or deleted, or that a nucleotide or nucleotide sequence is inserted or added between certain nucleotides on a DNA strand.
  • “modification” includes the substitution or deletion of one or more nucleotides at a targeted site of double-stranded DNA, or the insertion or addition of one or more nucleotides at a targeted site of double-stranded DNA.
  • the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA.
  • the "targeted site" of double-stranded DNA means all or a part of the “target nucleotide sequence” that the nucleic acid sequence recognition module specifically recognizes and binds to, or the vicinity of the target nucleotide sequence (either one or both of the 5' upstream and 3' downstream), and the range can be appropriately adjusted between one base and several hundred bases in length depending on the purpose.
  • the term "gene” is interpreted in the broadest sense to mean a character string of nucleic acid or a sequence of a substance that carries it (e.g., nucleotides such as DNA or RNA), and preferably a sequence or a substance that contains a sequence that exerts some function.
  • it also includes adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of the transcript as a transcription factor binding site, and adjacent transcriptional regulatory regions such as promoters and enhancers that control the timing and amount of transcription of the transcript as a transcription factor binding site.
  • essential gene refers to a gene or nucleic acid sequence, or a part thereof, whose functional expression is necessary to maintain viability under the target conditions.
  • essential genes can include genes encoding amino acid synthetases, nucleic acid synthetases, and vitamin synthases.
  • Exemplary essential genes include the gene encoding thymidylate synthase (thyA), 5-(carboxyamino)imidazole ribonucleotide synthase (purK), phosphoribosylglycinamide formyltransferase 1 (purN), phosphoribosylglycinamide formyltransferase 2 (purT), aspartate carbamoyltransferase catalytic subunit (as ...
  • thyA thymidylate synthase
  • purK 5-(carboxyamino)imidazole ribonucleotide synthase
  • purN phosphoribosylglycinamide formyltransferase 1
  • purT phosphoribosylglycinamide formyltransferase 2
  • aspartate carbamoyltransferase catalytic subunit as ...
  • nucleic acid synthesis enzymes such as dihydroorotase (pyrC), dihydroorotate dehydrogenase, type 2 (pyrD), and orotate phosphoribosyltransferase (pyrE), as well as the structural genes for thiamine biosynthesis enzymes (phosphomethylpyrimidine synthase: thiC, thiamine phosphate synthase: thiE, sulfur carrier protein: thiS, adenylyltransferase: thiF, sulfur carrier protein: thiS, 1-deoxy-D-xylulose 5-phosphate:thiol sulfurtransferase:thiG, 2-iminoacetate synthase:thiH), 3,4-dihydroxy-2-butanone-4-phosphatesynthase:ribB, riboflavin synthase:ribC, fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(
  • vitamin synthesis enzymes such as metE, and amino acid synthesis enzymes involved in the synthesis of one or more amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (e.g., genes encoding lysine biosynthetic enzymes (dapA), cysteine synthase (cysteine synthase A:cysK, cysteine synthase B:cysM), aminodeoxychorismate synthase (aminodeoxychorismate synthase subunit 2:pabA, aminodeoxychorismate synthase subunit 3:pabA, aminodeoxychorismate synthase sub
  • the term "medicinal effect-related gene” refers to a gene or nucleic acid sequence, or a part thereof, that can provide some medical or biological benefit to a provided organism (e.g., a human), and examples thereof include Type 1 fimbrin D-mannose specific adhesin (fimH) and Major curlin subunit gene A (csgA).
  • Medicinal effect-related genes include both genes that exert some medicinal effect when knocked out, and genes that exert medicinal effect when expression is improved.
  • Medicinal effect-related genes also include genes or nucleic acid sequences, or parts thereof, that can provide a microorganism or its biological component itself, or a composition containing these, that has functions such as acting on harmful microorganisms in a subject, forming a beneficial microbial flora, maintaining or improving health, and/or activating immunity, such as probiotics, which is a composition containing a biological component (e.g., a whole microorganism or a part thereof).
  • a biological component e.g., a whole microorganism or a part thereof.
  • morphology-related gene refers to a gene or nucleic acid sequence, or a part thereof, that can provide an element (e.g., a protein, a metabolite, etc.) that directly or indirectly determines the morphology of a microorganism, such as bacterial actin-like cytoskeleton protein (cell shape-determining protein: mreB).
  • element e.g., a protein, a metabolite, etc.
  • mreB cell shape-determining protein
  • colonization-related gene refers to a gene or nucleic acid sequence, or a part thereof, that codes for a protein that constitutes a mechanism for adhesion, adhesion, and/or attachment of a microorganism to an organ or cell of a host (e.g., a human), or a gene or nucleic acid sequence, or a part thereof, that codes for a factor that controls the expression of such a gene, such as a DNA-binding transcriptional activator (flhD), a DNA-binding iron uptake regulator (fur), a DNA-binding glucitol operon transcriptional repressor (glucitol operon transcriptional repressor), or a part thereof.
  • flhD DNA-binding transcriptional activator
  • fur DNA-binding iron uptake regulator
  • glucitol operon transcriptional repressor glucitol operon transcriptional repressor
  • sor myristoyl-acyl carrier protein (acp)-dependent acyltransferase (lpxM), DNA-binding transcriptional activator (ntrC), DNA-binding transcriptional repressor (cytR), DNA-binding mal regulon transcriptional repressor (malI), DNA-binding transcriptional dual regulator (DNA -binding transcriptional dual regulator: nagC), DNA-binding transcriptional activator (DNA-binding transcriptional activator: flhC), high-affinity gluconate transporter (High-affinity gluconate transporter: gntT), DNA-binding fructoselysine utilization operon transcriptional repressor (DNA-binding fructoselysine utilization operon transcriptional repressor: frlR), DNA-binding transcriptional repressor (DNA-binding transcriptional repressor: kdgR), DNA Examples of such regulators include DNA-binding transcriptional regulator (oxyR), o
  • function normally possessed by a protein or “activity normally possessed by a protein” refers to a function or activity that a protein can exhibit in an environment in which it normally exists.
  • function normally possessed by a protein is not substantially restored” refers to a situation in which a gene is modified such that the function and/or activity of the protein encoded by the gene is reduced or lost, and the function and/or activity does not subsequently return to the original function and/or activity compared to the protein encoded by the gene that has not been modified.
  • the term “reduced” or “lacking” a protein's function or activity means that, as a result of gene modification, the function and/or activity of the protein encoded by the gene is at least substantially reduced when compared to the function and/or activity of the original protein.
  • the term “reduced” or “lacking” a protein's function or activity includes cases where the function and/or activity is at least about 10% lower, at least about 25% lower, at least about 50% lower, at least about 75% lower, and/or at least about 90% lower than that of the original protein.
  • transmission refers to the movement of the microorganism from a first individual and/or cell to a second individual and/or cell through direct contact or indirectly through air, water, food, drink, objects, etc. Transmission also includes cases involving mobile genetic elements such as plasmids, transposons, and bacteriophages.
  • a microorganism in which at least one gene in the microorganism has been modified such that the function normally possessed by the protein encoded by the gene is not substantially restored.
  • the microorganism of the present disclosure is useful as a technology for preventing the diffusion of a bacterial therapeutic preparation into the environment.
  • the modified microorganism By modifying a microorganism in such a way that the normal function is not substantially restored, the modified microorganism will not have the normal function unless an external factor that restores the normal function (which could be, for example, nutrition (essential nutrients) or a certain type of drug) is provided, so the normal function (which could be, for example, survival, maintenance in a special state, etc.) can be controlled and managed depending on the presence or absence of the external factor, and this can be applied, for example, to environmental control.
  • an external factor that restores the normal function which could be, for example, nutrition (essential nutrients) or a certain type of drug
  • the normal function which could be, for example, survival, maintenance in a special state, etc.
  • auxotroph a microorganism known as an auxotroph. If the auxotrophic microorganism is to survive or grow, the amino acid required for growth must be provided from an external source.
  • the creation of auxotrophic microorganisms is well known, particularly for E. coli.
  • a microorganism when a microorganism is introduced into a target environment, it is desirable to be able to control the introduced microorganism after introduction into the target environment, for example, it is desirable that the introduced microorganism does not spread or spread to environments other than the target environment.
  • Such control can be achieved, for example, by imparting auxotrophy to the microorganism introduced into the target environment.
  • auxotrophy even when auxotrophy is imparted to a microorganism, it is also known that the auxotrophy disappears as the microorganism continues to grow, and this is thought to be due to the occurrence of modifications in the gene or protein modified to impart the auxotrophy that restore the function of the gene or protein.
  • a microorganism in which at least one gene has been modified, and the function normally possessed by the protein encoded by the modified gene is not substantially restored.
  • genes include, but are not limited to, essential genes, drug efficacy-related genes, morphology-related genes, and colonization-related genes.
  • the essential gene may be a gene or nucleic acid sequence, or a portion thereof, that will prevent the microorganism from substantially spreading in the environment or subject if the activity normally possessed by the protein encoded by the essential gene is reduced or eliminated, and examples of the essential gene include, but are not limited to, genes encoding amino acid synthetases, nucleic acid synthetases, and vitamin synthases.
  • the medicinal effect-related gene may be a gene or nucleic acid sequence, or a portion thereof, that can provide some medical or biological benefit to a provided organism (e.g., a human), such as, but not limited to, Type 1 fimbrin D-mannose specific adhesin (fimH) and Majorcurlin subunit gene A (csgA).
  • the medicinal effect-related gene also includes a gene or nucleic acid sequence, or a portion thereof, that can provide a microorganism or its biological component itself, or a composition containing the same, such as a probiotic, that has a function of acting on harmful microorganisms in a subject, forming a beneficial microbial flora, maintaining or improving health, and/or activating immunity.
  • the morphology-related gene may be a gene or nucleic acid sequence, or a part thereof, that can provide an element (e.g., a protein, a metabolite, etc.) that directly or indirectly determines the morphology of a microorganism, such as, but not limited to, a bacterial actin-like cytoskeleton protein (cell shape-determining protein: mreB).
  • an element e.g., a protein, a metabolite, etc.
  • mreB cell shape-determining protein
  • examples of colonization-related genes include genes or nucleic acid sequences, or parts thereof, that code for proteins that constitute mechanisms for adhesion, adhesion, and/or attachment of microorganisms to organs or cells of a host (e.g., human), or genes or nucleic acid sequences, or parts thereof, that code for factors that control the expression of such genes, such as DNA-binding transcriptional activator (flhD), DNA-binding iron uptake regulator (Ferric uptake regulator (fur), DNA-binding glucitol operon transcriptional repressor (g ...
  • ssor srlR
  • myristoyl-acyl carrier protein acp-dependent acyltransferase
  • DNA-binding transcriptional activator ntrC
  • DNA-binding transcriptional repressor cytR
  • DNA-binding mal regulon transcriptional repressor malI
  • DNA-binding transcriptional dual regulator DNA-bin ding transcriptional dual regulator (nagC)
  • DNA-binding transcriptional activator flhC
  • high-affinity gluconate transporter gntT
  • DNA-binding fructoselysine utilization operon transcriptional repressor frlR
  • DNA-binding transcriptional repressor kdgR
  • DNA-binding transcriptional regulator examples include, but are not limited to, DNA-binding transcriptional regulator (oxyR), osmolarity sensor protein (envZ), flagellar filament structural protein (fliC), tagatose 6-phosphate kinase (gatZ
  • the amino acid synthesis enzyme may include an enzyme involved in the synthesis of one or more amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • Exemplary amino acid synthesis enzymes include genes encoding lysine biosynthetic enzymes (dapA), cysteine synthase (cysteine synthase A: cysK, cysteine synthase B: cysM), aminodeoxychorismate synthase (aminodeoxychorismate synthase subunit 2: pabA, aminodeoxychorismate synthase subunit 1: pabB), aminodeoxychorismate lyase (aminodeoxychorismate lyase: pabC), serine hydroxymethyltransferase (serine hydroxymethyltransferase: glyA), and glutamate synthase (glyA).
  • glutamate 5-kinase proB
  • glutamate-5-semialdehyde dehydrogenase proA
  • 3-isopropylmalate dehydrogenase leuB
  • alanine racemase 1 alr, alanine racemase 2 (dadX)
  • phosphoserine/phosphohydroxythreonine aminotransferase serC
  • arginine Argininosuccinate lyase argH
  • histidinol dehydrogenase hisD
  • threonine deaminase ilvA
  • diaminopimelate decarboxylase lysA
  • O-succinylhomoserine(thiol)-lyase metalB
  • fused chorismate mutase/prephenate dehydratase FCH
  • the nucleic acid synthetase may be an enzyme involved in the synthesis of any nucleic acid. In one embodiment of the present disclosure, such a nucleic acid synthetase may be modified to produce a microorganism that is auxotrophic for the nucleic acid that is involved in the synthesis of the nucleic acid synthetase.
  • the nucleic acid synthetase may be, for example, a gene encoding thymidylate synthase (thyA), 5-(carboxyamino)imidazole ribonucleotide synthase (purK), phosphoribosylglycinamide formyltransferase 1 (purN), phosphoribosylglycinamide formyltransferase 2 (purN), or a gene encoding thymidylate synthase (thyA).
  • thyA thymidylate synthase
  • purK 5-(carboxyamino)imidazole ribonucleotide synthase
  • purN phosphoribosylglycinamide formyltransferase 1
  • purN phosphoribosylglycinamide formyltransferase 2
  • thyA a gene encoding thymidylate syntha
  • purT ormyltransferase 2
  • purT aspartate carbamoyltransferase catalytic subunit
  • pyrC dihydroorotase
  • pyrD dihydroorotate dehydrogenase
  • orotate phosphoribosyltransferase pyrE
  • the vitamin synthesis enzyme can be, for example, an enzyme involved in the synthesis of one or more vitamins selected from vitamin A group, vitamin B group, vitamin C group, vitamin D group, vitamin E group, and vitamin K group.
  • a vitamin synthesis enzyme can be modified to produce a microorganism that is auxotrophic for a vitamin whose synthesis is involved in the vitamin synthesis enzyme.
  • the vitamin synthesis enzyme can be, for example, a structural gene for a thiamine biosynthetic enzyme (phosphomethylpyrimidine synthase: thiC, thiamine phosphate synthase: thiE, sulfur carrier protein: thiS, adenylyltransferase: thiF, sulfur carrier protein thiS: thiG, 1-deoxy-D-xylulose 5-phosphate: thi ol sulfurtransferase: thiG, 2-iminoacetate synthase: thiH), 3,4-dihydroxy-2-butanone-4-phosphate synthase (ribB), riboflavin synthase (ribC), diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase (fu sed diaminohydroxyphosphoribosylaminopyrimidine deaminase/5
  • the modification of at least one gene in the microorganism of the present disclosure can be achieved by standard molecular biology techniques.
  • the modification can include a point mutation in the gene, and a method for site-specifically and precisely modifying a target double-stranded polynucleotide can be, for example, a method of contacting a target double-stranded polynucleotide with a Cas protein and a guide RNA, or a method of contacting a target double-stranded polynucleotide with a complex of a Cas protein and a nucleic acid base conversion enzyme and a guide RNA.
  • a complex is formed between the Cas9 protein and the guide RNA and binds to the target double-stranded polynucleotide.
  • the Cas9 protein modifies the base sequence in the target polynucleotide by not cleaving the target double-stranded polynucleotide or by cleaving only one strand, i.e., without causing a double-stranded cleavage.
  • the modification is preferably performed in single-base units.
  • the specific and precise modification of the single base unit is preferably performed using a nucleic acid base conversion enzyme in the complex.
  • the nucleic acid base conversion enzyme include deaminases.
  • deaminases that can be used include cytosine deaminase, cytidine deaminase, adenosine deaminase, and the like.
  • the complex in one embodiment may contain an Indel formation inhibitor such as uracil DNA glycosylase inhibitor (UGI) to inhibit Indel formation.
  • UBI uracil DNA glycosylase inhibitor
  • the specific and precise modification of the single base unit can also be achieved by using a method using a complex of a nucleic acid sequence recognition module and DNA glycosylase.
  • a complex of a nucleic acid sequence recognition module and DNA glycosylase is expressed from an expression vector or RNA molecule introduced into a cell
  • the nucleic acid sequence recognition module specifically recognizes and binds to a target nucleotide sequence in a double-stranded DNA of interest (e.g., genomic DNA)
  • the action of the DNA glycosylase linked to the nucleic acid sequence recognition module causes an abasic reaction in the sense or antisense strand of the targeted site (which can be appropriately adjusted within a range of several hundred bases including all or part of the target nucleotide sequence or their vicinity), resulting in an abasic site (AP site) in one strand of the double-stranded DNA.
  • the base excision repair (BER) system in the cell is activated, and first, an AP endonuclease recognizes the AP site and cuts the phosphate bond of one strand of DNA, and an exonuclease removes the abasic nucleotide. Next, a DNA polymerase inserts a new nucleotide using the opposite strand DNA as a template, and finally, a DNA ligase repairs the splice. When a repair error occurs at any stage of this BER, various mutations are introduced.
  • the CRISPR-Cas system recognizes the sequence of a double-stranded DNA of interest using a guide RNA complementary to the target nucleotide sequence, and therefore any sequence can be targeted simply by synthesizing an oligo-RNA and/or oligo-DNA capable of specifically hybridizing with the target nucleotide sequence. Moreover, at the targeted site, the double-stranded DNA is unwound to generate a single-stranded region and an adjacent region having a loosened double-stranded DNA structure, so that a nucleic acid base conversion enzyme or DNA glycosylase can be efficiently acted on the targeted site specifically without combining a factor that changes the structure of the double-stranded DNA.
  • a CRISPR-Cas system that does not have at least one DNA cleavage ability of Cas (CRISPR-mutant Cas) or a CRISPR-Cas system that does not have both DNA cleavage abilities of Cas (CRISPR-mutant Cas) can be preferably used as the nucleic acid sequence recognition module.
  • the nucleic acid sequence recognition module of the present disclosure using CRISPR-mutant Cas is provided as a complex of an RNA molecule consisting of a guide RNA complementary to a target nucleotide sequence and a tracrRNA required for recruiting the mutant Cas protein, and the mutant Cas protein.
  • the modification can be performed in any environment, in vivo or in vitro. In one embodiment, the modification can also be performed outside the body, i.e., ex vivo or in vitro.
  • the modification of at least one gene in the microorganism of the present disclosure can include a mutation that generates a stop codon using the techniques described above. This can reduce or eliminate the function or activity normally possessed by the protein encoded by the gene.
  • the modification of at least one gene in the microorganism of the present disclosure can include at least two, three, four, five, six, seven, or eight mutations in a gene. By creating at least two mutations in a gene, it is possible to ensure that the function normally possessed by the protein encoded by the gene is not substantially restored.
  • the modification of at least one gene in a microorganism is preferably performed by base editing, preferably single base editing.
  • base editing preferably single base editing.
  • a growth curve is drawn using the turbidity in a liquid medium to which growth essential substances (nucleic acid or amino acid) are not added for an essential gene-deficient E. coli strain produced using recombinant DNA technology, no growth is observed for a certain period of time from the start of culture.
  • growth essential substances nucleic acid or amino acid
  • Sci Transl Med. 2019 Jan 16;11(475):eaau7975 and Nat Biotechnol. 2018 Oct;36(9):857-864 show that the strain does not grow for at least 16 hours.
  • the present inventors have found through the process of this disclosure that when an E. coli strain produced using recombinant DNA technology that lacks a single essential gene is cultured for more than 100 hours, growth is observed.
  • the modification of at least one gene in the microorganism of the present disclosure can include at least one mutation in each of at least two genes, for example, the at least two genes can be independently selected from the group consisting of essential genes, drug efficacy-related genes, morphology-related genes, and colonization-related genes.
  • the modification of at least one gene in the microorganism of the present disclosure can include at least two mutations in each of at least two genes.
  • the present disclosure can, for example and without limitation, target biosynthetic genes for two independent growth-essential substances that are not physiologically complementary to one another, introduce a stop codon into each gene sequence, and create a dual biosynthetic gene knockout edited strain using base editing technology.
  • the present disclosure provides a modified microorganism that simultaneously meets two conditions: that it grows well in vivo, but cannot grow after excretion from the body.
  • the significance of dually deleting the functions of two genes is that even if a double mutant strain in which two stop codons are introduced into one gene is created, if one bypass pathway is complemented or substituted, the effect of imparting non-growth may disappear all at once, and this possibility is prevented as much as possible.
  • the frequency of mutations in microorganisms is about once per 10 6 to 10 7 colony forming units (CFU)
  • the probability of dual reversion mutations in two genes or mutations in alternative genes is theoretically about once per 10 12 to 10 14 CFU.
  • the number of bacteria expected to be administered in one clinical trial is a maximum of 10 9 to 10 10 CFU, it is impossible for dual mutations to occur in two genes at the same time, or the possibility can be reduced to an extremely low level.
  • the essential gene to be modified is a biosynthetic gene or a gene related thereto in the target microorganism, and that the two genes are not functionally complementary. This makes it possible to artificially create a situation in which a dual function-deficient strain of two genes cannot grow and dies in a normal environment, and it is possible to impose a condition in which the growth of the target microorganism is completely dependent on the simultaneous supply of two growth essential substances from the surrounding environment.
  • the place of survival can be limited so that the microorganism can survive only in an environment in which a nucleic acid-related substance derived from the living organism, an essential amino acid-related substance derived from food, or an essential vitamin-related substance is present.
  • the incidence of reversion mutations can be measured by any method.
  • the microorganism of the present disclosure is, for example, Escherichia coli, Lactococcus lactis, Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Members of the phyla Fusobacteria or Verrucomicrobia and the genera Bacteroides, Alistipes, Faecalibacterium, Parabacteroides, Prevotella, Roseburia, Ruminococcus, Clostridium, Oscillibacter, Gemmiger, Barnesiella, Dialister, Parasutterella, Phascolarctosporum, and the like.
  • Bifidobacterium animalis Bifidobacterium breve, Streptococcus cristatus, Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, Staphylococcus aureus, Staphylococcus epidermidis, Porphyromonas gingivalis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium sporogenes, Clostridium cocleatum, Clostridium saccharogumia, Clostridium spiroforme, Clostridium innocuum, Clostridium ramosum, Clostridium hathewayi, Clostridium saccharolyticum, Clostridium Sindens, Clostridium sp.
  • Clostridium bolteae Clostridium indolis, Clostridium lavalense, Clostridium asparagiforme, Clostridium symbiosum, Clostridiales bacterium, Fusobacterium nucleatum, Blautia hydrogenotrophic a, Blautia stercoris, Blautia wexlerae, Blautia producta, Blautia coccoides, Blautia hansenii, Blautia faecis, Blautia glucea, Blautia luti, Blautia schinkii, Megasphaera massiliensis, Megasp haera elsdenii, Megasphaera cerevisiae, Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis, Megasphaera micronuciformis, Megasphaera hexanoica, Eubacterium contortum, Eubacte rium fissicaten
  • the microorganisms of the present disclosure can inhabit the gut, oral cavity, and/or skin of a subject, e.g., of a genus that constitutes at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40% of the total culturable microorganisms in the feces of the subject.
  • the microorganisms in the gut or feces of a subject can be analyzed by any technique known in the art, including 16S ribosomal sequencing.
  • Bacteroides is the most naturally abundant genus in the human gut, and exemplary Bacteroides species include B. acidifaciens, B.
  • amylophilus B. asaccharolyticus, B. spp. ... B. barnesiaes, B. bivius, B. buccae, B. buccalis, B. caccae, B. caecicola, B. caecigallinarum, B. capillosus, B. capillus, B. cellulosilyticus, B. cellulosolvens, B. chinchilla, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. corporis, B. denticola, B. disiens, B. distasonis, B. dorei, B. eggerthii, B.
  • reticulothermitis B. rodentium, B. ruminicola, B. salanitronis, B. salivosus, B. saliersiae, B. B. sartorii, B. sediment, B. splanchnicus, B. stercoris, B. stercoris, B. succinogenes, B. suis, B. tectus, B. termitidis, B. thetaiotaomicron, B. ureformis, B. uniformis, B. ureolyticus, B. veroralis, B.
  • bacteria that may be present include B. vulgatus, B. xylanisolvens, B. xylanolyticus, and B.
  • bacteria that may be present in the oral cavity include Streptococcus gordonii, Streptococcus mutans, and Streptococcus salivarius, and examples of bacteria that may be present in the skin include Staphylococcus aureus and Staphylococcus epidermidis.
  • the microorganisms of the disclosure are capable of stably colonizing the human gut, oral cavity, and/or skin.
  • the microorganisms of the disclosure are capable of colonizing the gut of a subject with, for example, increased abundance, stability, or ease of initial colonization in the gut compared to the same or similar microorganisms that have not been modified.
  • the microorganism of the present disclosure may further include a gene related to a therapeutic agent.
  • a gene related to a therapeutic agent may be one that the microorganism originally has, or a gene that exerts a desired effect may be introduced as is or with a partial modification.
  • the gene related to a therapeutic agent may be a type 1 fimbrin D-mannose specific adhesin (fimH) or the like.
  • the microorganism of the present disclosure may further include a gene related to a diagnostic agent.
  • a gene related to a diagnostic agent may be one that the microorganism originally has, or a gene that exerts a desired effect may be introduced as is or with a partial modification.
  • the gene related to a diagnostic agent may be a bacterial actin-like cytoskeleton protein (cell shape-determining protein (mreB)) or the like.
  • the microorganism of the present disclosure may further include a gene related to a colonization property.
  • a gene related to a colonization property may be one that the microorganism originally has, or a gene that exerts a desired effect may be introduced as is or with a partial modification.
  • the gene associated with fixation can be the DNA-binding transcriptional activator flhD.
  • the effect of a stop codon introduced as a result of single base editing can inhibit functional expression of the target protein.
  • a microorganism contains genes or nucleic acids encoding multiple proteins, it is contemplated that open reading frames encoding two or more of the proteins can be present, for example, in a single operon.
  • the microorganisms of the present disclosure may be utilized as therapeutic preparations, and such therapeutic preparations may, for example, comprise a therapeutically effective amount of the microorganisms of the present disclosure, for example, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, or less by weight of the microorganism.
  • a method for producing a microorganism having at least one modified gene comprising modifying the at least one gene in the microorganism by converting or deleting one or more nucleotides of a target nucleic acid sequence of the gene to another nucleotide, or by inserting one or more nucleotides into the target nucleic acid sequence of the gene, the modification not substantially restoring a function normally possessed by a protein encoded by the gene.
  • the method of the present disclosure may comprise any of the features described elsewhere herein.
  • Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999).
  • gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) can be used, and other references include, for example, Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994).
  • Example 1 Non-proliferative Test Culture medium and culture conditions This example was carried out using LB Broth, Miller (LB liquid medium) (Nacalai Tesque, Inc.). Liquid culture of the knockout strain was carried out using LB liquid medium supplemented with a final concentration of 10 mM thymidine (Tokyo Chemical Industry Co., Ltd.) or 100 ⁇ g/mL 2,6-diaminopimelic acid (Tokyo Chemical Industry Co., Ltd.) at 37°C under shaking conditions at 200 rpm. Static culture was carried out using LB agar medium supplemented with a final concentration of 3 mM thymidine or 100 ⁇ g/mL 2,6-diaminopimelic acid.
  • ATCC American type culture collection
  • genome-edited strains strains were created in which the genes encoding thymidylate synthase (thyA) involved in nucleic acid synthesis and 4-hydroxy-tetrahydrodipicolinate synthase (dapA) involved in essential amino acid synthesis were knocked out by genome editing (Table 1).
  • Non-growth test 0.5 mL of the strain cultured overnight in LB liquid medium containing each additive was inoculated into 20 mL of freshly prepared medium. If the amount of bacteria cultured overnight was insufficient, 1 mL of the overnight cultured bacterial solution was inoculated into 40 mL of freshly prepared medium. The strain was cultured at 37°C with a rotation culture at 200 rpm until the turbidity OD600 of the bacterial solution reached 1.5 or more.
  • the cells were collected by centrifugation (20° C., 3,000 rpm, 15 min), washed with 10 mL of phosphate buffered saline (PBS solution), and then resuspended in 1.5 mL of PBS solution, and the turbidity OD 600 of the cell suspension was measured.
  • PBS solution phosphate buffered saline
  • 1 mL of the collected bacterial liquid was smeared on 10 LB agar plates at 100 ⁇ L each and cultured at 37°C.
  • 1 mL of the collected bacterial liquid of only the dapA knockout strain was diluted 2-fold with PBS solution, and 100 ⁇ L each was smeared on 20 LB agar plates and cultured at 37°C.
  • the remaining liquid of the smeared bacterial solution was diluted 107 times with PBS solution, and 100 ⁇ L of each was smeared on LB agar medium containing each additive. After overnight incubation at 37° C., the total number of bacteria in the smeared bacterial solution was calculated from the number of colonies that appeared.
  • Results Total number of bacteria in the recovered bacterial solution The total number of bacteria of the genome-edited bacteria subjected to the non-growth test is shown in Table 2. Each strain had 1 x 10 10 cfu or more.
  • Non-growth test results The results of confirming the non-growth of the thyA gene knockout strains (BP2037, BP2040, BP2043), dapA gene knockout strains (BP2067, BP2088, BP2239), and thyA and dapA gene double knockout strains (BP2230, BP2233) are shown in Figure 1.
  • the revertant appearance rate was calculated from the number of colonies that appeared on the LB agar medium (Table 3) ( Figure 1).
  • the revertant mutant occurrence rate was calculated from the number of colonies that appeared after 72 hours of culture. As a result, it was proven that the revertant mutant occurrence rate was reduced in the thyA-dapA double knockout strains (BP2230, BP2233) in which a stop codon was replaced at one site each in two genes with different physiological functions, compared to the thyA or dapA knockout strains (BP2037, BP2040, BP2067, BP2088) in which one amino acid was replaced with a stop codon.
  • microorganism disclosed herein can ensure the quality and safety of bacterial preparations and prevent their transmission and/or spread to the environment and humans, and is therefore expected to have a wide range of applications in the medical field.
  • SEQ ID NO:1 Nucleic acid sequence of thyA
  • SEQ ID NO:2 Amino acid sequence of thyA
  • SEQ ID NO:3 Nucleic acid sequence of dapA
  • SEQ ID NO:4 Amino acid sequence of dapA

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Abstract

La présente invention concerne la prévention de la transmission et/ou de la propagation à l'environnement et à des êtres humains lors de l'utilisation d'une préparation bactérienne. Plus spécifiquement, la présente invention concerne un micro-organisme dans lequel au moins un gène du micro-organisme a été modifié et la modification a pour effet que la protéine codée par le gène ne récupère pas sensiblement sa fonction normale.
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