WO2023085429A1 - Transformed cell and method for producing target protein - Google Patents

Transformed cell and method for producing target protein Download PDF

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WO2023085429A1
WO2023085429A1 PCT/JP2022/042284 JP2022042284W WO2023085429A1 WO 2023085429 A1 WO2023085429 A1 WO 2023085429A1 JP 2022042284 W JP2022042284 W JP 2022042284W WO 2023085429 A1 WO2023085429 A1 WO 2023085429A1
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gene
plasmid
gatc
transformed cell
gatb
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卓己 岩崎
愛 伊豫谷
伶 尾▲高▼
涼子 佐野
忍 高木
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合同酒精株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to transformed cells. Furthermore, the present invention also relates to a method for producing a target protein.
  • target gene products are produced by transformed cells obtained using recombinant DNA technology.
  • antibiotics such as tetracycline have been used for selection and maintenance of the transformed cells.
  • Non-Patent Document 1 discloses a chloramphenicol resistance gene in the Gram-positive bacterium Bacillus clausii.
  • Galopin, et al. "A chromosomal chloroamphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii", FEMS Microbiol Lett. , 2009, Jun, 296(2), 185-189
  • An object of the present invention is to provide transformed cells that do not require the addition of drugs such as antibiotics to maintain the transformed cells.
  • the present inventors have found a gene useful for a new method of maintaining transformed cells using host cell growth essential genes.
  • the present inventors have found that transformed cells can be obtained by a simple operation using the gene, and have completed the present invention.
  • a transformed cell that has, on its chromosome, at least the rest of the gatA gene, gatB gene, and gatC gene, which are not present in an expressible state on the plasmid, in an expressible state.
  • the transformed cell may not have one or two of the genes selected from the group in an expressible state on the chromosome.
  • the transformed cell may have the gatA gene, the gatB gene, and the gatC gene on the chromosome in an expressible state.
  • the plasmid does not have the gatA gene and the gatB gene in an expressible state, and The gatA gene, gatB gene, and gatC gene may be present on the chromosome in an expressible state.
  • the transformed cell may be a bacterium belonging to the genus Bacillus.
  • the transformed cell may be E. coli.
  • the plasmid may further contain the base sequence of the gene encoding the target protein.
  • transformed cells are provided that do not require the addition of drugs such as antibiotics for maintenance of the transformed cells.
  • a transformed cell can be obtained by a simple operation to produce a target protein.
  • FIG. 6 is a plasmid map in the process of constructing an E. coli-Bacillus subtilis shuttle vector containing the tetB and gatC genes.
  • FIG. 7 is a photograph showing the state of Bacillus subtilis having a plasmid containing or not containing the gatC gene, seeded on a medium containing or not containing tetracycline, at the end of the culture.
  • the transformed cell according to this embodiment has one or two genes selected from the group consisting of gatA gene, gatB gene, and gatC gene on a plasmid in an expressible state, and gatA gene and gatB gene , and the gatC gene, at least the rest of the genes that are not present on the plasmid in an expressible state are present on the chromosome in an expressible state.
  • Plasmid refers to an extrachromosomal DNA molecule, and a plasmid used for transformation is also particularly referred to as a plasmid vector.
  • a plasmid according to this embodiment may be circular or linear.
  • the plasmid according to this embodiment preferably further contains the tetB gene.
  • the plasmid according to this embodiment preferably has the tetB gene in an expressible state.
  • the nucleotide sequence of the tetB gene may have mutations to the extent that they do not substantially inhibit the function of the gene.
  • the tetB gene is a gene derived from Bacillus subtilis and encodes a drug efflux pump. Cells expressing drug efflux pumps can acquire drug resistance to antibiotics and the like.
  • the tetB gene is a gene derived from Bacillus subtilis, but even when a drug efflux pump encoded by the tetB gene is expressed in a bacterium belonging to a genus different from Bacillus subtilis, for example, in Escherichia coli, the Escherichia coli does not develop drug resistance. can get.
  • the plasmid according to this embodiment preferably does not contain a heterologous antibiotic resistance gene (heterologous gene having antibiotic resistance).
  • heterologous gene refers to a gene isolated from cells other than the genus to which the host cell belongs or a closely related genus with which the host cell can hybridize in nature. That is, a species belonging to the genus to which the host cell belongs and a species belonging to a closely related genus to which the host cell can hybridize in nature are regarded as the same species.
  • the plasmid according to this embodiment may contain the base sequence of the tetB gene, which is a drug resistance gene.
  • the tetB gene in this embodiment is a gene derived from Bacillus subtilis, which is one of bacteria belonging to the genus Bacillus.
  • Bacillus subtilis which is one of bacteria belonging to the genus Bacillus.
  • As a host cell when using a bacterium belonging to the genus Bacillus or a bacterium belonging to a closely related genus (for example, the genus Brevibacillus) with which Bacillus spp. Since it is a gene derived from the same species), it does not impose an environmental load greater than crossing in the natural world.
  • a plasmid containing the nucleotide sequences of the genes in which the gatA gene, gatB gene, gatC gene and tetB gene are combined in the above-described specific combination is produced by a known method. For example, it can be produced by chemical synthesis, genetic engineering techniques, mutagenesis, or the like, using the nucleotide sequence information of the gene. Genetic engineering techniques include, for example, a method of inserting the base sequence into a restriction enzyme-treated template plasmid.
  • the host cell has at least the rest of the gatA gene, gatB gene, and gatC gene, which are not present in an expressible state on the plasmid, in an expressible state on the chromosome. .
  • Table 1 shows host cell patterns when the plasmid has the gatC gene in an expressible state and does not have the gatA and gatB genes in an expressible state.
  • "O” indicates that the gene is present on the chromosome of the host cell in an expressible state
  • X indicates that the gene is not present on the chromosome of the host cell in an expressible state.
  • the host cell according to this embodiment may not have one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene on the chromosome in an expressible state.
  • the expression "gene cannot be expressed (gene is inactivated)" refers to a state in which gene function is lost or reduced.
  • the state in which the function of a gene is lost or decreased specifically means that the expression of mRNA, which is a transcription product of the gene, or a polypeptide, which is a translation product of the gene, is lost or decreased (for example, , mRNA or polypeptide expression level is 50% or less, preferably 30% or less, more preferably 10% or less of the normal), or a state where mRNA or protein does not function normally.
  • Gene inactivation by homologous recombination is carried out, for example, by the recombination of the homologous sequence on the host cell chromosome in the inserted DNA fragment with the homologous sequence. At this time, integration using one homologous region may be performed, or double-crossover type integration using two homologous regions may be performed. Cells in which the target site has been replaced by such a method can be selected using the marker gene added to the introduced DNA fragment as an indicator.
  • a marker gene for example, an upp gene having sensitivity to fluorouracil can be used.
  • the host cell according to the present embodiment is not particularly limited as long as it can stably maintain the plasmid, but is preferably a bacterium, more preferably a bacterium of the genus Bacillus or a bacterium of the genus Brevibacillus, and particularly preferably a bacterium of the genus Bacillus.
  • Bacillus bacteria include Bacillus subtilis, Bacillus liqueniformis, Bacillus amyliquefaciens, Bacillus clausii, and Bacillus megaterium, with Bacillus subtilis being particularly preferred.
  • transformation refers to introducing the above plasmid into a host cell.
  • a known method can be appropriately used, and examples thereof include electroporation method, lipofection method, calcium ion method, competent cell method, protoplast method, and the like.
  • the host cell is a bacterium belonging to the genus Bacillus, the competent cell method or the protoplast method is preferred.
  • the transformed cell according to the present embodiment has one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene in an expressible state on a plasmid, and gatA At least the rest of the genes, the gatB gene, and the gatC gene, which are not present on the plasmid in an expressible state, are present on the chromosome in an expressible state.
  • a transformed cell according to this embodiment may not have one or two genes selected from the above group in an expressible state on the chromosome (for example, pattern 1 in Table 1). At this time, host cells in which one or two genes selected from the group have been inactivated cannot grow as they are, as described above. On the other hand, the host cell transformed with a plasmid containing the gene inactivated in the host cell can be complemented by the plasmid-derived gene product and grow. This allows transformed cells to be selected without traditional selection pressures such as antibiotics or auxotrophy. Furthermore, at this time, if the plasmid is lost from the host cell, the host cell can no longer grow.
  • the plasmid retention rate of the transformed cells according to this embodiment is 100%.
  • the gatA gene, the gatB gene, and the gatC gene only the gatC gene is present on the plasmid in an expressible state, and among the gatA gene, the gatB gene, and the gatC gene, only the gatA gene and the gatB gene are present.
  • Transformed cells that have expression on their chromosomes are particularly preferred. According to this, the plasmid designability is improved, and transformed cells with a plasmid retention rate of 100% can be obtained.
  • the transformed cell according to this embodiment may have the gatA gene, gatB gene, and gatC gene on the chromosome in an expressible state (eg, pattern 2 in Table 1). Unlike the host cells described above in which any of these genes are inactivated, the plasmid is not required for growth. Normally, when such cells are passaged repeatedly, the plasmid is lost from the host cells at a certain rate. Surprisingly, the transformed cells according to this embodiment have a high plasmid retention rate. In one embodiment, plasmid retention may be 90% or greater, preferably 95% or greater. That is, in this case, transformed cells having a high plasmid retention rate can be obtained without requiring inactivation of chromosomal genes.
  • the gatA gene, the gatB gene, and the gatC gene only the gatC gene is present on the plasmid in an expressible state, and the gatA gene, the gatB gene, and the gatC gene are present on the chromosome in an expressible state.
  • Particularly preferred are transformed cells with According to this method, transformed cells with a high plasmid retention rate can be easily obtained without complicated genetic manipulations.
  • selection genes one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene, which the plasmid according to this embodiment has, are referred to as "selection genes".
  • the heterologous drug resistance gene may be used in the intermediate process.
  • the host cell is a bacterium of the genus Bacillus
  • the host cell is transformed with a plasmid containing a heterologous kanamycin resistance gene to obtain a transformed cell, and then the transformed cell is selected by the selection gene.
  • the tetB gene and the gene of interest. Cells having no kanamycin resistance and tetracycline resistance can be selected from the obtained cell group to obtain final transformed cells.
  • the target protein can be produced by culturing the transformed cells obtained by the above method by a known method, and preferably by purifying the culture by a known method. In this production, transformed cells with a high plasmid retention rate can be obtained without relying on conventional selection pressures such as antibiotics and auxotrophy, so production costs can be kept low. Moreover, in one embodiment, the culture obtained by the production method does not contain a heterologous drug resistance gene, so that the environmental load is small.
  • pMDgatC2F was obtained.
  • the Escherichia coli-Bacillus subtilis shuttle vector pHY300PLK (TakaraBio) was digested with restriction enzymes XbaI and HindIII, and the HindIII ends were blunt-ended.
  • a DNA fragment containing the gatC gene obtained by digesting the pMDgatC2F obtained above with the restriction enzymes XbaI and PvuII (blunt ends) was inserted into this shuttle vector, resulting in a shuttle vector containing the gatC gene: pBK-ProgatC1 (Fig. 1). reference) was obtained.
  • Both fragments were prepared at a concentration of 200 fmol/50 ⁇ L, and two-fragment PCR was performed using primers 3 and 6 to ligate both fragments.
  • the obtained DNA fragment was subjected to TA cloning in the same manner as described above to obtain a plasmid for disrupting the upp gene: pMD ⁇ uppKm.
  • Bacillus subtilis strain 168 was transformed using the pMD ⁇ uppKm obtained above. Transformed cells exhibiting kanamycin resistance were selected, and the genome (chromosomal DNA) of the obtained strain was subjected to PCR to confirm that pMD ⁇ uppKm was inserted near the upp gene in the strain genome. The strain was cultured in an inorganic salt medium containing 5-fluorouracil to obtain a growing strain. Among these strains, strains that lost kanamycin resistance and exhibited 5-fluorouracil resistance were selected, and disruption of the upp gene on the chromosome was confirmed by PCR.
  • a DNA fragment containing a kanamycin resistance gene amplified by PCR from pUB110 using primers 13 (SEQ ID NO: 13) and primer 8, and genomic DNA of Bacillus subtilis strain 168 as a template primers 7 and 14 (SEQ ID NO: 14) were used.
  • a DNA fragment containing the uppp gene amplified by PCR was prepared to have a concentration of 100 fmol/50 ⁇ L, respectively, and two-fragment PCR was performed using primer 13 and primer 14 to ligate both fragments.
  • primers 9 and 14 were used to prepare each DNA fragment at a concentration of 100 fmol/50 ⁇ L. Fragment PCR was performed.
  • the DNA fragment of interest was TA cloned in the same manner as above to obtain a plasmid for disrupting the gatC gene: pMD ⁇ gatC101R (see FIG. 2).
  • the pBK-ProgatC1 prepared in 1 above was introduced into the strain using tetracycline resistance as an index.
  • the resulting transformed cells were cultured in a medium containing 5-fluorouracil and tetracycline to select strains that acquired 5-fluorouracil resistance, lost kanamycin resistance, and maintained tetracycline resistance.
  • PCR was performed on the genome of the strain to confirm disruption of the gatC gene on the chromosome.
  • the strain has only the gatC gene among the gatA gene, the gatB gene, and the gatC gene in an expressible state on the plasmid, and is capable of expressing only the gatA gene and the gatB gene on the chromosome. Had in the state.
  • the resulting strain was named 168 ⁇ upp, gatC (pBK-ProgatC1) strain.
  • the comparative strain and the 168 ⁇ upp, gatC (pBK-ProgatC1) strain thus obtained were each streaked on a tetracycline-containing LB agar medium and cultured at 30°C.
  • One colony of each strain was inoculated into 3 mL of tetracycline-free LB liquid medium and cultured overnight at 30°C.
  • the obtained culture solution was spread on a tetracycline-free LB agar medium and cultured at 30°C. After culturing, 100 or more single colonies of each strain were obtained.
  • Fig. 3 shows the appearance of each plate at the end of the culture.
  • 168 ⁇ upp,gatC pBK-ProgatC1
  • the control strain grew 69 colonies on tetracycline-containing medium (69% plasmid retention). From the above, it was confirmed that 168 ⁇ upp,gatC (pBK-ProgatC1) retained the pBK-ProgatC1 plasmid even after subculturing in an antibiotic-free medium.
  • the resulting fragment was subcloned into pMD20 T-Vector in the same manner as above to obtain pMDKmupp1F.
  • the resulting pMDKmupp1F was digested with restriction enzymes EcoRI and PvuII, and the resulting DNA fragment containing the kanamycin resistance gene Kmr was introduced into the EcoRI-HincII site of pHY300PLK ⁇ tetrcII obtained above to obtain pBK-PreKm2.
  • the pMDtetB2-1R obtained above was digested with restriction enzymes HindIII and EcoRI, and the resulting DNA fragment containing the tetB gene was inserted into the HindIII-EcoRI site of pBK-PreKm2.
  • a shuttle vector pBK-PreKmtetB7 (see FIG. 4) containing the tetB gene and the kanamycin resistance gene Kmr was obtained.
  • the pBK-PreKmtetB7 obtained above was introduced into the Bacillus subtilis 168 ⁇ upp strain using kanamycin resistance as an index.
  • Bacillus subtilis strain 168 ⁇ upp was transformed with commercially available pHY300PLK to prepare strain 168 ⁇ upp (pHY300PLK). Each strain was streaked on LB agar medium containing 0 mg/L, 7.5 mg/L, 70 mg/L, and 100 mg/L of tetracycline, and cultured at 30° C. for 36 hours. The results are shown in FIG.
  • the shuttle vector pBK-PreKmtetB7 obtained above the commercially available E. coli JM109 strain was transformed with kanamycin resistance as an indicator.
  • the transformant grew even on an LB agar medium containing 10 mg/L of tetracycline, on which non-transformed E. coli cannot grow.
  • the tetB gene functions as a marker gene not only in Bacillus subtilis but also in E. coli.
  • the DNA fragment of interest was TA cloned in the same manner as above to obtain a plasmid: pMDtetB18F having a fragment for disrupting the tetB gene. Subsequently, a DNA fragment containing a tetB-disrupting fragment obtained by digesting pMDtetB18F with restriction enzymes EcoRI and XbaI; A DNA fragment containing the kanamycin resistance gene Kmr and the upp gene obtained by digesting pMDKmupp1F prepared in 3 with restriction enzymes XbaI and BamHI was introduced into the EcoRI-BamHI site of pHSG298 (TakaraBio) to disrupt the tetB gene. : pHSG ⁇ tetB1 was obtained.
  • pHY300PLK was used as a template for primer 22 (SEQ ID NO: 22) and primer 23 (SEQ ID NO: 23). 23) to obtain a vector fragment obtained by removing the TcR gene from pHY300PLK. PCR was performed using primer 24 (SEQ ID NO: 24) and primer 25 (SEQ ID NO: 25) using genomic DNA of Bacillus subtilis strain 168 as a template to amplify the tetB gene (ACCESSION No. NC 000964: 4187681 to 4189153).

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Abstract

Provided is a transformed cell that does not require addition of a drug such as an antibiotic for maintenance of the transformed cell. One embodiment of the present invention is a transformed cell that has one or two genes selected from the group consisting of a gatA gene, gatB gene, and gatC gene in an expressible state on a plasmid and includes at least the remaining genes among the gatA gene, gatB gene, and gatC gene that are not in an expressible state on the plasmid in an expressible state on a chromosome.

Description

形質転換細胞及び目的タンパク質の生産方法Transformed cell and method for producing target protein
　本発明は、形質転換細胞に関する。さらに、本発明は目的タンパク質の生産方法にも関する。 The present invention relates to transformed cells. Furthermore, the present invention also relates to a method for producing a target protein.
　有用な遺伝子産物を取得するために、組み換えＤＮＡ技術を利用して得られた形質転換細胞による目的遺伝子産物の生産が行われている。従来、当該形質転換細胞の選抜及び維持にはテトラサイクリン等の抗生物質が利用されてきた。 In order to obtain useful gene products, target gene products are produced by transformed cells obtained using recombinant DNA technology. Conventionally, antibiotics such as tetracycline have been used for selection and maintenance of the transformed cells.
　しかしながら、抗生物質を用いた生産はコストがかかり、且つ一部の抗生物質には免疫原性があるため除去工程を要する。さらに、用いられる抗生物質耐性遺伝子は宿主と異なる生物に由来するものが多く、環境中への拡散の観点から、異種由来抗生物質耐性遺伝子の製品中での残存が問題となっている。この問題に対し、宿主由来の抗生物質耐性遺伝子を利用した形質転換細胞の選抜及び維持方法が模索されている。非特許文献１には、グラム陽性菌Ｂａｃｉｌｌｕｓ　ｃｌａｕｓｉｉにおける、クロラムフェニコール耐性遺伝子が開示されている。 However, production using antibiotics is costly, and some antibiotics are immunogenic and require a removal process. Furthermore, many of the antibiotic resistance genes used are derived from organisms different from the host, and from the viewpoint of diffusion into the environment, the survival of heterologous antibiotic resistance genes in products is a problem. To address this problem, methods for selecting and maintaining transformed cells using host-derived antibiotic resistance genes have been sought. Non-Patent Document 1 discloses a chloramphenicol resistance gene in the Gram-positive bacterium Bacillus clausii.
　従来の形質転換細胞の維持方法は、抗生物質の添加を必要とするため、生産コストが高く、さらに抗生物質の除去工程を要するという問題があった。本発明は、形質転換細胞の維持に抗生物質等の薬剤添加を必須としない形質転換細胞を提供することを目的とする。 　Conventional methods for maintaining transformed cells required the addition of antibiotics, which posed the problem of high production costs and the need for an antibiotic removal process. An object of the present invention is to provide transformed cells that do not require the addition of drugs such as antibiotics to maintain the transformed cells.
　本発明者らは、宿主細胞の生育必須遺伝子を利用した、新たな形質転換細胞の維持方法に有用な遺伝子を見出した。また、本発明者らは、当該遺伝子を使用すれば、簡便な操作で形質転換細胞を取得できることを見出し、本発明を完成させた。 The present inventors have found a gene useful for a new method of maintaining transformed cells using host cell growth essential genes. In addition, the present inventors have found that transformed cells can be obtained by a simple operation using the gene, and have completed the present invention.
　すなわち、本発明の第一の態様によれば、
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子をプラスミド上に発現可能な状態で有し、且つ、
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、少なくとも、前記プラスミド上に発現可能な状態で有さない残余の遺伝子を、染色体上に発現可能な状態で有する、形質転換細胞が提供される。 That is, according to the first aspect of the present invention,
Having one or two genes selected from the group consisting of gatA gene, gatB gene, and gatC gene on a plasmid in an expressible state, and
A transformed cell is provided that has, on its chromosome, at least the rest of the gatA gene, gatB gene, and gatC gene, which are not present in an expressible state on the plasmid, in an expressible state.
　前記第一の態様において、前記形質転換細胞は、前記群より選択される一又は二の前記遺伝子を、前記染色体上に発現可能な状態で有さなくてもよい。 In the first aspect, the transformed cell may not have one or two of the genes selected from the group in an expressible state on the chromosome.
　前記第一の態様において、前記形質転換細胞は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を前記染色体上に発現可能な状態で有してもよい。 In the first aspect, the transformed cell may have the gatA gene, the gatB gene, and the gatC gene on the chromosome in an expressible state.
　前記第一の態様において、前記形質転換細胞は、ｇａｔＣ遺伝子を前記プラスミド上に発現可能な状態で有してもよい。 In the first aspect, the transformed cell may have the gatC gene on the plasmid in an expressible state.
　前記第一の態様において、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を前記プラスミド上に発現可能な状態で有さず、且つ、
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子のみを前記染色体上に発現可能な状態で有してもよい。 In the first aspect, the plasmid does not have the gatA gene and the gatB gene in an expressible state, and
Of the gatA gene, the gatB gene, and the gatC gene, only the gatA gene and the gatB gene may be present on the chromosome in an expressible state.
　前記第一の態様において、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を前記プラスミド上に発現可能な状態で有さず、且つ、
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を前記染色体上に発現可能な状態で有してもよい。 In the first aspect, the plasmid does not have the gatA gene and the gatB gene in an expressible state, and
The gatA gene, gatB gene, and gatC gene may be present on the chromosome in an expressible state.
　前記第一の態様において、前記形質転換細胞は、ｔｅｔＢ遺伝子を前記プラスミド上に発現可能な状態で有してもよい。 In the first aspect, the transformed cell may have the tetB gene on the plasmid in an expressible state.
　前記第一の態様において、前記形質転換細胞は、バチルス属に属する細菌であってもよい。 In the first aspect, the transformed cell may be a bacterium belonging to the genus Bacillus.
　前記第一の態様において、前記形質転換細胞は、枯草菌であってもよい。 In the first aspect, the transformed cell may be Bacillus subtilis.
　前記第一の態様において、前記形質転換細胞は、大腸菌であってもよい。 In the first aspect, the transformed cell may be E. coli.
　前記第一の態様において、前記プラスミドが、目的タンパク質をコードする遺伝子の塩基配列をさらに含んでもよい。 In the first aspect, the plasmid may further contain the base sequence of the gene encoding the target protein.
　前記第一の態様において、前記形質転換細胞を用いて目的タンパク質を生産する方法であってもよい。 In the first aspect, the method may be a method of producing the target protein using the transformed cell.
　本発明によれば、形質転換細胞の維持に抗生物質等の薬剤添加を必須としない、形質転換細胞が提供される。また、本発明によれば、簡便な操作で形質転換細胞を取得し、目的タンパク質を生産できる。 According to the present invention, transformed cells are provided that do not require the addition of drugs such as antibiotics for maintenance of the transformed cells. Moreover, according to the present invention, a transformed cell can be obtained by a simple operation to produce a target protein.
図１は、ｇａｔＣ遺伝子を含む大腸菌－枯草菌のシャトルベクター構築過程におけるプラスミドマップである。FIG. 1 is a plasmid map in the process of constructing an E. coli-Bacillus subtilis shuttle vector containing the gatC gene. 図２は、ｇａｔＣ遺伝子破壊用プラスミドの構築方法を示す図である。FIG. 2 shows a method for constructing a plasmid for disrupting the gatC gene. 図３は、テトラサイクリン含有又は不含培地に播種した枯草菌の培養終了時の様子を示す写真である。FIG. 3 is a photograph showing the appearance of Bacillus subtilis seeded on a medium containing or not containing tetracycline at the end of culture. 図４は、ｔｅｔＢ遺伝子を含む大腸菌－枯草菌のシャトルベクター構築過程におけるプラスミドマップである。FIG. 4 is a plasmid map in the process of constructing an E. coli-Bacillus subtilis shuttle vector containing the tetB gene. 図５は、所定濃度のテトラサイクリン含有培地に播種した枯草菌の培養終了時の様子を示す写真である。FIG. 5 is a photograph showing the state of Bacillus subtilis seeded in a medium containing tetracycline at a predetermined concentration at the end of culturing. 図６は、ｔｅｔＢ遺伝子及びｇａｔＣ遺伝子を含む大腸菌－枯草菌のシャトルベクター構築過程におけるプラスミドマップである。FIG. 6 is a plasmid map in the process of constructing an E. coli-Bacillus subtilis shuttle vector containing the tetB and gatC genes. 図７は、テトラサイクリン含有又は不含培地に播種した、ｇａｔＣ遺伝子を含む、又は含まないプラスミドを有する枯草菌の培養終了時の様子を示す写真である。FIG. 7 is a photograph showing the state of Bacillus subtilis having a plasmid containing or not containing the gatC gene, seeded on a medium containing or not containing tetracycline, at the end of the culture.
　以下、本発明について詳述する。 The present invention will be described in detail below.
　「宿主細胞」とは、プラスミドが導入され形質転換される細胞を意味し、形質転換前後を問わない。宿主細胞を単に「宿主」と称することもある。形質転換後の細胞を特に、「形質転換細胞」と称する。 "Host cell" means a cell into which a plasmid is introduced and transformed, regardless of whether it is before or after transformation. A host cell is sometimes simply referred to as a "host". Cells after transformation are specifically referred to as "transformed cells".
　「形質転換細胞を選抜する」とは、プラスミドによって形質転換された形質転換細胞及び形質転換されていない非形質転換細胞を含む群から、形質転換細胞を選択的に取得することを意味する。
　「形質転換細胞を維持する」とは、プラスミドによって形質転換された形質転換細胞において、当該プラスミドを細胞内に保持することを意味する。 "Selecting transformed cells" means selectively obtaining transformed cells from a group comprising transformed cells that have been transformed with a plasmid and non-transformed cells that have not been transformed.
"Maintaining a transformed cell" means retaining a plasmid in a transformed cell transformed with the plasmid.
　本実施形態に係る形質転換細胞は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子をプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、少なくとも、前記プラスミド上に発現可能な状態で有さない残余の遺伝子を、染色体上に発現可能な状態で有する。まず、本実施形態に係るプラスミド及び宿主細胞について説明し、次いで本実施形態に係る形質転換細胞について説明する。 The transformed cell according to this embodiment has one or two genes selected from the group consisting of gatA gene, gatB gene, and gatC gene on a plasmid in an expressible state, and gatA gene and gatB gene , and the gatC gene, at least the rest of the genes that are not present on the plasmid in an expressible state are present on the chromosome in an expressible state. First, the plasmid and host cell according to this embodiment will be described, and then the transformed cell according to this embodiment will be described.
（プラスミド）
　本実施形態に係るプラスミドは、染色体外に存在するＤＮＡ分子を指し、形質転換に用いられるプラスミドは特にプラスミドベクターともいう。本実施形態に係るプラスミドは環状又は直鎖状であり得る。 (plasmid)
A plasmid according to the present embodiment refers to an extrachromosomal DNA molecule, and a plasmid used for transformation is also particularly referred to as a plasmid vector. A plasmid according to this embodiment may be circular or linear.
　本実施形態に係るプラスミドは、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子を含む。具体的には、本実施形態に係るプラスミドは、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群から選択される一又は二の遺伝子を発現可能な状態で有する。「遺伝子が発現可能な状態」とは、前記遺伝子が転写され、前記遺伝子にコードされたタンパク質が発現可能な状態であることを意味する。これらの遺伝子は、プロモーター領域が明確なため、安定的な遺伝子発現を実現できる。なお、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子の塩基配列は、変異を有していてもよい。ここで変異とは、天然に存在する変異及び天然に存在しない改変による変異を含み、コードする遺伝子の機能を実質的に阻害しない変異を意味する。 The plasmid according to this embodiment contains one or two genes selected from the group consisting of gatA gene, gatB gene and gatC gene. Specifically, the plasmid according to this embodiment has one or two genes selected from the group consisting of gatA gene, gatB gene, and gatC gene in an expressible state. The expression "gene can be expressed" means that the gene is transcribed and the protein encoded by the gene can be expressed. Since these genes have well-defined promoter regions, stable gene expression can be achieved. The nucleotide sequences of the gatA gene, gatB gene, and gatC gene may have mutations. The term "mutation" as used herein means a mutation that does not substantially inhibit the function of the encoding gene, including naturally occurring mutations and non-naturally occurring mutations due to modifications.
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子は、それぞれグルタミンｔＲＮＡ合成酵素をコードする遺伝子である。グルタミンｔＲＮＡ合成酵素は３つのサブユニットからなり、これらが協働して細胞のタンパク質合成に寄与する。グルタミンｔＲＮＡ合成酵素は細胞の生育に必須の遺伝子（生育必須遺伝子ともいう）である。ｇａｔＣ遺伝子はｇａｔＡ遺伝子及びｇａｔＢ遺伝子に比べて遺伝子配列が短いため、プラスミドの設計性に優れる。このため、前記群より選択される一又は二の遺伝子は特に限定されないが、前記群より選択される一の遺伝子がｇａｔＣ遺伝子であること、又は前記群より選択される二の遺伝子のうち、いずれか一方がｇａｔＣ遺伝子であることが好ましい。 The gatA gene, gatB gene, and gatC gene are genes that encode glutamine tRNA synthetase, respectively. Glutamine tRNA synthetase consists of three subunits that cooperate to contribute to cellular protein synthesis. Glutamine-tRNA synthetase is a gene essential for cell growth (also called growth-essential gene). Since the gatC gene has a shorter gene sequence than the gatA gene and the gatB gene, it is superior in plasmid designability. Therefore, one or two genes selected from the group are not particularly limited, but one gene selected from the group is the gatC gene, or two genes selected from the group. Preferably, one of them is the gatC gene.
　本実施形態に係るプラスミドは、さらにｔｅｔＢ遺伝子を含むことが好ましい。具体的には、本実施形態に係るプラスミドは、ｔｅｔＢ遺伝子を発現可能な状態で有することが好ましい。ここで、ｔｅｔＢ遺伝子の塩基配列は、遺伝子の機能を実質的に阻害しない範囲で変異を有していてもよい。 The plasmid according to this embodiment preferably further contains the tetB gene. Specifically, the plasmid according to this embodiment preferably has the tetB gene in an expressible state. Here, the nucleotide sequence of the tetB gene may have mutations to the extent that they do not substantially inhibit the function of the gene.
　ｔｅｔＢ遺伝子は、枯草菌由来の遺伝子であり、薬剤排出ポンプをコードしている。薬剤排出ポンプが発現した細胞は、抗生物質等に対する薬剤耐性を獲得し得る。ｔｅｔＢ遺伝子は枯草菌由来の遺伝子であるが、枯草菌とは異なる属に属する菌、例えば、大腸菌でｔｅｔＢ遺伝子がコードする薬剤排出ポンプを発現させた場合であっても、当該大腸菌は薬剤耐性を獲得し得る。 The tetB gene is a gene derived from Bacillus subtilis and encodes a drug efflux pump. Cells expressing drug efflux pumps can acquire drug resistance to antibiotics and the like. The tetB gene is a gene derived from Bacillus subtilis, but even when a drug efflux pump encoded by the tetB gene is expressed in a bacterium belonging to a genus different from Bacillus subtilis, for example, in Escherichia coli, the Escherichia coli does not develop drug resistance. can get.
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、ｇａｔＣ遺伝子、及びｔｅｔＢ遺伝子の遺伝子断片は、それぞれ、公知の方法により得ることができる。具体的には、染色体ＤＮＡを含む抽出液を用いて、ＰＣＲ反応により当該遺伝子部分を含む断片を増幅することができる。ここで、ＰＣＲ反応に用いるプライマーは、公知の全ゲノム配列を利用して、公知の方法により設計することができる。 Gene fragments of the gatA gene, gatB gene, gatC gene, and tetB gene can be obtained by known methods. Specifically, an extract containing chromosomal DNA can be used to amplify a fragment containing the gene portion by PCR reaction. Here, the primers used in the PCR reaction can be designed by a known method using a known whole genome sequence.
　本実施形態に係るプラスミドは、前記遺伝子が発現可能であれば、他の任意の配列を含んでもよい。含み得る他の配列としては、例えば、プロモーター配列、エンハンサー配列、ターミネーター配列、開始コドン、終始コドン、制限酵素認識部位などが挙げられる。 The plasmid according to this embodiment may contain any other sequence as long as the gene can be expressed. Other sequences that may be included include, for example, promoter sequences, enhancer sequences, terminator sequences, initiation codons, termination codons, restriction enzyme recognition sites, and the like.
　プロモーター及びターミネーターは特に限定されない。プロモーターとして、例えば、前記遺伝子由来のプロモーター、前記遺伝子由来以外のプロモーター、又はこれらのプロモーターの変異体を用いることで、目的遺伝子の発現量を調節することができる。 The promoter and terminator are not particularly limited. By using, for example, a promoter derived from the gene, a promoter not derived from the gene, or a mutant of these promoters, the expression level of the target gene can be regulated.
　本実施形態に係るプラスミドは、目的タンパク質をコードする遺伝子の塩基配列を含んでもよい。目的タンパク質をコードする遺伝子の塩基配列としては、宿主由来に限られず、あらゆる生物由来の任意の遺伝子の塩基配列を利用することができる。目的タンパク質としては、例えば、工業又は医療に利用できるタンパク質が挙げられ、より具体的には、酵素、受容体、ホルモン、サイトカイン、膜タンパク質、抗体、抗原、その他の生体因子等がある。これらのタンパク質は、例えば、ワクチン等の医薬品等に使用できる。本実施形態に係るｇａｔＣ遺伝子は、プラスミドの設計性の向上に寄与するため、ｇａｔＣ遺伝子を含むプラスミドの場合、多様な塩基配列を採用することができる。 The plasmid according to this embodiment may contain the base sequence of the gene encoding the target protein. The nucleotide sequence of the gene encoding the target protein is not limited to that derived from the host, and any nucleotide sequence of any gene derived from any organism can be used. Target proteins include, for example, proteins that can be used industrially or medically, and more specifically include enzymes, receptors, hormones, cytokines, membrane proteins, antibodies, antigens, and other biological factors. These proteins can be used, for example, in medicines such as vaccines. Since the gatC gene according to the present embodiment contributes to the improvement of plasmid designability, various base sequences can be employed for plasmids containing the gatC gene.
　ただし、本実施形態に係るプラスミドは、異種由来抗生物質耐性遺伝子（抗生物質耐性を有する異種由来遺伝子）を含まないことが好ましい。ここで、本明細書において「異種由来遺伝子」とは、宿主細胞の属する属又は宿主細胞が自然界で交雑可能な近縁属以外の細胞から分離された遺伝子を指す。すなわち、宿主細胞の属する属に属する種、及び宿主細胞が自然界で交雑可能な近縁属に属する種は同種とみなす。 However, the plasmid according to this embodiment preferably does not contain a heterologous antibiotic resistance gene (heterologous gene having antibiotic resistance). As used herein, the term "heterologous gene" refers to a gene isolated from cells other than the genus to which the host cell belongs or a closely related genus with which the host cell can hybridize in nature. That is, a species belonging to the genus to which the host cell belongs and a species belonging to a closely related genus to which the host cell can hybridize in nature are regarded as the same species.
　本実施形態に係るプラスミドは、薬剤耐性遺伝子であるｔｅｔＢ遺伝子の塩基配列を含み得る。本実施形態におけるｔｅｔＢ遺伝子は、バチルス属細菌の一つである枯草菌由来の遺伝子である。宿主細胞として、バチルス属細菌又はバチルス属細菌が自然界で交雑可能な近縁属（例えば、ブレビバチルス属）に属する細菌を使用した場合、ｔｅｔＢ遺伝子は同種由来抗生物質耐性遺伝子（抗生物質耐性を有する同種由来遺伝子）であるため、自然界での交雑以上の環境負荷を与えることはない。 The plasmid according to this embodiment may contain the base sequence of the tetB gene, which is a drug resistance gene. The tetB gene in this embodiment is a gene derived from Bacillus subtilis, which is one of bacteria belonging to the genus Bacillus. As a host cell, when using a bacterium belonging to the genus Bacillus or a bacterium belonging to a closely related genus (for example, the genus Brevibacillus) with which Bacillus spp. Since it is a gene derived from the same species), it does not impose an environmental load greater than crossing in the natural world.
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、ｇａｔＣ遺伝子及びｔｅｔＢ遺伝子を上述した特定の組み合わせとした遺伝子の塩基配列を含むプラスミドは、公知の方法により作製される。例えば、前記遺伝子の塩基配列情報を用いて、化学合成、遺伝子工学的手法、又は突然変異誘発等により作製できる。遺伝子工学的手法としては、例えば、制限酵素処理した鋳型プラスミドに前記塩基配列を挿入する方法が挙げられる。 A plasmid containing the nucleotide sequences of the genes in which the gatA gene, gatB gene, gatC gene and tetB gene are combined in the above-described specific combination is produced by a known method. For example, it can be produced by chemical synthesis, genetic engineering techniques, mutagenesis, or the like, using the nucleotide sequence information of the gene. Genetic engineering techniques include, for example, a method of inserting the base sequence into a restriction enzyme-treated template plasmid.
（宿主細胞）
　本実施形態に係る宿主細胞は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、少なくとも、上述したプラスミド上に発現可能な状態で有さない残余の遺伝子を、染色体上に発現可能な状態で有する。例として、プラスミドがｇａｔＣ遺伝子を発現可能な状態で有し、且つ、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を発現可能な状態で有さない場合の、宿主細胞のパターンを表１に示す。表１において、「〇」は当該遺伝子を宿主細胞の染色体上に発現可能な状態で有すること、「×」は当該遺伝子を宿主細胞の染色体上に発現可能な状態で有さないことを示す。このとき、宿主細胞は、少なくともｇａｔＡ遺伝子及びｇａｔＢ遺伝子を染色体上に発現可能な状態で有する。すなわち、宿主細胞は、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を染色体上に発現可能な状態で有し、且つ、ｇａｔＣ遺伝子を染色体上に発現可能な状態で有さない場合（表１におけるパターン１）もあり得る。あるいは、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子をすべて有する場合（表１におけるパターン２）もあり得る。 (host cell)
The host cell according to this embodiment has at least the rest of the gatA gene, gatB gene, and gatC gene, which are not present in an expressible state on the plasmid, in an expressible state on the chromosome. . As an example, Table 1 shows host cell patterns when the plasmid has the gatC gene in an expressible state and does not have the gatA and gatB genes in an expressible state. In Table 1, "O" indicates that the gene is present on the chromosome of the host cell in an expressible state, and "X" indicates that the gene is not present on the chromosome of the host cell in an expressible state. At this time, the host cell has at least the gatA gene and the gatB gene on the chromosome in an expressible state. That is, the host cell may have the gatA gene and the gatB gene on the chromosome in an expressible state, and may not have the gatC gene on the chromosome in an expressible state (pattern 1 in Table 1). . Alternatively, there may be cases where the gatA gene, gatB gene, and gatC gene are all present (pattern 2 in Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　本実施形態に係る宿主細胞は、染色体上のｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子を発現可能な状態で有さなくてもよい。「遺伝子が発現可能な状態でない（遺伝子が不活性化した状態）」とは、遺伝子の機能が失われている状態、又は機能が減少している状態を指す。なお、遺伝子の機能が失われている状態、又は減少している状態とは、具体的には、当該遺伝子の転写産物であるｍＲＮＡや翻訳産物であるポリペプチドの発現が消失、又は減少（例えば、ｍＲＮＡ又はポリペプチドの発現量が通常の５０％以下、好ましくは３０％以下、さらに好ましくは１０％以下）している状態や、ｍＲＮＡやタンパク質として正常に機能しない状態を指す。 The host cell according to this embodiment may not have one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene on the chromosome in an expressible state. The expression "gene cannot be expressed (gene is inactivated)" refers to a state in which gene function is lost or reduced. In addition, the state in which the function of a gene is lost or decreased specifically means that the expression of mRNA, which is a transcription product of the gene, or a polypeptide, which is a translation product of the gene, is lost or decreased (for example, , mRNA or polypeptide expression level is 50% or less, preferably 30% or less, more preferably 10% or less of the normal), or a state where mRNA or protein does not function normally.
　遺伝子の不活性化は、特に限定されないが、薬剤や紫外線を用いたＤＮＡ変異処理、ＰＣＲを用いた部分特異的変異導入、ＲＮＡｉ、プロテアーゼ、相同組み換え等の手法を利用することができる。遺伝子の不活性化は、例えば、前記遺伝子のＯＲＦ内の塩基配列の改変、及び／又はプロモーター領域、エンハンサー領域、ターミネーター領域などの転写開始又は停止を制御する領域内の塩基配列の改変により行うことができる。塩基配列の改変は、欠失（破壊ともいう）、置換、付加、又は挿入等により行うことができる。欠失、置換、付加、又は挿入の標的となる塩基配列の部位は、前記遺伝子の正常な機能が欠損し得る限り、特に限定されない。 Gene inactivation is not particularly limited, but methods such as DNA mutation treatment using drugs or ultraviolet rays, partial specific mutagenesis using PCR, RNAi, protease, and homologous recombination can be used. Gene inactivation is carried out, for example, by modifying the base sequence within the ORF of the gene and/or modifying the base sequence within the regions controlling transcription initiation or termination such as promoter regions, enhancer regions and terminator regions. can be done. A nucleotide sequence can be modified by deletion (also referred to as disruption), substitution, addition, insertion, or the like. The site of the base sequence targeted for deletion, substitution, addition, or insertion is not particularly limited as long as the normal function of the gene can be lost.
　相同組み換えによる遺伝子の不活性化は、例えば、挿入したＤＮＡ断片における、宿主細胞染色体上の相同性がある配列が、その相同性のある配列と組み換わることにより行われる。このとき、１箇所の相同領域を利用した組み込みでもよいし、２箇所の相同領域を利用したダブルクロスオーバー型の組み込みでもよい。このような方法で標的部位を置換した細胞は、導入ＤＮＡ断片に付加したマーカー遺伝子を指標に選択することができる。マーカー遺伝子としては、例えば、フルオロウラシル感受性を有するｕｐｐ遺伝子を利用することができる。 Gene inactivation by homologous recombination is carried out, for example, by the recombination of the homologous sequence on the host cell chromosome in the inserted DNA fragment with the homologous sequence. At this time, integration using one homologous region may be performed, or double-crossover type integration using two homologous regions may be performed. Cells in which the target site has been replaced by such a method can be selected using the marker gene added to the introduced DNA fragment as an indicator. As a marker gene, for example, an upp gene having sensitivity to fluorouracil can be used.
　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子は宿主細胞の生育に必須な遺伝子であるため、例えば、「ｇａｔＣ遺伝子が不活性化された」とは、宿主細胞が生育できない程度にまで当該遺伝子の発現を減少又は消失させた状態を指す。以上から、例えば、染色体上のｇａｔＣ遺伝子が不活性化された宿主細胞が生育するためには、ｇａｔＣ遺伝子の塩基配列を含むプラスミドによる形質転換が必須となる。 Since the gatA gene, the gatB gene, and the gatC gene are genes essential for the growth of host cells, for example, "the gatC gene is inactivated" means that the expression of the gene is inhibited to the extent that the host cell cannot grow. Refers to the state of being reduced or eliminated. From the above, for example, in order to grow host cells in which the gatC gene on the chromosome is inactivated, transformation with a plasmid containing the base sequence of the gatC gene is essential.
　本実施形態に係る宿主細胞は、上記プラスミドを安定的に維持できるものであれば、特に限定されないが、細菌が好ましく、バチルス属細菌又はブレビバチルス属細菌がさらに好ましく、バチルス属細菌が特に好ましい。バチルス属細菌としては、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ（枯草菌）、Ｂａｃｉｌｌｕｓ　ｌｉｑｕｅｎｉｆｏｒｍｉｓ、Ｂａｃｉｌｌｕｓ　ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ、Ｂａｃｉｌｌｕｓ　ｃｌａｕｓｉｉ、Ｂａｃｉｌｌｕｓ　ｍｅｇａｔｅｒｉｕｍ等が挙げられるが、枯草菌が特に好ましい。 The host cell according to the present embodiment is not particularly limited as long as it can stably maintain the plasmid, but is preferably a bacterium, more preferably a bacterium of the genus Bacillus or a bacterium of the genus Brevibacillus, and particularly preferably a bacterium of the genus Bacillus. Bacillus bacteria include Bacillus subtilis, Bacillus liqueniformis, Bacillus amyliquefaciens, Bacillus clausii, and Bacillus megaterium, with Bacillus subtilis being particularly preferred.
（形質転換細胞）
　本実施形態において、「形質転換」とは、上記プラスミドを宿主細胞に導入することを指す。形質転換は、公知の方法を適宜利用することができ、例えば、エレクトロポレーション法、リポフェクション法、カルシウムイオン法、コンピテントセル法、プロトプラスト法による方法等が挙げられる。宿主細胞がバチルス属細菌である場合、コンピテントセル法、又はプロトプラスト法が好ましい。 (transformed cell)
In this embodiment, "transformation" refers to introducing the above plasmid into a host cell. For transformation, a known method can be appropriately used, and examples thereof include electroporation method, lipofection method, calcium ion method, competent cell method, protoplast method, and the like. When the host cell is a bacterium belonging to the genus Bacillus, the competent cell method or the protoplast method is preferred.
　本実施形態に係る形質転換細胞は、上述の通り、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子をプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、少なくとも、前記プラスミド上に発現可能な状態で有さない残余の遺伝子を、染色体上に発現可能な状態で有する。 As described above, the transformed cell according to the present embodiment has one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene in an expressible state on a plasmid, and gatA At least the rest of the genes, the gatB gene, and the gatC gene, which are not present on the plasmid in an expressible state, are present on the chromosome in an expressible state.
　本実施形態に係る形質転換細胞は、前記群より選択される一又は二の遺伝子を、染色体上に発現可能な状態で有さなくてもよい（例えば、表１におけるパターン１）。このとき、前記群より選択される一又は二の遺伝子が不活性化された宿主細胞は、上述の通り、そのままでは生育できない。一方、宿主細胞で不活性化された当該遺伝子を含むプラスミドによって形質転換された当該宿主細胞は、プラスミド由来の当該遺伝子産物により相補され、生育することができる。このことから、抗生物質や栄養要求性といった従来の選択圧によらず、形質転換細胞を選択することができる。さらに、このとき、当該宿主細胞から当該プラスミドが脱落した場合、当該宿主細胞はもはや生育できない。そのため、上述の形質転換細胞の選択と同様に、抗生物質や栄養要求性といった従来の選択圧によらず、形質転換細胞を維持することができる。この場合において、本実施形態に係る形質転換細胞のプラスミド保持率は１００％となる。なお、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＣ遺伝子のみをプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子のみを染色体上に発現可能な状態で有する形質転換細胞が特に好ましい。これによれば、プラスミドの設計性が向上し、且つ、プラスミド保持率が１００％の形質転換細胞を取得できる。 A transformed cell according to this embodiment may not have one or two genes selected from the above group in an expressible state on the chromosome (for example, pattern 1 in Table 1). At this time, host cells in which one or two genes selected from the group have been inactivated cannot grow as they are, as described above. On the other hand, the host cell transformed with a plasmid containing the gene inactivated in the host cell can be complemented by the plasmid-derived gene product and grow. This allows transformed cells to be selected without traditional selection pressures such as antibiotics or auxotrophy. Furthermore, at this time, if the plasmid is lost from the host cell, the host cell can no longer grow. Therefore, as in the selection of transformed cells described above, transformed cells can be maintained without conventional selection pressures such as antibiotics and auxotrophy. In this case, the plasmid retention rate of the transformed cells according to this embodiment is 100%. Among the gatA gene, the gatB gene, and the gatC gene, only the gatC gene is present on the plasmid in an expressible state, and among the gatA gene, the gatB gene, and the gatC gene, only the gatA gene and the gatB gene are present. Transformed cells that have expression on their chromosomes are particularly preferred. According to this, the plasmid designability is improved, and transformed cells with a plasmid retention rate of 100% can be obtained.
　一方、本実施形態に係る形質転換細胞は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を染色体上に発現可能な状態で有してもよい（例えば、表１におけるパターン２）。上述した、これらいずれかの遺伝子が不活性化された宿主細胞とは異なり、生育にプラスミドを必須としない。通常、このような細胞の継代を重ねた場合、一定の割合で宿主細胞からプラスミドが脱落してしまうが、驚くべきことに、本実施形態に係る形質転換細胞は高いプラスミド保持率を有する。一実施形態において、プラスミド保持率は９０％以上であり、好ましくは９５％以上であり得る。すなわち、この場合において、染色体上遺伝子の不活性化を要さずに高いプラスミド保持率を有する形質転換細胞を取得することができる。なお、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＣ遺伝子のみをプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を前記染色体上に発現可能な状態で有する形質転換細胞が特に好ましい。これによれば、高いプラスミド保持率の形質転換細胞を、煩雑な遺伝子操作を介さず簡便に取得できる。 On the other hand, the transformed cell according to this embodiment may have the gatA gene, gatB gene, and gatC gene on the chromosome in an expressible state (eg, pattern 2 in Table 1). Unlike the host cells described above in which any of these genes are inactivated, the plasmid is not required for growth. Normally, when such cells are passaged repeatedly, the plasmid is lost from the host cells at a certain rate. Surprisingly, the transformed cells according to this embodiment have a high plasmid retention rate. In one embodiment, plasmid retention may be 90% or greater, preferably 95% or greater. That is, in this case, transformed cells having a high plasmid retention rate can be obtained without requiring inactivation of chromosomal genes. Among the gatA gene, the gatB gene, and the gatC gene, only the gatC gene is present on the plasmid in an expressible state, and the gatA gene, the gatB gene, and the gatC gene are present on the chromosome in an expressible state. Particularly preferred are transformed cells with According to this method, transformed cells with a high plasmid retention rate can be easily obtained without complicated genetic manipulations.
（目的タンパク質の生産）
　最後に、本実施形態に係る形質転換細胞を利用した目的タンパク質の生産について説明する。なお、ここでは本実施形態に係るプラスミドが有する、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子を、「選択遺伝子」と称する。 (Production of target protein)
Finally, the production of the target protein using the transformed cells according to this embodiment will be described. Here, one or two genes selected from the group consisting of the gatA gene, gatB gene, and gatC gene, which the plasmid according to this embodiment has, are referred to as "selection genes".
　上述の通り、形質転換に使用するプラスミドには、目的タンパク質をコードする遺伝子（以下、目的遺伝子とも称する）の塩基配列を含んでもよい。なお、目的タンパク質を生産するための形質転換細胞は、本実施形態に係る選択遺伝子及び目的遺伝子を共に含むプラスミドで、本実施形態に係る宿主細胞を形質転換することにより得られる。このとき、宿主細胞は所望するプラスミド保持率に応じて染色体上の選択遺伝子を不活性化したもの（表１におけるパターン１）、及び不活性化していないもの（表１におけるパターン２）から選択できる。当該目的遺伝子を含むプラスミドが導入されているかは、マーカー遺伝子により判断してもよい。このとき、好ましくは最終的な形質転換細胞に異種由来薬剤耐性遺伝子を含まない。ただし、この場合に、途中の過程において異種由来薬剤耐性遺伝子を用いてもよい。具体的には、例えば、宿主細胞がバチルス属細菌である場合、まず、宿主細胞を異種由来カナマイシン耐性遺伝子を含むプラスミドにより形質転換して形質転換細胞を得た後、当該形質転換細胞を選択遺伝子、ｔｅｔＢ遺伝子、及び目的遺伝子を含むプラスミドにより再度形質転換する。得られた細胞群から、カナマイシン耐性を有さず、且つテトラサイクリン耐性を有する細胞を選択し、最終的な形質転換細胞を取得することができる。 As described above, the plasmid used for transformation may contain the nucleotide sequence of the gene encoding the target protein (hereinafter also referred to as the target gene). A transformed cell for producing the target protein is obtained by transforming the host cell according to the present embodiment with a plasmid containing both the selection gene and the target gene according to the present embodiment. At this time, host cells can be selected from those with inactivated selection genes on the chromosome (pattern 1 in Table 1) and those without inactivation (pattern 2 in Table 1) depending on the desired plasmid retention rate. . A marker gene may be used to determine whether a plasmid containing the gene of interest has been introduced. At this time, the final transformed cell preferably does not contain a heterologous drug resistance gene. However, in this case, the heterologous drug resistance gene may be used in the intermediate process. Specifically, for example, when the host cell is a bacterium of the genus Bacillus, first, the host cell is transformed with a plasmid containing a heterologous kanamycin resistance gene to obtain a transformed cell, and then the transformed cell is selected by the selection gene. , the tetB gene, and the gene of interest. Cells having no kanamycin resistance and tetracycline resistance can be selected from the obtained cell group to obtain final transformed cells.
　目的タンパク質の製造は、上記の方法により得られた形質転換細胞を、公知の方法により培養して、好適には、当該培養物から公知の方法により精製することにより行うことができる。当該製造においては、抗生物質や栄養要求性といった従来の選択圧によらず、高いプラスミド保持率を有する形質転換細胞を取得できるため、生産コストを低く抑えることができる。また、一実施形態において、当該製造方法によって得られた培養物は、異種由来薬剤耐性遺伝子を含まないため、環境負荷が少ない。 The target protein can be produced by culturing the transformed cells obtained by the above method by a known method, and preferably by purifying the culture by a known method. In this production, transformed cells with a high plasmid retention rate can be obtained without relying on conventional selection pressures such as antibiotics and auxotrophy, so production costs can be kept low. Moreover, in one embodiment, the culture obtained by the production method does not contain a heterologous drug resistance gene, so that the environmental load is small.
　本発明は、上述した実施の形態に限定されるものではなく、当業者の知識に基づいて各種の設計変更などの変形を加えることも可能であり、そのような変形が加えられた実施の形態も本発明の範囲に含まれる。 The present invention is not limited to the above-described embodiments, and modifications such as various design changes can be made based on the knowledge of those skilled in the art. are also included in the scope of the present invention.
１．ｇａｔＣ遺伝子を含む大腸菌－枯草菌のシャトルベクターの構築
　プロモーター領域を含むｇａｔＣ遺伝子（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＮＣ　０００９６４：７２８７３２～７２９０２２）を、プライマー１（配列番号１）及びプライマー２（配列番号２）を用いて枯草菌１６８株（ＮＲＢＣ１１１４７０）の染色体ＤＮＡからＰＣＲで増幅した。取得したＰＣＲ産物の末端にアデニンを付加するためＴａｑ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（Ｂｉｏ　ａｃａｄｅｍｉｃ社）で処理（７２℃、１５分間反応）した後、ｐＭＤ２０　Ｔ－Ｖｅｃｔｏｒ（Ｔａｋａｒａ社）にサブクローニングし、ｇａｔＣ遺伝子ＴＡクローニングプラスミド：ｐＭＤｇａｔＣ２Ｆを得た。 1. Construction of E. coli-Bacillus subtilis Shuttle Vector Containing the gatC Gene The gatC gene containing the promoter region (ACCESSION No. NC 000964:728732-729022) was constructed using primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2). It was amplified by PCR from chromosomal DNA of Bacillus subtilis strain 168 (NRBC111470). In order to add adenine to the end of the obtained PCR product, it was treated with Taq DNA polymerase (Bioacademic) (reacted at 72°C for 15 minutes), then subcloned into pMD20 T-Vector (Takara) to create a gatC gene TA cloning plasmid. : pMDgatC2F was obtained.
　大腸菌－枯草菌のシャトルベクターｐＨＹ３００ＰＬＫ（ＴａｋａｒａＢｉｏ社）を制限酵素ＸｂａＩ及びＨｉｎｄＩＩＩで消化し、さらにＨｉｎｄＩＩＩ末端については平滑末端処理をした。このシャトルベクターに、上記で取得したｐＭＤｇａｔＣ２Ｆを制限酵素ＸｂａＩ及びＰｖｕＩＩ（平滑末端）で消化して得られたｇａｔＣ遺伝子を含むＤＮＡ断片を挿入し、ｇａｔＣ遺伝子を含むシャトルベクター：ｐＢＫ－ＰｒｏｇａｔＣ１（図１参照）を取得した。 The Escherichia coli-Bacillus subtilis shuttle vector pHY300PLK (TakaraBio) was digested with restriction enzymes XbaI and HindIII, and the HindIII ends were blunt-ended. A DNA fragment containing the gatC gene obtained by digesting the pMDgatC2F obtained above with the restriction enzymes XbaI and PvuII (blunt ends) was inserted into this shuttle vector, resulting in a shuttle vector containing the gatC gene: pBK-ProgatC1 (Fig. 1). reference) was obtained.
２．枯草菌のｕｐｐ遺伝子破壊株の作製
　枯草菌由来ｕｐｐ遺伝子をカウンターセレクションマーカーとして利用するために、枯草菌１６８株のｕｐｐ遺伝子破壊を行った。ｕｐｐ遺伝子（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＮＣ　０００９６４：３７８８４２６～３７８９０５５）の上流及び下流それぞれ約０．２５ｋｂの領域を、プライマー３（配列番号３）及びプライマー４（配列番号４）、プライマー５（配列番号５）及びプライマー６（配列番号６）を用いて、それぞれＰＣＲで増幅し、回収した。両断片を２００ｆｍｏｌ／５０μＬの濃度になるように調製して、プライマー３及びプライマー６を用いて二断片ＰＣＲを行い、両断片を連結した。連結されたＤＮＡ断片と、別途ｐＵＢ１１０（Ｇｅｎｂａｎｋ：Ｘ０３８８５，Ｍ１９４６５）からプライマー７（配列番号７）及びプライマー８（配列番号８）を用いてＰＣＲ増幅したカナマイシン耐性遺伝子を含むＤＮＡ断片とを連結するため、両断片１００ｆｍｏｌ／５０μＬに調製し、プライマー３及びプライマー８を用いて、再度二断片ＰＣＲを行った。得られたＤＮＡ断片を上記と同様にＴＡクローニングし、ｕｐｐ遺伝子破壊用プラスミド：ｐＭＤΔｕｐｐＫｍを取得した。 2. Preparation of Bacillus subtilis upp gene-disrupted strain In order to use the Bacillus subtilis-derived upp gene as a counter-selection marker, the upp gene of Bacillus subtilis strain 168 was disrupted. upp gene (ACCESSION No. NC 000964: 3788426 to 3789055) upstream and downstream of about 0.25 kb region, primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), primer 5 (SEQ ID NO: 5) and Each was amplified by PCR using primer 6 (SEQ ID NO: 6) and collected. Both fragments were prepared at a concentration of 200 fmol/50 μL, and two-fragment PCR was performed using primers 3 and 6 to ligate both fragments. To ligate the ligated DNA fragment and a DNA fragment containing the kanamycin resistance gene separately PCR-amplified from pUB110 (Genbank: X03885, M19465) using primer 7 (SEQ ID NO: 7) and primer 8 (SEQ ID NO: 8) , Both fragments were prepared to 100 fmol/50 μL, and using primer 3 and primer 8, two-fragment PCR was performed again. The obtained DNA fragment was subjected to TA cloning in the same manner as described above to obtain a plasmid for disrupting the upp gene: pMDΔuppKm.
　上記で取得したｐＭＤΔｕｐｐＫｍを用いて、枯草菌１６８株を形質転換した。カナマイシン耐性を示す形質転換細胞を選抜し、得られた菌株のゲノム（染色体ＤＮＡ）についてＰＣＲを行い、当該菌株ゲノム中のｕｐｐ遺伝子の近傍に、ｐＭＤΔｕｐｐＫｍが挿入されていることを確認した。当該菌株を５－フルオロウラシルを含む無機塩培地で培養し、生育する株を取得した。このうち、カナマイシン耐性を失い、且つ５－フルオロウラシル耐性を示す菌を選抜し、ＰＣＲによって染色体上のｕｐｐ遺伝子が破壊されていることを確認し、得られたｕｐｐ遺伝子破壊株を１６８Δｕｐｐ株とした。 　Bacillus subtilis strain 168 was transformed using the pMDΔuppKm obtained above. Transformed cells exhibiting kanamycin resistance were selected, and the genome (chromosomal DNA) of the obtained strain was subjected to PCR to confirm that pMDΔuppKm was inserted near the upp gene in the strain genome. The strain was cultured in an inorganic salt medium containing 5-fluorouracil to obtain a growing strain. Among these strains, strains that lost kanamycin resistance and exhibited 5-fluorouracil resistance were selected, and disruption of the upp gene on the chromosome was confirmed by PCR.
３．枯草菌ｇａｔＣ遺伝子破壊株の作製
　ｇａｔＣ遺伝子の上流及び下流それぞれ約１．０ｋｂの領域を、プライマー９（配列番号９）及びプライマー１０（配列番号１０）、プライマー１１（配列番号１１）及びプライマー１２（配列番号１２）を用いて、それぞれＰＣＲで増幅し、回収した。両断片を２００ｆｍｏｌ／５０μＬの濃度になるように調製して、プライマー９とプライマー１２を用いて二断片ＰＣＲを行い、両断片を連結した。また、ｐＵＢ１１０からプライマー１３（配列番号１３）及びプライマー８を用いてＰＣＲ増幅したカナマイシン耐性遺伝子を含むＤＮＡ断片と、枯草菌１６８株のゲノムＤＮＡをテンプレートとしてプライマー７及びプライマー１４（配列番号１４）を用いてＰＣＲ増幅したｕｐｐ遺伝子を含むＤＮＡ断片を、各々１００ｆｍｏｌ／５０μＬの濃度になるように調製して、プライマー１３及びプライマー１４を用いて二断片ＰＣＲを行い、両断片を連結した。これを、先に増幅したｇａｔＣ遺伝子の上流・下流を含むＤＮＡ断片と連結するため、プライマー９とプライマー１４を用いて、各々のＤＮＡ断片が１００ｆｍｏｌ／５０μＬの濃度になるように調製して、二断片ＰＣＲを行った。目的のＤＮＡ断片を上記と同様にＴＡクローニングし、ｇａｔＣ遺伝子破壊用プラスミド：ｐＭＤΔｇａｔＣ１０１Ｒを取得した（図２参照）。 3. Preparation of gatC gene-disrupted strain of Bacillus subtilis About 1.0 kb region upstream and downstream of the gatC gene, primer 9 (SEQ ID NO: 9) and primer 10 (SEQ ID NO: 10), primer 11 (SEQ ID NO: 11) and primer 12 ( Using SEQ ID NO: 12), each was amplified by PCR and collected. Both fragments were prepared at a concentration of 200 fmol/50 μL, and two-fragment PCR was performed using primers 9 and 12 to ligate both fragments. In addition, a DNA fragment containing a kanamycin resistance gene amplified by PCR from pUB110 using primers 13 (SEQ ID NO: 13) and primer 8, and genomic DNA of Bacillus subtilis strain 168 as a template, primers 7 and 14 (SEQ ID NO: 14) were used. A DNA fragment containing the uppp gene amplified by PCR was prepared to have a concentration of 100 fmol/50 μL, respectively, and two-fragment PCR was performed using primer 13 and primer 14 to ligate both fragments. In order to ligate this with the previously amplified DNA fragment containing the upstream and downstream of the gatC gene, primers 9 and 14 were used to prepare each DNA fragment at a concentration of 100 fmol/50 μL. Fragment PCR was performed. The DNA fragment of interest was TA cloned in the same manner as above to obtain a plasmid for disrupting the gatC gene: pMDΔgatC101R (see FIG. 2).
　得られたｐＭＤΔｇａｔＣ１０１Ｒを用いて、上記２．で取得した枯草菌１６８Δｕｐｐ株を形質転換した。カナマイシン耐性を示す形質転換細胞を選抜し、得られた菌株のゲノムについてＰＣＲを行い、当該菌株ゲノム中のｇａｔＣ遺伝子の近傍にｐＭＤΔｇａｔＣ１０１Ｒが挿入されていることを確認した。 Using the obtained pMDΔgatC101R, the above 2. The Bacillus subtilis 168Δupp strain obtained in 1 was transformed. Transformed cells exhibiting kanamycin resistance were selected, and the genome of the obtained strain was subjected to PCR to confirm that pMDΔgatC101R was inserted near the gatC gene in the strain genome.
　当該菌株に、テトラサイクリン耐性を指標に上記１.で作製したｐＢＫ－ＰｒｏｇａｔＣ１を導入した。得られた形質転換細胞を、５－フルオロウラシル及びテトラサイクリン含有培地で培養して、５－フルオロウラシル耐性を獲得し、カナマイシン耐性を喪失し、且つテトラサイクリン耐性を維持する菌株を選抜した。当該菌株のゲノムについてＰＣＲを行い、染色体上のｇａｔＣ遺伝子の破壊を確認した。換言すると、当該菌株は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＣ遺伝子のみをプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子のみを前記染色体上に発現可能な状態で有していた。得られた菌株を１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）株と命名した。 The pBK-ProgatC1 prepared in 1 above was introduced into the strain using tetracycline resistance as an index. The resulting transformed cells were cultured in a medium containing 5-fluorouracil and tetracycline to select strains that acquired 5-fluorouracil resistance, lost kanamycin resistance, and maintained tetracycline resistance. PCR was performed on the genome of the strain to confirm disruption of the gatC gene on the chromosome. In other words, the strain has only the gatC gene among the gatA gene, the gatB gene, and the gatC gene in an expressible state on the plasmid, and is capable of expressing only the gatA gene and the gatB gene on the chromosome. Had in the state. The resulting strain was named 168Δupp, gatC (pBK-ProgatC1) strain.
４．ｇａｔＣ遺伝子を含むシャトルベクター保持の確認
　上記で取得した１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）株が、テトラサイクリンを含まない培地においてもｇａｔＣ遺伝子を含むプラスミドの脱落を起こさず、保持できるか確認した。まず、比較対象株として、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ．ｆａｅｃａｌｉｓ由来のテトラサイクリン耐性遺伝子：ＴｃＲを有し、且つｇａｔＣ遺伝子を有さない、ｐＨＹ３００ＰＬＫの派生プラスミド：ｐＢＫ－Ｂ３（ｐＨＹ３００ＰＬＫ　のＥｃｏＲＩ－ＸｂａＩ部位間に酵素遺伝子Ｂ３を挿入して構築した）を用いて１６８Δｕｐｐ株を形質転換した。得られた比較対象株と１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）株をそれぞれテトラサイクリン含有ＬＢ寒天培地にストリークして３０℃で培養した。各株１コロニーを、各々テトラサイクリン不含のＬＢ液体培地３ｍＬに植菌し、３０℃で一晩培養した。続いて、取得した培養液をテトラサイクリン不含のＬＢ寒天培地に塗布し、３０℃で培養した。培養後、生じたシングルコロニーを各株１００個以上取得した。これらのシングルコロニー各１００個について、テトラサイクリン含有ＬＢ培地及びテトラサイクリン不含ＬＢ培地にそれぞれ植え継ぎ、３０℃で培養した。テトラサイクリン不含ＬＢ培地において植菌した全コロニーが生育していることを確認して培養終了とし、培養終了時のテトラサイクリン含有ＬＢ培地における生育コロニー数を、プラスミド保持率として算出した。 4. Confirmation of Retention of Shuttle Vector Containing gatC Gene It was confirmed whether the 168Δupp, gatC (pBK-ProgatC1) strain obtained above could maintain the plasmid containing the gatC gene without dropping it even in a medium containing no tetracycline. First, as a comparative strain, Streptococcus. faecalis-derived tetracycline resistance gene: pHY300PLK derivative plasmid having Tc R and no gatC gene: pBK-B3 (constructed by inserting enzyme gene B3 between EcoRI-XbaI sites of pHY300PLK) was transformed into the 168Δupp strain. The comparative strain and the 168Δupp, gatC (pBK-ProgatC1) strain thus obtained were each streaked on a tetracycline-containing LB agar medium and cultured at 30°C. One colony of each strain was inoculated into 3 mL of tetracycline-free LB liquid medium and cultured overnight at 30°C. Subsequently, the obtained culture solution was spread on a tetracycline-free LB agar medium and cultured at 30°C. After culturing, 100 or more single colonies of each strain were obtained. 100 of each of these single colonies were transferred to tetracycline-containing LB medium and tetracycline-free LB medium, respectively, and cultured at 30°C. After confirming that all colonies inoculated in the tetracycline-free LB medium had grown, the culture was terminated.
　培養終了時の各プレートの様子を図３に示す。１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）はテトラサイクリン含有培地において１００個すべてのコロニーが生育したのに対し、比較対象株はテトラサイクリン含有培地において６９個のコロニーが生育した（プラスミド保持率６９％）。以上から、１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）は抗生物質不含培地での継代培養後もｐＢＫ－ＰｒｏｇａｔＣ１プラスミドを保持していることが確認された。 Fig. 3 shows the appearance of each plate at the end of the culture. 168Δupp,gatC (pBK-ProgatC1) grew all 100 colonies on tetracycline-containing medium, whereas the control strain grew 69 colonies on tetracycline-containing medium (69% plasmid retention). From the above, it was confirmed that 168Δupp,gatC (pBK-ProgatC1) retained the pBK-ProgatC1 plasmid even after subculturing in an antibiotic-free medium.
５．ｔｅｔＢ遺伝子のマーカー遺伝子としての有効性の確認
　ｔｅｔＢ遺伝子が、細菌の形質転換マーカー遺伝子として利用可能かどうかを評価するため、ｔｅｔＢ遺伝子を含む大腸菌－枯草菌のシャトルベクターを構築した。 5. Confirmation of the effectiveness of the tetB gene as a marker gene To evaluate whether the tetB gene can be used as a bacterial transformation marker gene, an E. coli-Bacillus subtilis shuttle vector containing the tetB gene was constructed.
　プロモーター領域を含むｔｅｔＢ遺伝子（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＮＣ　０００９６４：４１８７６８１～４１８９０５７）を枯草菌１６８株のゲノムＤＮＡからプライマー１５（配列番号１５）及びプライマー１６（配列番号１６）を用いてＰＣＲによって増幅した。得られたＰＣＲ産物をＴａｑ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（Ｂｉｏ　ａｃａｄｅｍｉｃ社）で７２℃, １５分間処理した後、ｐＭＤ２０　Ｔ－Ｖｅｃｔｏｒにサブクローニングし、ｐＭＤｔｅｔＢ－１Ｒを得た。 The tetB gene (ACCESSION No. NC 000964: 4187681-4189057) containing the promoter region was amplified by PCR from the genomic DNA of Bacillus subtilis strain 168 using primers 15 (SEQ ID NO: 15) and 16 (SEQ ID NO: 16). The resulting PCR product was treated with Taq DNA polymerase (Bioacademic) at 72°C for 15 minutes and then subcloned into pMD20 T-Vector to obtain pMDtetB-1R.
　次に、大腸菌－枯草菌のシャトルベクターｐＨＹ３００ＰＬＫ上にあるテトラサイクリン耐性遺伝子：ＴｃＲを、当該遺伝子内部に２箇所あるＨｉｎｃＩＩ部位を用いて破壊した（ｐＨＹ３００ＰＬＫΔtｅｔｒｃＩＩ）。また、ｐＵＢ１１０からプライマー１３及びプライマー１７（配列番号１７）を用いてカナマイシン耐性遺伝子を含むＤＮＡ断片、枯草菌１６８株のゲノムＤＮＡからプライマー７及びプライマー１４を用いてｕｐｐ遺伝子を含むＤＮＡ断片を、それぞれＰＣＲ増幅し、両遺伝子断片を１００ｆｍｏｌ／５０μＬの濃度になるように調製して、プライマー１３及びプライマー１４を用いて二断片ＰＣＲを行い、両断片を連結した。得られた断片を、上記と同様にｐＭＤ２０　Ｔ－Ｖｅｃｔｏｒにサブクローニングし、ｐＭＤＫｍｕｐｐ１Ｆを得た。得られたｐＭＤＫｍｕｐｐ１Ｆを制限酵素ＥｃｏＲＩ及びＰｖｕＩＩで消化し、得られたカナマイシン耐性遺伝子Ｋｍｒを含むＤＮＡ断片を、上記で取得したｐＨＹ３００ＰＬＫΔｔｅｔｒｃＩＩのＥｃｏＲＩ－ＨｉｎｃＩＩ部位に導入し、ｐＢＫ－ＰｒｅＫｍ２を得た。 Next, the tetracycline resistance gene: TcR on the E. coli-Bacillus subtilis shuttle vector pHY300PLK was disrupted using two HincII sites within the gene (pHY300PLKΔtetrcII). In addition, a DNA fragment containing the kanamycin resistance gene from pUB110 using primers 13 and 17 (SEQ ID NO: 17), and a DNA fragment containing the upp gene from genomic DNA of Bacillus subtilis strain 168 using primers 7 and 14, respectively. PCR amplification was performed, both gene fragments were adjusted to a concentration of 100 fmol/50 μL, two-fragment PCR was performed using primer 13 and primer 14, and both fragments were ligated. The resulting fragment was subcloned into pMD20 T-Vector in the same manner as above to obtain pMDKmupp1F. The resulting pMDKmupp1F was digested with restriction enzymes EcoRI and PvuII, and the resulting DNA fragment containing the kanamycin resistance gene Kmr was introduced into the EcoRI-HincII site of pHY300PLKΔtetrcII obtained above to obtain pBK-PreKm2.
　上記で取得したｐＭＤｔｅｔＢ２－１Ｒを制限酵素ＨｉｎｄＩＩＩ及びＥｃｏＲＩで消化し、得られたｔｅｔＢ遺伝子を含むＤＮＡ断片をｐＢＫ－ＰｒｅＫｍ２のＨｉｎｄＩＩＩ－ＥｃｏＲＩ部位に挿入した。これにより、ｔｅｔＢ遺伝子及びカナマイシン耐性遺伝子Ｋｍｒを有するシャトルベクター：ｐＢＫ－ＰｒｅＫｍｔｅｔＢ７（図４参照）を取得した。 The pMDtetB2-1R obtained above was digested with restriction enzymes HindIII and EcoRI, and the resulting DNA fragment containing the tetB gene was inserted into the HindIII-EcoRI site of pBK-PreKm2. As a result, a shuttle vector pBK-PreKmtetB7 (see FIG. 4) containing the tetB gene and the kanamycin resistance gene Kmr was obtained.
　上記で取得したｐＢＫ－ＰｒｅＫｍｔｅｔＢ７を、カナマイシン耐性を指標に枯草菌１６８Δｕｐｐ株に導入した。比較対象として、枯草菌１６８Δｕｐｐ株を市販のｐＨＹ３００ＰＬＫで形質転換し、菌株１６８Δｕｐｐ（ｐＨＹ３００ＰＬＫ）を作製した。各菌株を、テトラサイクリンの含有量が０ｍｇ／Ｌ、７．５ｍｇ／Ｌ、７０ｍｇ／Ｌ、及び１００ｍｇ／ＬのＬＢ寒天培地にそれぞれストリークし、３０℃で３６時間培養した結果を図５に示す。ｔｅｔＢ遺伝子含有シャトルベクターｐＢＫ－ＰｒｅＫｍｔｅｔＢ７を有する株は、市販のテトラサイクリン耐性遺伝子ＴｃＲを有する１６８Δｕｐｐ（ｐＨＹ３００ＰＬＫ）と同等のテトラサイクリン耐性を示した。 The pBK-PreKmtetB7 obtained above was introduced into the Bacillus subtilis 168Δupp strain using kanamycin resistance as an index. For comparison, Bacillus subtilis strain 168Δupp was transformed with commercially available pHY300PLK to prepare strain 168Δupp (pHY300PLK). Each strain was streaked on LB agar medium containing 0 mg/L, 7.5 mg/L, 70 mg/L, and 100 mg/L of tetracycline, and cultured at 30° C. for 36 hours. The results are shown in FIG. A strain carrying the tetB gene-containing shuttle vector pBK-PreKmtetB7 exhibited tetracycline resistance equivalent to that of the commercially available 168Δupp (pHY300PLK) carrying the tetracycline resistance gene TcR .
　上記で取得したシャトルベクターｐＢＫ－ＰｒｅＫｍｔｅｔＢ７を用いて、カナマイシン耐性を指標として市販の大腸菌ＪＭ１０９株を形質転換した。当該形質転換株は、形質転換されていない大腸菌が生育できないテトラサイクリン１０ｍｇ／Ｌ含有ＬＢ寒天培地においても生育した。これにより、ｔｅｔＢ遺伝子は、枯草菌に加え大腸菌でもマーカー遺伝子として機能することを確認した。 Using the shuttle vector pBK-PreKmtetB7 obtained above, the commercially available E. coli JM109 strain was transformed with kanamycin resistance as an indicator. The transformant grew even on an LB agar medium containing 10 mg/L of tetracycline, on which non-transformed E. coli cannot grow. As a result, it was confirmed that the tetB gene functions as a marker gene not only in Bacillus subtilis but also in E. coli.
６．枯草菌ｔｅｔＢ遺伝子破壊株の作製
　ｔｅｔＢ遺伝子の上流及び下流それぞれ約１．０ｋｂの領域を、プライマー１８（配列番号１８）及びプライマー１９（配列番号１９）、プライマー２０（配列番号２０）及びプライマー２１（配列番号２１）を用いて、それぞれＰＣＲで増幅し、回収した。両断片を２００ｆｍｏｌ／５０μＬの濃度になるように調製して、プライマー１８及びプライマー２１を用いて二断片ＰＣＲを行い、両断片を連結した。目的のＤＮＡ断片を上記と同様にＴＡクローニングし、ｔｅｔＢ遺伝子破壊用断片を有するプラスミド：ｐＭＤｔｅｔＢ１８Ｆを取得した。続いて、ｐＭＤｔｅｔＢ１８Ｆを制限酵素ＥｃｏＲＩ及びＸｂａＩで消化して得られたｔｅｔＢ破壊用断片を含むＤＮＡ断片と、上記５．で作製したｐＭＤＫｍｕｐｐ１Ｆを制限酵素ＸｂａＩ及びＢａｍＨＩで消化して得られたカナマイシン耐性遺伝子Ｋｍｒ及びｕｐｐ遺伝子を含むＤＮＡ断片を、ｐＨＳＧ２９８（ＴａｋａｒａＢｉｏ社）のＥｃｏＲＩ－ＢａｍＨＩ部位に導入し、ｔｅｔＢ遺伝子破壊用プラスミド：ｐＨＳＧΔｔｅｔＢ１を得た。 6. Preparation of Bacillus subtilis tetB gene-disrupted strain About 1.0 kb region upstream and downstream of the tetB gene, primer 18 (SEQ ID NO: 18) and primer 19 (SEQ ID NO: 19), primer 20 (SEQ ID NO: 20) and primer 21 ( Using SEQ ID NO: 21), each was amplified by PCR and collected. Both fragments were prepared at a concentration of 200 fmol/50 μL, and two-fragment PCR was performed using primer 18 and primer 21 to ligate both fragments. The DNA fragment of interest was TA cloned in the same manner as above to obtain a plasmid: pMDtetB18F having a fragment for disrupting the tetB gene. Subsequently, a DNA fragment containing a tetB-disrupting fragment obtained by digesting pMDtetB18F with restriction enzymes EcoRI and XbaI; A DNA fragment containing the kanamycin resistance gene Kmr and the upp gene obtained by digesting pMDKmupp1F prepared in 3 with restriction enzymes XbaI and BamHI was introduced into the EcoRI-BamHI site of pHSG298 (TakaraBio) to disrupt the tetB gene. : pHSGΔtetB1 was obtained.
　得られたｐＨＳＧΔｔｅｔＢ１を用いて、２．で取得した枯草菌１６８Δｕｐｐ株をコンピテントセル法で形質転換した。カナマイシン耐性を示す形質転換細胞を選抜し、得られた菌株についてＰＣＲを行い、当該菌株ゲノム中のｔｅｔＢ遺伝子の近傍にｐＨＳＧΔｔｅｔＢ１が挿入されていることを確認した。当該菌株を５－フルオロウラシル入り培地で培養し、５－フルオロウラシル耐性を獲得し、且つカナマイシン耐性を喪失した菌株を選抜、ＰＣＲにより染色体上のｔｅｔＢ遺伝子の破壊を確認した。得られた菌株を１６８Δｕｐｐ，ｔｅｔＢ株と命名した。 Using the obtained pHSGΔtetB1, 2. The Bacillus subtilis 168Δupp strain obtained in 1 was transformed by the competent cell method. Transformed cells exhibiting kanamycin resistance were selected, PCR was performed on the resulting strain, and it was confirmed that pHSGΔtetB1 was inserted near the tetB gene in the genome of the strain. The strains were cultured in a medium containing 5-fluorouracil, strains that had acquired 5-fluorouracil resistance and lost kanamycin resistance were selected, and disruption of the tetB gene on the chromosome was confirmed by PCR. The resulting strain was named 168Δupp, tetB strain.
７．ｔｅｔＢ遺伝子とｇａｔＣ遺伝子を用いた酵素発現系の構築
　異種由来の抗生物質耐性遺伝子をもたない枯草菌用ベクターを構築するため、ｐＨＹ３００ＰＬＫを鋳型にプライマー２２（配列番号２２）及びプライマー２３（配列番号２３）を用いてＰＣＲを行い、ｐＨＹ３００ＰＬＫからＴｃＲ遺伝子を除いたベクター断片を得た。枯草菌１６８株のゲノムＤＮＡを鋳型にプライマー２４（配列番号２４）及びプライマー２５（配列番号２５）を用いてＰＣＲを行い、ｔｅｔＢ遺伝子（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＮＣ　０００９６４：４１８７６８１～４１８９１５３）を増幅した。上記ベクター断片と当該ｔｅｔＢ遺伝子断片を、制限酵素ＭｌｕＩとＥｃｏＲＩで消化した上で連結し、ｐＨＹ３００ＰＬＫ－ｔｅｔＢΔｔｅｔｒを構築した。次いで、ｐＨＹ３００ＰＬＫ－ｔｅｔＢΔｔｅｔｒを鋳型に、プライマー２６（配列番号２６）及びプライマー２７（配列番号２７）を用いてＰＣＲを行い、ｐＨＹ３００ＰＬＫ－ｔｅｔＢΔｔｅｔｒからアンピシリン耐性遺伝子を除いたベクター断片を得た。また、枯草菌１６８株のゲノムＤＮＡを鋳型にプライマー２８（配列番号２８）及びプライマー２９（配列番号２９）を用いてプロモーター領域を含むｇａｔＣ遺伝子（ＡＣＣＥＳＳＩＯＮ　Ｎｏ．ＮＣ　０００９６４：７２９７２９～７２９０２２）を増幅し、これらの断片を制限酵素ＣｌａＩとＸｈｏＩで消化して、それぞれの断片を連結し、ｐＨＹＴＢＧＣ２を構築した（図６参照）。 7. Construction of enzyme expression system using tetB gene and gatC gene In order to construct a vector for Bacillus subtilis that does not have a heterologous antibiotic resistance gene, pHY300PLK was used as a template for primer 22 (SEQ ID NO: 22) and primer 23 (SEQ ID NO: 23). 23) to obtain a vector fragment obtained by removing the TcR gene from pHY300PLK. PCR was performed using primer 24 (SEQ ID NO: 24) and primer 25 (SEQ ID NO: 25) using genomic DNA of Bacillus subtilis strain 168 as a template to amplify the tetB gene (ACCESSION No. NC 000964: 4187681 to 4189153). The above vector fragment and the tetB gene fragment were digested with restriction enzymes MluI and EcoRI and ligated to construct pHY300PLK-tetBΔtetr. Next, PCR was performed using pHY300PLK-tetBΔttr as a template and primer 26 (SEQ ID NO: 26) and primer 27 (SEQ ID NO: 27) to obtain a vector fragment from pHY300PLK-tetBΔttr without the ampicillin resistance gene. In addition, the gatC gene (ACCESSION No. NC 000964: 729729 to 729022) containing the promoter region was amplified using primer 28 (SEQ ID NO: 28) and primer 29 (SEQ ID NO: 29) using genomic DNA of Bacillus subtilis strain 168 as a template. , these fragments were digested with restriction enzymes ClaI and XhoI, and the respective fragments were ligated to construct pHYTBGC2 (see FIG. 6).
　得られたｐＨＹＴＢＧＣ２を用いて１６８Δｕｐｐ，ｔｅｔＢ株を形質転換したところ、テトラサイクリン１０ｍｇ／Ｌを含むＬＢ寒天培地でテトラサイクリン耐性を示す形質転換体を得ることが出来た。 When the obtained pHYTBGC2 was used to transform the 168Δupp, tetB strain, a transformant exhibiting tetracycline resistance could be obtained on an LB agar medium containing 10 mg/L of tetracycline.
８．ｇａｔＣ非破壊株でのｇａｔＣ遺伝子を含むシャトルベクター保持の確認
　上記３．の１６８Δｕｐｐ，ｇａｔＣ（ｐＢＫ－ＰｒｏｇａｔＣ１）株を選抜する過程において、テトラサイクリンを含まない５－フルオロウラシル入り培地を用いて培養した際にも、５－フルオロウラシル耐性を獲得し、カナマイシン耐性を喪失し、且つテトラサイクリン耐性を維持する菌株が多数得られていた。これらの菌株について、ｇａｔＣ破壊の確認をＰＣＲで行ったところ、確認した全ての菌株はｇａｔＣ非破壊株であった。この結果は、ｇａｔＣ非破壊株においてもプラスミド上にｇａｔＣ遺伝子が存在していれば、安定的にプラスミドが保持される可能性を示していると考えられたことから、これを検証した。７．で作製したｐＨＹＴＢＧＣ２のＥｃｏＲＩ－ＸｂａＩ部位に酵素遺伝子Ｂ３を導入し、ｐＨＹＴＢＧＣ２－Ｂ３を構築した。６．で作製した枯草菌１６８Δｕｐｐ，ｔｅｔＢ株に、ｐＢＫ－Ｂ３及びｐＨＹＴＢＧＣ２－Ｂ３を、それぞれ導入し、プラスミド上にｇａｔＣ遺伝子を有する形質転換体：１６８Δｕｐｐ，ｔｅｔＢ（ｐＨＹＴＢＧＣ２－Ｂ３）株とプラスミド上にｇａｔＣ遺伝子を有さない形質転換体：１６８Δｕｐｐ，ｔｅｔＢ（ｐＢＫ－Ｂ３）株を得た。得られた各形質転換体を、それぞれテトラサイクリン不含のＬＢ液体培地に植菌し、３０℃で一晩培養した。取得した培養液をテトラサイクリン不含のＬＢ寒天培地に塗布して、３０℃で培養した。培養後、生じたシングルコロニー各株５０個をテトラサイクリン含有ＬＢ培地及びテトラサイクリン不含ＬＢ培地に植え継ぎ、３０℃で培養した。テトラサイクリン不含ＬＢ培地において植菌した全コロニーが生育していることを確認して培養終了とし、培養終了時のテトラサイクリン含有ＬＢ培地における生育コロニー数を、プラスミド保持率として算出した。 8. Confirmation of retention of shuttle vector containing gatC gene in gatC non-disrupted strain 3 above. In the process of selecting the 168 Δupp, gatC (pBK-ProgatC1) strain, even when cultured using a medium containing 5-fluorouracil that does not contain tetracycline, it acquired 5-fluorouracil resistance, lost kanamycin resistance, and tetracycline A number of strains that maintained resistance were obtained. When confirmation of gatC disruption was performed on these strains by PCR, all strains confirmed were gatC non-disruption strains. This result was considered to indicate the possibility that the plasmid could be stably maintained even in the gatC non-disrupted strain if the gatC gene was present on the plasmid, and this was verified. 7. The enzyme gene B3 was introduced into the EcoRI-XbaI site of pHYTBGC2 prepared in 1. to construct pHYTBGC2-B3. 6. pBK-B3 and pHYTBGC2-B3 are respectively introduced into the Bacillus subtilis 168Δupp, tetB strain prepared in , and a transformant having the gatC gene on the plasmid: 168Δupp, tetB (pHYTBGC2-B3) strain and the gatC gene on the plasmid A transformant without : 168Δupp, tetB (pBK-B3) strain was obtained. Each transformant thus obtained was inoculated into a tetracycline-free LB liquid medium and cultured overnight at 30°C. The obtained culture solution was spread on a tetracycline-free LB agar medium and cultured at 30°C. After culturing, 50 single colonies of each strain were transferred to tetracycline-containing LB medium and tetracycline-free LB medium, and cultured at 30°C. After confirming that all colonies inoculated in the tetracycline-free LB medium had grown, the culture was terminated.
　培養終了時の各プレートにおけるコロニーの様子を図７に示す。プラスミド上にｇａｔＣ遺伝子を有する形質転換体：１６８Δｕｐｐ，ｔｅｔＢ（ｐＨＹＴＢＧＣ２－Ｂ３）株はテトラサイクリン含有培地において４９個のコロニーが生育（プラスミド保持率９８％）したのに対し、プラスミド上にｇａｔＣ遺伝子を有さない形質転換体：１６８Δｕｐｐ，ｔｅｔＢ（ｐＢＫ－Ｂ３）株ではテトラサイクリン含有培地において６個のコロニーが生育した（プラスミド保持率１２％）。これにより、プラスミド上にｇａｔＣ遺伝子を有する形質転換体株は、染色体上ｇａｔＣ非破壊株を宿主としたとき（換言すると、当該菌株は、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＣ遺伝子のみをプラスミド上に発現可能な状態で有し、且つ、ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子をいずれも前記染色体上に発現可能な状態で有する）も、抗生物質不含培地での継代培養後も保有するプラスミドを安定的に保持することが分かった。 Figure 7 shows the state of the colonies on each plate at the end of the culture. Transformant having gatC gene on plasmid: 168Δupp, tetB (pHYTBGC2-B3) strain grew 49 colonies (plasmid retention rate 98%) in tetracycline-containing medium, whereas it had gatC gene on plasmid. Transformant: 168Δupp, tetB(pBK-B3) strain grew 6 colonies on tetracycline-containing medium (12% plasmid retention). As a result, the transformant strain having the gatC gene on the plasmid, when the gatC non-disrupted strain on the chromosome is used as the host (in other words, the strain has only the gatC gene among the gatA gene, the gatB gene, and the gatC gene in an expressible state on a plasmid, and having the gatA gene, gatB gene, and gatC gene in an expressible state on the chromosome) also after subculturing in an antibiotic-free medium was found to stably retain the plasmid harboring it.

Claims (12)

  1. 　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子からなる群より選択される一又は二の遺伝子をプラスミド上に発現可能な状態で有し、且つ、
    　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、少なくとも、前記プラスミド上に発現可能な状態で有さない残余の遺伝子を、染色体上に発現可能な状態で有する、形質転換細胞。     Having one or two genes selected from the group consisting of gatA gene, gatB gene, and gatC gene on a plasmid in an expressible state, and
    A transformed cell having, on its chromosome, at least the rest of the gatA gene, gatB gene, and gatC gene, which are not present in an expressible state on the plasmid, in an expressible state.
  2. 　前記群より選択される一又は二の前記遺伝子を、前記染色体上に発現可能な状態で有さない、請求項１に記載の形質転換細胞。 The transformed cell according to claim 1, which does not have one or two of said genes selected from said group in an expressible state on said chromosome.
  3. 　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を前記染色体上に発現可能な状態で有する、請求項１に記載の形質転換細胞。 The transformed cell according to claim 1, which has the gatA gene, the gatB gene, and the gatC gene on the chromosome in an expressible state.
  4. 　ｇａｔＣ遺伝子を前記プラスミド上に発現可能な状態で有する、請求項１～３のいずれか１項に記載の形質転換細胞。 The transformed cell according to any one of claims 1 to 3, which has the gatC gene on the plasmid in an expressible state.
  5. 　ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を前記プラスミド上に発現可能な状態で有さず、且つ、
    　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子のうち、ｇａｔＡ遺伝子及びｇａｔＢ遺伝子のみを前記染色体上に発現可能な状態で有する、請求項４に記載の形質転換細胞。     does not have the gatA gene and the gatB gene on the plasmid in an expressible state, and
    5. The transformed cell according to claim 4, which has only the gatA gene and the gatB gene among the gatA gene, the gatB gene, and the gatC gene in an expressible state on the chromosome.
  6. 　ｇａｔＡ遺伝子及びｇａｔＢ遺伝子を前記プラスミド上に発現可能な状態で有さず、且つ、
    　ｇａｔＡ遺伝子、ｇａｔＢ遺伝子、及びｇａｔＣ遺伝子を前記染色体上に発現可能な状態で有する、請求項４に記載の形質転換細胞。     does not have the gatA gene and the gatB gene on the plasmid in an expressible state, and
    5. The transformed cell according to claim 4, which has the gatA gene, the gatB gene, and the gatC gene on the chromosome in an expressible state.
  7. 　ｔｅｔＢ遺伝子を前記プラスミド上に発現可能な状態で有する、請求項１～６のいずれか１項に記載の形質転換細胞。 The transformed cell according to any one of claims 1 to 6, which has the tetB gene on the plasmid in an expressible state.
  8. 　前記形質転換細胞がバチルス属に属する細菌である、請求項１～７のいずれか１項に記載の形質転換細胞。 The transformed cell according to any one of claims 1 to 7, wherein the transformed cell is a bacterium belonging to the genus Bacillus.
  9. 　前記形質転換細胞が枯草菌である、請求項１～８のいずれか１項に記載の形質転換細胞。 The transformed cell according to any one of claims 1 to 8, wherein the transformed cell is Bacillus subtilis.
  10. 　前記形質転換細胞が大腸菌である、請求項７に記載の形質転換細胞。 The transformed cell according to claim 7, wherein said transformed cell is Escherichia coli.
  11. 　前記プラスミドが、目的タンパク質をコードする遺伝子の塩基配列をさらに含む、請求項１～１０のいずれか１項に記載の形質転換細胞。 The transformed cell according to any one of claims 1 to 10, wherein the plasmid further contains a nucleotide sequence of a gene encoding a target protein.
  12. 　請求項１１に記載の形質転換細胞を用いて目的タンパク質を生産する方法。

          A method for producing a target protein using the transformed cell according to claim 11 .
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Citations (1)

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JP2009017840A (en) * 2007-07-13 2009-01-29 Japan Agengy For Marine-Earth Science & Technology Method for stably holding extraneous gene in cell

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Publication number Priority date Publication date Assignee Title
JP2009017840A (en) * 2007-07-13 2009-01-29 Japan Agengy For Marine-Earth Science & Technology Method for stably holding extraneous gene in cell

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