WO2021187560A1 - Preparation method for genetically modified cells having enhanced endogenous gene expression - Google Patents

Preparation method for genetically modified cells having enhanced endogenous gene expression Download PDF

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WO2021187560A1
WO2021187560A1 PCT/JP2021/011093 JP2021011093W WO2021187560A1 WO 2021187560 A1 WO2021187560 A1 WO 2021187560A1 JP 2021011093 W JP2021011093 W JP 2021011093W WO 2021187560 A1 WO2021187560 A1 WO 2021187560A1
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seq
base sequence
sequence represented
same
nucleic acid
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洋一郎 伊藤
石井　純
近藤　昭彦
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国立大学法人神戸大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—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
    • 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
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell.
  • the present invention relates to a method for screening an endogenous gene that enhances.
  • the gene recombination method is widely used for the production of industrially useful biomaterials such as antibodies, enzymes, and cytokines for medical and diagnostic purposes.
  • Hosts for producing the target protein by the gene recombination method include animals such as chickens, animal cells such as CHO, insects such as silk moth, insect cells such as sf9, and microorganisms such as yeast, Escherichia coli, and actinomycetes. It is used.
  • yeast can be cultivated on a large scale and at high density in an inexpensive medium, so that the target protein can be produced at low cost, and if a signal peptide or the like is used, the target protein is secreted into the culture solution.
  • Komagataella pastoris a type of yeast, is a methanol-utilizing (Mut + ) yeast that has excellent protein expression capacity and can utilize an inexpensive carbon source that is advantageous for industrial production.
  • Non-Patent Document 1 reports a method for producing a heterologous protein such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataera pastris. ing.
  • a heterologous protein such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataera pastris. ing.
  • When heterologous proteins are produced in yeast in order to improve their productivity, addition of signal sequences, utilization of strong promoters, codon modification, co-expression of chaperon genes, co-expression of transcription factor genes, proteases derived from host yeast Various attempts have been made such as gene inactivation and examination of culture conditions.
  • the present invention is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell. It is an object of the present invention to provide a screening method for an endogenous gene that enhances the protein.
  • a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced which comprises the following steps.
  • (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the start codon of the endogenous gene.
  • a step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
  • the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). the method of. (1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46. (2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47. (3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48. (4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
  • nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238, (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239, (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240, (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253, (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254, (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255, (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256, (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
  • (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258, (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259, (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260, (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
  • a gene set containing an endogenous gene in which a highly expressive promoter is operably linked in which a plasmid containing a nucleic acid fragment is homologously recombined with a linear nucleic acid in which a nucleic acid fragment is cleaved with a restriction enzyme.
  • the nucleic acid fragment was a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, and the partial sequence into which the restriction enzyme recognition site was inserted and the end codon were sequentially linked.
  • a recombinant cell containing a base sequence.
  • the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). Genetically modified cells.
  • nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238, (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239, (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240, (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253, (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254, (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255, (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256, (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
  • (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258, (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259, (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260, (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
  • [13] The genetically modified cell according to any one of [7] to [12], which comprises a base sequence encoding a target protein in its genome.
  • a method for producing a target protein which comprises the step of culturing the recombinant cells according to [13] or [14].
  • a method for screening an endogenous gene that enhances the production of a target protein which comprises the following steps.
  • a method for screening an endogenous gene that enhances the production of a target protein which comprises the following steps.
  • each endogenous gene By providing a method for enhancing the expression of each endogenous gene, it has become possible to easily construct a transgenic cell library in which the expression of each endogenous gene is enhanced. In addition, the use of the library has made it possible to screen for endogenous genes that increase the production of the target protein.
  • the present invention is a method for producing a recombinant cell with enhanced expression of an endogenous gene (hereinafter, the method for producing a recombinant cell of the present invention). I will provide a.
  • the recombinant cell refers to a cell in which a linear nucleic acid described later is introduced and the expression of an endogenous gene is enhanced by the gene recombination.
  • the recombinant cell before the introduction of the linear nucleic acid may be referred to as a host cell.
  • the host cell is not particularly limited as long as it is a cell into which a linear nucleic acid can be introduced.
  • the biological species of the recombinant cell and the host cell are not particularly limited, and examples thereof include yeast, bacteria, fungi, insect cells, animal cells and plant cells, with yeast being preferred and methanol assimilation. Sex yeast, fission yeast, and germination yeast are more preferable, and methanol-utilizing yeast is even more preferable.
  • methanol-utilizing yeast is defined as yeast that can be cultivated using methanol as the only carbon source. Originally, it was methanol-utilizing yeast, but it was assimilated into methanol by artificial modification or mutation. Yeasts that have lost their performance are also included in the methanol-utilizing yeasts of the present invention.
  • yeasts belonging to the genus Pichia, Ogataea, Komagataella, etc. yeasts of the genus Komagataella or yeasts of the genus Ogataea are preferable, and yeasts of the genus Komagataela are particularly preferable. ..
  • a strain derived from these yeast strains of the genus Komagataera can also be used, and examples thereof include Komagataera pastris GS115 strain (available from Thermo Fisher Scientific Co., Ltd.) as a histidine requirement.
  • a non-homologous recombination mechanism disrupting strain ( ⁇ ku70, ⁇ dnl4) of Komagataera fafi can also be used.
  • strains derived from these strains and the like can also be used.
  • the endogenous gene includes not only DNA but also its mRNA and cDNA among the nucleic acids possessed by the cell of the present invention, but can be typically DNA. In particular, it can be genomic DNA. Further, the endogenous gene does not ask the distinction of the functional region, and may contain, for example, only exons, or may contain exons and introns.
  • the endogenous gene is not particularly limited as long as it is a gene contained in the cell of the present invention.
  • it is composed of the following endogenous genes (1) to (32) for the purpose of enhancing the production of the target protein described later.
  • At least one endogenous gene selected from the group is exemplified.
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49 (EF1st-5).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50 (EF1st-6).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51 (EF1st-7).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52 (EF1st-8).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53 (EF1st-9).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54 (EF1st-10).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55 (EF1st-11).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56 (EF1st-12).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57 (EF1st-13).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58 (EF1st-14).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59 (EF1st-15).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60 (EF1st-16).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61 (EF1st-17).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76 (EF3rd-6).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77 (EF3rd-7).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78 (EF3rd-8), and (32) the base represented by SEQ ID NO: 79 (EF3rd-9).
  • At least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32) is preferably selected from the group consisting of the following (a) to (g) endogenous genes. It may be at least two endogenous genes.
  • C An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
  • the endogenous gene may further include the following endogenous genes in addition to at least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32). (33) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • Examples of the endogenous gene include the following combinations.
  • A An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • B An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71 (EF3rd-1), and the same or substantially the same as the base sequence represented by SEQ ID NO: 75 (EF3rd-5).
  • An endogenous gene containing the same base sequence. (C) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
  • An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
  • D An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4), and the same base sequence represented by SEQ ID NO: 73 (EF3rd-3). Or an endogenous gene containing substantially the same base sequence.
  • the base sequence substantially the same as the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79 is about 85% or more of the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79. , Preferably about 90% or more, most preferably about 95% or more base sequences and the like.
  • identity means the optimum alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is used for the optimum alignment of sequences. It means the ratio (%) of the same base sequence to the total base sequence that overlaps in (which can consider the introduction of a gap in one or both).
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • enhanced expression of an endogenous gene means that the expression level of mRNA, which is a transcript of the endogenous gene, or polypeptide, which is a translation product, is enhanced.
  • the expression level of mRNA can be quantified by using a real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using a DNA array, or the like, and the expression level of a polypeptide recognizes a polypeptide. It can be quantified using a staining compound or the like having binding property to an antibody or a polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
  • the degree of enhancement of the expression of the endogenous gene is not particularly limited as long as the production amount of the target protein described later is enhanced, but the transcript or translation product of the endogenous gene is not particularly limited.
  • the expression level is enhanced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more. Is preferable.
  • the method for producing a recombinant cell of the present invention includes the following steps. (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked. (2) The step of introducing the linear nucleic acid into a host cell, and (3) the endogenous gene is homologously recombined by the linear nucleic acid, and a highly expressive promoter is operably linked. A step of selecting transgenic cells containing a sex gene.
  • the method for producing a transgenic cell of the present invention includes a step (step (1)) of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme.
  • the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked. It is a nucleic acid fragment.
  • the highly expressive promoter (hereinafter, the highly expressive promoter of the present invention) is not particularly limited as long as it is a promoter that enhances the expression of the endogenous gene of the cell of the present invention.
  • the type of the highly expressive promoter of the present invention may be any promoter suitable for the cells of the present invention.
  • the highly expressive promoter of the present invention is preferably the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like.
  • the cell of the present invention is a bacterium of the genus Escherichia
  • high expression promoter of the present invention trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
  • the highly expressive promoter of the present invention is preferably the SPO1 promoter, SPO2 promoter, penP promoter or the like.
  • the highly expressive promoter of the present invention is preferably an ADH promoter, a CMV (cytomegalovirus) promoter or the like.
  • the highly expressive promoter of the present invention is preferably a polyhedrin promoter, a P10 promoter or the like.
  • the highly expressive promoters of the present invention are SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, HSV. -TK (herpes simplex virus thymidine kinase) promoter and the like are preferred.
  • the highly expressive promoter of the present invention is preferably the CaMV (cauliflower mosaic virus) 35S promoter or the like.
  • the partial sequence (hereinafter referred to as the partial sequence of the present invention) is a partial sequence starting from the start codon of the endogenous gene of the cell of the present invention and into which a restriction enzyme recognition site is inserted.
  • the nucleotide sequence information of the partial sequence starting from the start codon of the endogenous gene of the cell of the present invention can be obtained from the nucleotide sequence information described in a known database.
  • the nucleotide sequence information starting from the start codon of all endogenous genes of Komagataera fafi is the nucleotide sequence information (ACCESSION No.) of the four chromosomal DNAs of the Komagataera fafi CBS7435 strain. It can be obtained from FR839628 to FR839631 (J. Biotechnol.154 (4), 312-320 (2011)).
  • the nucleotide sequence information starting from the starting codon of the entire endogenous gene of Komagataera pastris is the nucleotide sequence information of the four chromosomal DNAs of the Komagataera pastris NBRC 0948 strain (Mattanovich). Et al., Microbial Cell Factories 8, 29 (2009)), and the nucleotide sequence information of the four chromosomal DNAs of the Komagataera Pastris GS115 strain (ACCESSION No. FN392319 to FN392322 (Nat. Biotechnol. 27 (6), 561- It can be obtained from 566 (2009))). Based on the base sequence information obtained in this way, a partial sequence starting from the start codon of the endogenous gene can be designed.
  • the length of the partial sequence of the present invention is not particularly limited as long as it is the length at which homologous recombination occurs between the endogenous gene and the linear nucleic acid, and is, for example, 20 bases or more, 50 bases or more, and 100 bases. Long or longer, 150 bases or longer.
  • the length of the partial sequence of the present invention is, for example, 1000 bases or less, 750 bases or less, 500 bases or less, or 250 bases or less.
  • the partial sequence of the present invention is a partial sequence in which a restriction enzyme recognition site is inserted.
  • the restriction enzyme recognition site is not particularly limited as long as it is a restriction enzyme recognition site present only in the partial sequence in the entire base sequence of the plasmid containing the above nucleic acid fragment.
  • Examples of restriction enzyme recognition sites inserted into the partial sequences of the present invention include sites recognized by type IIS type restriction enzymes, such as BspQI, BbsI, BsaI, and BsmBI, whose recognition sites and cleavage sites are different.
  • the above restriction enzyme recognition site may be inserted at any site within the partial sequence of the present invention, but the partial sequence of the present invention is divided to such an extent that recombination occurs homologously with the endogenous gene. It is preferably inserted in position.
  • the insertion position of the restriction enzyme recognition site in such a partial sequence is usually inserted in the middle of the partial sequence of the present invention.
  • the restriction enzyme recognition site is inserted by substituting the 92nd base.
  • the number of the restriction enzyme recognition sites inserted in the partial sequence of the present invention is not particularly limited as long as it is present only in the partial sequence in the entire base sequence of the plasmid containing the nucleic acid fragment, but is usually limited.
  • the number is one to several, preferably one or two.
  • the stop codon (hereinafter referred to as the stop codon of the present invention) is either TAA, TAG, or TGA.
  • the nucleic acid fragment (hereinafter referred to as the nucleic acid fragment of the present invention) includes a highly expressive promoter of the present invention, a partial sequence of the present invention, and a base sequence in which the stop codon of the present invention is linked in this order. That is, in the nucleic acid fragment of the present invention, the carbon at the 3'end of the highly expressive promoter of the present invention and the carbon at the 5'end of the partial sequence of the present invention form a phosphodiester bond, and the 3'of the partial sequence of the present invention. The terminal carbon and the 5'terminal carbon of the termination codon of the present invention form a phosphodiester bond.
  • a spacer sequence may be inserted between the highly expressive promoter of the present invention and the partial sequence of the present invention, and between the partial sequence of the present invention and the stop codon of the present invention.
  • the length of the spacer sequence may be appropriately determined by those skilled in the art, and may be, for example, 15 to 25 bases in length.
  • the nucleic acid fragment of the present invention uses the genomic DNA fraction prepared from the cells of the present invention as a template, and a primer is used from the base sequence information of the highly expressive promoter and the endogenous gene described in a known database. It can be designed and directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method").
  • PCR method Polymerase Chain Reaction
  • the restriction enzyme recognition site into which the partial sequence of the present invention is inserted can be introduced into the partial sequence by a known site-specific mutagenesis method.
  • the nucleic acid fragment of the present invention can be obtained by outsourcing the production to Agilent Technologies.
  • the length of the nucleic acid fragment of the present invention is not particularly limited, but is, for example, 20 bases or more, 50 bases or more, 100 bases or more, 150 bases or more, or 200 bases or more.
  • the length of the partial sequence of the present invention is, for example, 1000 bases or less, 500 bases or less, or 400 bases or less.
  • the nucleic acid fragment of the present invention is the following (i) to (xxxii). ) Is at least one nucleic acid fragment selected from the group consisting of nucleic acid fragments.
  • nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (EF1st-2 OLS), (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS), (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), (Iv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241 (EF1st-5 OLS), (V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242 (EF1st-6 OLS), (Vi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243 (EF1st-7 OLS), (Vii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244 (EF1st-8 OLS), (
  • At least one nucleic acid fragment selected from the group consisting of the above nucleic acid fragments (i) to (xxxii) is preferably selected from the group consisting of the following nucleic acid fragments (a') to (g'). It may be at least two nucleic acid fragments.
  • A' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS)
  • B' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS)
  • C' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS)
  • D' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS)
  • E' Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS)
  • nucleic acid fragment may further contain the following nucleic acid fragments in addition to at least one nucleic acid fragment selected from the group consisting of the above-mentioned nucleic acid fragments (i) to (xxxii).
  • Xxxiii A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 237 (EF1st-1 OLS).
  • nucleic acid fragment examples include the following combinations.
  • A Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS), Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS).
  • D A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS).
  • a plasmid is an artificially constructed nucleic acid molecule.
  • the nucleic acid molecule that constitutes the plasmid is usually DNA, preferably double-stranded DNA.
  • plasmids include YEp vector, YRp vector, YCp vector, pPICHOLI, pHIP (Journal of General Microbioiogy (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha), pHRP (pHIP).
  • pHARS Molecular and General Genetics MGG February1986, Volume 202, Issue 2, pp 302-308, Transformation of the methylotrophic yeast Hansenula polymorpha by automatic replication and integration vector UC), derived from E. coli pBR322, pBluescript, pQE), plasmid vector derived from bacillus (pHY300PLK, pMTLBS72) and the like can be used.
  • the above plasmids further utilize cloning sites containing one or more restriction enzyme recognition sites, Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, and the like.
  • auxotrophic complementary genes include URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene and the like.
  • drug resistance genes include G418 resistance gene, Zeocin TM resistance gene, hyglomycin resistance gene, Clone NAT resistance gene, blastsaidin S resistance gene, noseoslisin resistance gene and the like.
  • step (1) the plasmid containing the nucleic acid fragment obtained as described above is cleaved with a restriction enzyme capable of cleaving the restriction enzyme recognition site contained in the partial sequence of the present invention to form a linear nucleic acid (hereinafter referred to as the present invention).
  • Linear nucleic acid is prepared.
  • the latter half of the partial sequence of the present invention (homologous sequence 1) linked to the stop codon is placed at the 5'end and highly expressed at the 3'end.
  • a linear nucleic acid can be prepared in which the first half (homologous sequence 2) of the partial sequence of the present invention linked to the sex promoter is arranged.
  • the method for producing a recombinant cell of the present invention includes a step (step (2)) of introducing the linear nucleic acid of the present invention into a host cell.
  • a method for introducing the linear nucleic acid of the present invention into a host cell that is, a transformation method
  • a known method can be appropriately used.
  • yeast cells are used as host cells
  • an electroporation method, a lithium acetate method, or a spheroplast method can be used. Examples thereof include the spheroplast method, but the method is not particularly limited thereto.
  • the method for producing a recombinant cell of the present invention is a gene recombination containing an endogenous gene in which an endogenous gene is homologously recombined by the linear nucleic acid of the present invention and a highly expressive promoter is operably linked.
  • the step of selecting cells (step (3)) is included.
  • the linear nucleic acid of the present invention introduced into the host cell is the latter half (homologous sequence 1) and 3'of the partial sequence of the present invention linked to the stop codon located at the 5'end.
  • Homologous recombination single crossover recombination
  • the first half homologous sequence 2 of the partial sequence of the present invention linked to the highly expressive promoter located at the end as a homologous region for the endogenous gene.
  • the linear nucleic acid of the present invention is inserted between the homologous sequence 1 and the homologous sequence 2 contained in the endogenous gene, and the endogenous gene originally provided in the host cell is the linear of the present invention.
  • step (3) when a transgenic cell containing an endogenous gene operably linked to a highly expressive promoter (hereinafter referred to as the recombinant cell of the present invention) is selected, a auxotrophic complementary gene or drug resistance is selected. It is preferable to use a selectable marker gene such as a gene.
  • the selection marker is not particularly limited, but if the host cell is yeast of the genus Komagataera, if it is an auxotrophic complementary gene such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, uracil, leucine, adenin, respectively.
  • the genetically modified cells of the present invention can be selected by restoring the auxotrophic strain phenotype in the auxotrophic strains of yeast and arginine.
  • it is a drug resistance gene such as G418 resistance gene, Zeocin TM resistance gene, Hyglomycin resistance gene, Clone NAT resistance gene, Blasticidin S resistance gene, G418, Zeocin TM, Hyglomycin, Clone, respectively.
  • the recombinant cells of the present invention can be selected by resistance on a medium containing NAT and Blasticidin S.
  • the auxotrophic selectable marker used when producing the recombinant yeast cannot be used if the selectable marker is not destroyed in the host yeast. In this case, the selectable marker may be destroyed in the host yeast, and a method known to those skilled in the art can be used as the method.
  • the present invention provides transgenic cells with enhanced expression of endogenous genes (hereinafter, the recombinant cells of the present invention).
  • the genetically modified cell of the present invention can be prepared by the method for producing a genetically modified cell of the present invention.
  • the recombinant cell of the present invention can function as a highly expressive promoter in which an endogenous gene is homologously recombined with a linear nucleic acid in which a plasmid containing a nucleic acid fragment is cleaved with a limiting enzyme.
  • the nucleic acid fragment is a highly expressive promoter, a partial sequence starting from the starting codon of the endogenous gene, and the restriction enzyme recognition site is inserted.
  • a recombinant cell containing a base sequence in which a partial sequence and a termination codon are sequentially linked.
  • the gene-recombinant cell, endogenous gene, nucleic acid fragment, plasmid, restriction enzyme, highly expressive promoter, partial sequence, etc. in the gene-recombinant cell of the present invention are described in the method for producing a gene-recombinant cell of the present invention. May be the same as.
  • the recombinant cell of the present invention may contain a base sequence encoding a target protein in its genome.
  • the target protein is a protein produced by a cell whose genome contains a base sequence encoding the target protein, and may be an endogenous protein of the cell or a heterologous protein.
  • the target protein include enzymes derived from microorganisms, proteins produced by animals and plants that are multicellular organisms, and the like.
  • Examples thereof include phytase, protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, fluorescent protein and the like.
  • phytase protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, fluorescent protein and the like.
  • human and / or animal therapeutic proteins are preferred.
  • hepatitis B virus surface antigen As proteins for human and / or animal treatment, specifically, hepatitis B virus surface antigen, hirudin, antibody, human antibody, partial antibody, human partial antibody, serum albumin, human serum albumin, epithelial growth factor, human epithelial growth.
  • Factors insulin, growth hormone, erythropoetin, interferon, blood coagulation factor VIII, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), thrombopoetin, IL-1, IL-6, Tissue plasminogen activator (TPA), urokinase, leptin, stem cell growth factor (SCF) and the like.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • TPA Tissue plasminogen activator
  • urokinase urokinase
  • the antibody refers to a heterotetramer protein composed of two polypeptide chains, an L chain and an H chain, formed by disulfide bonds, especially if it has the ability to bind to a specific antigen. Not limited.
  • the partial antibody refers to a Fab antibody, (Fab) 2 antibody, scFv antibody, diabody antibody, camel VHH antibody, derivatives thereof, etc., as long as it has the ability to bind to a specific antigen.
  • Fab antibody refers to a heteromer protein in which the L chain and Fd chain of an antibody are bound by an SS bond, or a heteromer protein in which the L chain and Fd chain of an antibody are associated without an SS bond. It is not particularly limited as long as it has the ability to combine.
  • amino acids constituting the above-mentioned target protein may be natural, non-natural, or modified.
  • amino acid sequence of the protein may be artificially modified or may be designed by de-novo.
  • the base sequence encoding the above-mentioned target protein is contained in an expression vector, and is integrated by homologous recombination into an arbitrary site in the genome of the recombinant cell of the present invention.
  • the expression vector can be produced, for example, by cutting out a DNA fragment containing a base sequence encoding the above-mentioned target protein and linking the DNA fragment downstream of a promoter in an appropriate expression vector.
  • Expression vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacteriophage-derived plasmids (eg, pUB110, pTP5, pC194); Yeast-derived plasmids (eg, pSH19, pSH15); Insect cell expression.
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacteriophage-derived plasmids eg, pUB110, pTP5, pC194
  • Yeast-derived plasmids eg, pSH19, pSH15
  • Insect cell expression include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacteriophage-derived plasmids
  • Plasmid eg pFast-Bac
  • animal cell expression plasmid eg pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • bacteriophage such as ⁇ phage
  • insect virus vector such as baculovirus (eg) Example: BmNPV, AcNPV)
  • Animal virus vectors such as retrovirus, vaccinia virus, and adenovirus are used.
  • the promoter may be any promoter as long as it is suitable for the host used for gene expression, and for example, the highly expressive promoter of the present invention is preferably mentioned.
  • a vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication, or the like can be used, if desired.
  • the selectable marker include a dihydrofolate reductase (dhfr) gene, an ampicillin resistance gene, a neomycin resistance gene and the like.
  • dhfr gene-deficient Chinese hamster cells when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selectable marker, the target gene can also be selected using a thymidine-free medium.
  • a base sequence (signal codon) encoding a signal sequence suitable for the host cell is added (or replaced with a native signal codon) to the 5'end side of the base sequence encoding the target protein. May be good.
  • the host cell is a bacterium of the genus Escherichia, the PhoA signal sequence, the OmpA signal sequence, etc .; if the host cell is a bacterium of the genus Bacillus, the ⁇ -amylase signal sequence, the subtilisin signal sequence, etc.; If the host cell is an animal cell, the insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. are used, respectively.
  • the expression vector is a partial sequence of the genome of the recombinant cell of the present invention, and contains a partial sequence in which a restriction enzyme recognition site is inserted.
  • the linear expression vector cleaved by the restriction enzyme was introduced into the recombinant cell of the present invention and placed at the latter half (homologous sequence 1) and 3'end of the partial sequence arranged at the 5'end.
  • Homologous recombination single crossover recombination
  • the base sequence encoding the target protein of the present invention is inserted into the genome of the recombinant cell of the present invention.
  • a method for producing a target protein (hereinafter, a method for producing a target protein of the present invention), which comprises a step of culturing a recombinant cell of the present invention containing a base sequence encoding the target protein in the genome.
  • the cell culture conditions are not particularly limited and may be appropriately selected according to the cells.
  • any medium containing a nutrient source capable of assimilating cells can be used.
  • the nutrient source include sugars such as glucose, shoe cloth and maltose, organic acids such as lactic acid, acetic acid, citric acid and propionic acid, alcohols such as methanol, ethanol and glycerol, hydrocarbons such as paraffin and soybean oil. Oils such as rapeseed oil, carbon sources such as mixtures thereof, nitrogen sources such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, and other inorganic salts, vitamins, etc.
  • a normal medium in which nutrient sources are appropriately mixed can be used.
  • the culture can be either batch culture or continuous culture.
  • the carbon source may be one kind of glucose, glycerol, and methanol, or two or more kinds. Further, these carbon sources may be present from the initial stage of culturing, or may be added during culturing.
  • the target protein By culturing the recombinant cell of the present invention containing the base sequence encoding the target protein in the genome, the target protein can be accumulated and recovered in the cell or in the culture medium.
  • a known purification method can be used in an appropriate combination. For example, first, the recombinant cells of the present invention containing the nucleotide sequence encoding the target protein in the genome are cultured in an appropriate medium, and the cells are removed from the culture supernatant by centrifugation or filtration of the culture medium.
  • the obtained culture supernatant is subjected to salting (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone, ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography.
  • the target protein is recovered from the culture supernatant by using techniques such as imaging, affinity chromatography, reverse phase chromatography, and ultrafiltration alone or in combination.
  • the cells can usually be cultured under general conditions.
  • the cells are aerobically cultured for 10 hours to 10 days in a pH range of 2.5 to 10.0 and a temperature range of 10 ° C to 48 ° C. It can be done by.
  • the recovered target protein can be used as it is, but it can also be used afterwards with modifications that bring about pharmacological changes such as PEGylation and modifications that add functions such as enzymes and isotopes. Moreover, various formulation treatments may be used.
  • the endogenous gene originally provided in the host cell loses its function due to the stop codon contained in the linear nucleic acid of the present invention. Instead, downstream of the dysfunctional gene results in a new gene (the same gene as the endogenous gene) operably linked to the highly expressive promoter contained in the linear nucleic acid of the invention.
  • the expression of the endogenous gene is enhanced by the highly expressive promoter. Therefore, the recombinant cell population of the present invention in which the expression of each gene of all endogenous genes of the host cell is enhanced can be used as a cell library in which the expression of each endogenous gene is enhanced.
  • the screening method 1 of the present invention includes the following steps. (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked. (2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding a target protein in the genome.
  • the screening method 1 of the present invention is a step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a stop codon of the endogenous gene.
  • the step (step (1)) is included, which comprises a partial sequence starting from, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked.
  • the step (1) in the screening method 1 of the present invention may be the same as the step (1) in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention is a step of introducing the linear nucleic acid of the present invention into a host cell (hereinafter, a cell expressing the target protein of the present invention) containing a base sequence encoding the target protein in the genome (step (2)). )including.
  • the method for introducing the linear nucleic acid of the present invention into the target protein-expressing cell of the present invention may be the same as that described in step (2) in the method for producing a recombinant cell of the present invention.
  • the target protein may be the same as the target protein contained in the genome of the recombinant cell of the present invention.
  • the host cell may be the same as the host cell used in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention selects transgenic cells containing an endogenous gene operably linked with a highly expressive promoter, in which the endogenous gene is homologously recombined by the linear nucleic acid of the present invention. Includes step (step (3)).
  • the step (3) in the screening method 1 of the present invention may be the same as the step (3) in the method for producing a recombinant cell of the present invention.
  • the screening method 1 of the present invention includes a step (step (4)) of culturing the cells obtained in the step (3) and the target protein-expressing cells of the present invention.
  • the method for culturing the cells obtained in step (3) and the target protein-expressing cells of the present invention may be the same as the culturing method described in the method for culturing the recombinant cells of the present invention.
  • the screening method 1 of the present invention includes a step (step (5)) of measuring the production amount of the target protein by the cells obtained in the step (3) and the target protein-expressing cells, respectively.
  • the amount of target protein produced by the cells obtained in step (3) and the target protein-expressing cells of the present invention is measured by measuring the expression level of mRNA, which is a transcript of the target protein, or polypeptide, which is a translation product. It can be carried out.
  • the expression level of mRNA can be quantified by using real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using DNA array, etc., and the expression level of polypeptide can be determined by an antibody that recognizes a polypeptide or It can be quantified using a staining compound or the like having binding property to the polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
  • the screening method 1 of the present invention includes a step (step (6)) of identifying an endogenous gene that increases the production amount of the target protein.
  • step (6) if the production amount of the target protein in the cells obtained in step (3) is higher than the production amount of the target protein in the target protein-expressing cells of the present invention, the cells increase the production amount of the target protein. It can be determined that the cell contains an endogenous gene.
  • the increase in the production amount of the target protein in the cells obtained in step (3) is, for example, 1.01 times, 1.02 times, 1.03 times, 1.04 times, 1.05 times the production amount of the target protein in the target protein-expressing cells of the present invention.
  • the amount of total protein secreted and produced from the cells can be easily determined by a method known to those skilled in the art, such as the Bladeford method, the Lowry method, the BCA method, etc., using the cell culture supernatant or the like. Can be decided.
  • the amount of secreted production of a specific target protein from cells can be easily determined by an ELISA method or the like using a cell culture supernatant or the like.
  • the endogenous gene whose expression is enhanced by a known means.
  • the genome is extracted from cells, fragmented with a restriction enzyme, and then the fragmented genome is self-ligated. Genome fragments containing the endogenous gene of interest cyclized by self-ligation can be screened by drug resistance genes.
  • the endogenous gene can be specifically identified by specifying the nucleotide sequence of the screened genomic fragment containing the target endogenous gene by the Sanger method.
  • the screening method 2 of the present invention includes the following steps. (1) A step of introducing an expression vector containing a base sequence encoding a target protein into a cell library overexpressing an endogenous gene of the present invention and a host cell and culturing the cells. (2) A step of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell, and (3) a step of identifying the endogenous gene that increases the production amount of the target protein.
  • the screening method 2 of the present invention includes a step (step (1)) of introducing an expression vector containing a base sequence encoding a target protein into a host cell and an endogenous gene overexpressing cell library of the present invention and culturing the cells.
  • the method of introducing the endogenous gene overexpressing cell library of the present invention and the expression vector containing the base sequence encoding the target protein into the host cell and culturing the method is the method of introducing the linear nucleic acid of the present invention into the host cell and the present invention. It may be the same as that described in the method for culturing transgenic cells of the present invention.
  • the screening method 2 of the present invention includes a step (step (2)) of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell.
  • the method for measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell may be the same as the method for measuring the production amount of the target protein in the screening method 1 of the present invention.
  • the screening method 2 of the present invention includes a step (step (3)) of identifying an endogenous gene that increases the production of the target protein.
  • the method for identifying the endogenous gene that increases the production of the target protein may be the same as the method for identifying the endogenous gene in the screening method 1 of the present invention.
  • GAP promoter SEQ ID NO: 1
  • AOX1 promoter SEQ ID NO: 2
  • AOX1 terminator SEQ ID NO: 3
  • CCA38473 terminator SEQ ID NO: 4
  • ARG4 gene SEQ ID NO: 5
  • the downstream sequence (SEQ ID NO: 6), GUT1 gene (SEQ ID NO: 7), the gene encoding KAR2 (SEQ ID NO: 8) derived from Komagataera fafi to which the terminator is linked, and the gene encoding PDI1 (SEQ ID NO: 9) are Komagataera.
  • the chromosomal DNA of the Faffy CBS7435 strain (the base sequence is described in EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) was prepared by PCR using a mixture as a template.
  • the gene (SEQ ID NO: 45-79) encoding the antibody expression-promoting protein group (amino acid sequence shown by SEQ ID NOs: 10 to 44) derived from Komagataera fafi to which the terminator is linked is the genome of Komagataera fafi obtained by screening. Prepared by PCR as a template.
  • the GAP promoter is Primer 1 (SEQ ID NO: 80) and Primer 2 (SEQ ID NO: 81)
  • the AOX1 promoter is Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83)
  • the AOX1 terminator is Primer 5 (SEQ ID NO: 84) and Primer.
  • CCA38473 terminator is primer 7 (SEQ ID NO: 86) and primer 8 (SEQ ID NO: 87), promoter-regulated ARG4 gene is primer 9 (SEQ ID NO: 88) and primer 10 (SEQ ID NO: 89),
  • the URA3 gene lacking the starting codon and having the AscI-PmeI recognition site added inside is Primer 11 (SEQ ID NO: 90), Primer 12 for mutagenesis (SEQ ID NO: 91), and Primer 13 for mutagenesis (SEQ ID NO: 92).
  • primer 14 (SEQ ID NO: 93), the GUT1 gene lacking the starting codon and having the AscI-PmeI recognition site added internally is primer 15 (SEQ ID NO: 94) and primer 16 for mutagenesis (SEQ ID NO: 95),
  • the gene encoding the mutation-introducing primer 17 (SEQ ID NO: 96) and primer 18 (SEQ ID NO: 97), and the antibody expression-promoting protein (amino acid sequence shown by SEQ ID NOs: 10 to 44) to which the terminator is linked is a primer for forward. It was prepared by PCR using 19 (SEQ ID NO: 98) and primers 20 to 55 (SEQ ID NO: 99 to 134) for each reverse.
  • the secretory signal MF ⁇ gene (SEQ ID NO: 135) used in the construction of the vector is the chromosomal DNA of the Saccharomyces cerevisiae BY4741 strain (the base sequence is described in ACCESSION No. BK006934 to BK006949). It was prepared by PCR using Primer 57 (SEQ ID NO: 137).
  • the diamino acid substitution (L42S / V50A) (SEQ ID NO: 138) of the secretory signal MF ⁇ gene was prepared by PCR using synthetic DNA as a template and primers 56 (SEQ ID NO: 136) and 57 (SEQ ID NO: 137).
  • the promoter-controlled Zeocin TM resistance gene (SEQ ID NO: 139) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-regulated G418 resistance gene (SEQ ID NO: 140) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-controlled hygromycin resistance gene (SEQ ID NO: 141) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-regulated noseoslisin resistance gene (SEQ ID NO: 142) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the promoter-controlled blastidin resistance gene (SEQ ID NO: 143) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
  • the anti-lysothium single-chain antibody gene (SEQ ID NO: 144), tandem scFv226 antibody gene (SEQ ID NO: 145), blinatumomab antibody gene (SEQ ID NO: 146) and minibody antibody (SEQ ID NO: 147) used in the construction of the vector are synthetic DNAs. Was used as a template and prepared by PCR.
  • the KAR2 gene (SEQ ID NO: 8) and PDI1 gene (SEQ ID NO: 9) used in the construction of the vector were prepared by PCR using the genome of the Komagataera fafi CBS7435 strain as a template.
  • Prime STAR HS DNA Polymerase (manufactured by Takara Bio Inc.) was used for PCR, and the reaction conditions were as described in the attached manual.
  • the chromosomal DNA was prepared from the Komagataera pastris ATCC76273 strain or the Saccharomyces cerevisiae BY4741 strain using Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka Corporation) under the conditions described therein.
  • pUC_G418 a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using Primer 7 (SEQ ID NO: 86) and Primer 8 (SEQ ID NO: 87), and the above pUC_G418 was mixed with the XbaI-treated nucleic acid fragment and In. -pUC_T38473_G418 was constructed by connecting using the fusion HD Cloning Kit (manufactured by Clontech).
  • nucleic acid fragment having a BamHI recognition sequence and a SpeI recognition sequence added to the end of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83). BamHI and SpeI treatment, pUC_T38473_G418 was inserted between the BamHI-BglII sites to construct pUC_Paox1_T38473_G418.
  • nucleic acid fragment having the MluI recognition sequence and the BglII recognition sequence added to the end of the AOX1 terminator (SEQ ID NO: 3) was prepared by PCR using Primer 5 (SEQ ID NO: 84) and Primer 6 (SEQ ID NO: 85).
  • pUC_Paox1_T38473_G418 was inserted between the MluI-BglII sites to construct pUC_Paox1_Taox1_T38473_G418.
  • nucleic acid fragment having a SpeI recognition sequence and a BglII recognition sequence added to the ends of the secretory signal MF ⁇ gene (SEQ ID NO: 135) and its two amino acid substitutions (L42S / V50A) (SEQ ID NO: 138) was added to Primer 56 (SEQ ID NO: 136).
  • primer 57 SEQ ID NO: 137, respectively, after treatment with SpeI and BglII, each was inserted between the MluI-BglII sites of pUC_Paox1_Taox1_T38473_G418 to construct pUC_Paox1_MF ⁇ _Taox1_T38473_G418 and pUC_Paox1_MF ⁇ (mut) _43.
  • nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the anti-lysozyme antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Pgap_MF ⁇ _Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the anti-lysodium single-chain antibody prepared by the above PCR, and the In-fusion HD Cloning Kit was used.
  • Bispecific antibody tandem sc Fv226 expression vector Bispecific antibody (tandem scFv226; taFv226) gene (SEQ ID NO:) in which an anti-CD3 single-chain antibody and an anti-EGFR single-chain antibody are fused. 145) was prepared by PCR using synthetic DNA as a template and Primer 62 (SEQ ID NO: 154) and Primer 63 (SEQ ID NO: 155).
  • This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substitution (L42S / V50A)) as an overlapping region upstream of the base sequence encoding the taFv226 antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream.
  • the terminal region is added.
  • a nucleotide sequence encoding a c-Myc tag (SEQ ID NO: 156) and a His tag (SEQ ID NO: 153) are added between the nucleotide sequence encoding the taFv226 antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Paox1_MF ⁇ (mut) _Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the bispecific antibody (taFv226) prepared by PCR above.
  • a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using primer 64 (SEQ ID NO: 157) and primer 65 (SEQ ID NO: 158), and the above pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_T38473_G418 was treated with the nucleic acid fragment treated with XbaI.
  • the mixture was mixed and spliced using an In-fusion HD Cloning Kit (manufactured by Clontech) to construct pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_loxP_T38473_loxP_G418.
  • This vector is designed for bispecific antibody (taFv226) to be expressed under AOX1 promoter control.
  • the blinatumomab gene (SEQ ID NO: 146), which is the only bispecific low molecular weight antibody currently on the market, is used as a template for primer 66 (SEQ ID NO: 159) and primer. Prepared by PCR using 67 (SEQ ID NO: 160).
  • This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the brinatsumomab antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream. The terminal region is added.
  • nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the blinatumomab antibody and the upstream terminal region of the AOX1 terminator sequence.
  • the pUC_Paox1_MF ⁇ (mut) _taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above was treated with XhoI and MluI to prepare a nucleic acid fragment, mixed with the nucleic acid fragment having the nucleotide sequence encoding the blinatumomab antibody prepared by the above PCR, and the In-fusion HD Cloning Kit.
  • minibody antibody expression vector Primer 68 of the minibody gene (SEQ ID NO: 147), which is a small antibody fused with an anti-EGFR single-chain antibody gene and a part of the Fc region, using synthetic DNA as a template. It was prepared by PCR using (SEQ ID NO: 161) and primer 69 (SEQ ID NO: 162). This nucleic acid fragment is the downstream terminal region of the secretory signal MF ⁇ gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the minibody antibody, and the AOX1 terminator sequence downstream as an overlapping region. The upstream end region is added.
  • a nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the minibody antibody and the upstream terminal region of the AOX1 terminator sequence.
  • Nucleic acid fragments were prepared by treating pPGP_EGFP with BamHI and SpeI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pPGPdel_EGFP.
  • a nucleic acid fragment was prepared by PCR using primer 76 (SEQ ID NO: 169) and primer 77 (SEQ ID NO: 170) using pPGPdel_EGFP as a template. ..
  • nucleic acid fragment of CYC1 terminator (SEQ ID NO: 171) derived from Saccharomyces cerevisiae
  • PCR using Saccharomyces cerevisiae BY4741 strain as a template and Primer 78 (SEQ ID NO: 172) and Primer 79 (SEQ ID NO: 173) was performed.
  • Nucleic acid fragments were prepared after treating pUC_del_Zeo with SacI and BamHI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-EGFP-CYC1t.
  • this vector can be used as a vector for producing a gene overexpressing cell library using Komagataera fafi. Is designed for.
  • KAR2 and PDI1 gene overexpression vector Primer 70 (SEQ ID NO: 163) and primer were added to the nucleic acid fragment in which the EcoRI recognition sequence was added to both ends of the Zeocin TM resistance gene (SEQ ID NO: 139) controlled by the promoter. It was prepared by PCR using 71 (SEQ ID NO: 164) and inserted between the EcoRI sites of pUC19 to construct pUC_Zeo.
  • nucleic acid fragments of the first half and the second half of the ARG4 gene (SEQ ID NO: 5) regulated by the promoter are subjected to Primer 9 (SEQ ID NO: 88), Primer 80 for mutation insertion (SEQ ID NO: 174), and Primer 81 (SEQ ID NO: 81).
  • Primer 9 SEQ ID NO: 88
  • Primer 80 for mutation insertion SEQ ID NO: 174
  • Primer 81 SEQ ID NO: 81
  • a pUC_Arg4_Zeo having a sequence into which a PmeI restriction enzyme site (SEQ ID NO: 176) was introduced was constructed.
  • a nucleic acid fragment having a HindIII recognition sequence added to one end of the EGFP expression cassette (GAPDH promoter-EGFP gene-AOX1t) was added to pPGP_EGFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018).
  • nucleic acid fragment in which the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene encoding the KAR2 and PDI1 proteins to which the terminator was ligated was added to Primer 84 (SEQ ID NO: 181) and Prepared by PCR using Primer 85 (SEQ ID NO: 182), Primer 86 (SEQ ID NO: 183) and Primer 87 (SEQ ID NO: 184), mixed with SpeI and HindIII treated pUC_Arg4_Pgap_EGFP_Taox1_Zeo, and used with In-fusion HD Cloning Kit.
  • This vector is designed to overexpress KAR2 or PDI1 as a model protein under the control of the GAPDH promoter.
  • pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo 36 types of pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (amino acid sequences shown in SEQ ID NOs: 10 to 44) are each expressed under the control of the GAPDH promoter.
  • nucleic acid fragments of the first half and the second half of the URA3 gene (SEQ ID NO: 6) from which the starting codon was deleted were subjected to primer 90 (SEQ ID NO: 187), primer 91 for mutagenesis (SEQ ID NO: 188), and mutagenesis, respectively.
  • primer 90 SEQ ID NO: 187
  • primer 91 for mutagenesis SEQ ID NO: 188
  • mutagenesis respectively.
  • Primer 92 SEQ ID NO: 189
  • Primer 93 SEQ ID NO: 190
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_URA3_Pgap_EGFP_Taox1_Hyg and mixed.
  • pUC_URA3_Pgap_EF2nd-4_Hyg Eight types of pUC_URA3_Pgap_EF2nd-4_Hyg were constructed from pUC_URA3_Pgap_EF1st-1_Hyg by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • nucleic acid fragments of the first half and the second half of the GUT1 gene (SEQ ID NO: 7) from which the starting codon was deleted were used as primer 95 (SEQ ID NO: 193), primer 96 for mutagenesis (SEQ ID NO: 194), and mutagenesis, respectively.
  • primer 95 SEQ ID NO: 193
  • primer 96 for mutagenesis SEQ ID NO: 194
  • mutagenesis respectively.
  • pUC_Gut1_Pgap_EGFP_Taox1_NAT having a sequence in which the AscI-PmeI restriction enzyme site (SEQ ID NO: 191) was introduced in the center of the downstream sequence (0.7Kb).
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_Gut1_Pgap_EGFP_Taox1_NAT and mixed.
  • pUC_GUT1_Pgap_EF2nd-4_NAT Eight types of pUC_GUT1_Pgap_EF2nd-4_NAT were constructed from pUC_GUT1_Pgap_EF1st-1_NAT by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • nucleic acid fragment of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 100 (SEQ ID NO: 198) and Primer 101 (SEQ ID NO: 199), mixed with NheI and PstI-treated pUC_Arg4_Pgap_EGFP_Taox1_bsd, and In- By connecting using the fusion HD Cloning Kit, pUC_Paox1_Pgap_EGFP_Taox1_bsd having the AOX1 promoter sequence as the genome transfer site was constructed.
  • the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked.
  • Nucleic acid fragments were prepared by PCR using forward primers 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NOs: 99-106) and mixed with SpeI and HindIII treated pUC_Paox1_Pgap_EGFP_Taox1_bsd.
  • pUC_Paox1_Pgap_EF2nd-4_bsd Eight types were constructed from pUC_Paox1_Pgap_EF1st-1_bsd by connecting them using the In-fusion HD Cloning Kit.
  • This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
  • Blinatumomab antibody expression vector pUC_Paox1_MF ⁇ (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and the minibody antibody vector pUC_Paox1_MF ⁇ (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above.
  • Komagataera fafi Dnl4 deficient / histidine auxotrophic strain (Ito et al., FEMS Yeast Research, Vol. 18, No.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (Manufactured by Becton Dickinson), 2% glucose) 2 ml, shake-cultured at 30 ° C. for 16 hours, subcultured in fresh YPD medium at 10-fold dilution, and further shake-cultured at 30 ° C. for 4 hours. After culturing yeast cells are collected by centrifugation, the yeast cells are washed (suspended by adding 6 ml of sterilized water, and the yeast cells are collected by centrifugation), and then the yeast cells are re-suspended with the sterilized water remaining on the test tube wall.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (Manufactured by Becton Dickinson), 2% glucose) 2 ml
  • yeast cells are collected, suspended in 500 ⁇ l of YPD medium (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose), and then 30 The mixture was allowed to stand at ° C for 2 hours. After standing for 2 hours, yeast cells were subjected to YPDG418 selection agar plate (1% dried yeast extract (manufactured by Nakaraitesk), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate.
  • YPD medium 1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate.
  • a strain that is applied to salt (manufactured by Nakaraitesk Co., Ltd.) and grows in a static culture at 30 ° C. for 3 days is selected, and anti-lysodium single-stranded antibody-expressing yeast, tandem scFv226 antibody-expressing yeast, brinatsumomab antibody-expressing yeast, and Minibody antibody-expressing yeast was obtained.
  • Gene activation was confirmed by PCR using the chromosomal DNA of transformed yeast as a template for fragment length and internal sequence sequence analysis of amplified nucleic acid fragments, gene expression analysis, and the like. As a result, it was confirmed that the desired gene was activated in the transformed yeast obtained in Example 7.
  • bacto manufactured by Becton Dickinson
  • polypeptone manufactured by Nippon Pharmaceutical Co., Ltd.
  • yeast nitrogen base Without Amino Acid and Ammonium Sulfate manufactured by Becton Dickinson
  • 1% Ammonium Sulfate 0.4 mg / l Biotin, 100 mM phosphate Potassium (pH 6.0), 2% Glycerol
  • BMMY medium 1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 200 ⁇ l of preculture solution was subcultured in 0.4 mg / l Biotin, 100 mM potassium phosphate (pH 6.0), 2% Methanol), and this was cultured with shaking at 30 ° C., 170 rpm, 48 hours, and then centrifuged (12,000 rpm, 5 minutes). , 4 ° C) to collect the culture supernatant.
  • Protein L dissolved in (for Ray Biotech, tandem scFv226 antibody, brinattumomab antibody, minibody antibody) was added in 50 ⁇ l of each well and incubated overnight at 4 ° C. After incubation, the solution in the well was removed, blocked with 200 ⁇ l of immunoblock (manufactured by Sumitomo Dainippon Pharma), and allowed to stand at room temperature for 1 hour.
  • immunoblock manufactured by Sumitomo Dainippon Pharma
  • TMB-1 Component Microwell Peroxidase Substrate SureBlue manufactured by KPL
  • the absorbance at 450 nm was measured with a microplate reader (Envison; manufactured by PerkinElmer). The quantification of each small molecule antibody in the culture supernatant was performed using the calibration curve of each standard antibody.
  • Nucleic acid fragments with EcoRI recognition sequences added to both ends of the promoter-controlled noseoslisin resistance gene (SEQ ID NO: 142) and hyglomycin resistance gene (SEQ ID NO: 141) were added to Primer 88 (SEQ ID NO: 185) and Primer 94 (SEQ ID NO: 141), respectively. 192) and Primer 88 (SEQ ID NO: 185) and Primer 89 (SEQ ID NO: 186) were prepared by PCR and inserted into the EcoRI site of pUC-2 after EcoRI treatment to construct pUC2_NAT and pUC2_Hyg.
  • nucleic acid fragments of the first half and the second half of the MRP40 terminator were subjected to primer 116 (SEQ ID NO: 220), primer 117 for mutagenesis (SEQ ID NO: 221), and primer 118 for mutagenesis (SEQ ID NO: 221), respectively.
  • primer 116 SEQ ID NO: 220
  • primer 117 for mutagenesis SEQ ID NO: 221
  • primer 118 for mutagenesis SEQ ID NO: 221)
  • nucleic acid fragments of the first half and the second half of the ARG83 terminator were subjected to primer 120 (SEQ ID NO: 225), primer 121 for mutagenesis (SEQ ID NO: 226), and primer 122 for mutagenesis (SEQ ID NO: 226), respectively.
  • primer 120 SEQ ID NO: 225
  • primer 121 for mutagenesis SEQ ID NO: 226)
  • primer 122 for mutagenesis SEQ ID NO: 226)
  • pUC2_Hyg_TARG83 with a sequence into which the site (GGCGCGCC) was introduced.
  • Example 1 Preliminary study for preparation of Komagataera fafi gene overexpressing cell library As a gene overexpressing cell library that replaces the conventional cDNA library and genome library, the endogenous promoters of all yeast genes are highly expressed (GAPDH promoter).
  • GPDH promoter highly expressed
  • a plasmid library is prepared by treating pUC_del_Zeo_Pgap-EGFP-CYC1t prepared in (7) above with SpeI and XhoI, mixing with the above double-stranded OLS sequence, and connecting them using the In-fusion HD Cloning Kit.
  • the plasmid of the above library is linearly converted with the restriction enzyme BspQI, and the linear plasmid is inserted into the target position (each GeneX) on the genome of Komagataera fafi by homologous recombination by single crossover integration. ..
  • the base sequence from the start codon to the 91st and the base sequence from the 93rd to the 183rd of the Komagataera fafi gene KAR2 or PDI1 are extracted, and the 3'end sequence of the GAPDH promoter, the restriction enzyme SpeI recognition site, and the start codon to the 91st.
  • TGA termination codon
  • the KAR2 overexpression OLS sequence and the PDI1 overexpression OLS sequence linked in the order of the restriction enzyme recognition cleavage site XhoI and the CYC1 terminator derived from Saccharomyces cerevisiae were prepared (Fig. 2, SEQ ID NO: 200 (KAR2)). , SEQ ID NO: 201 (PDI1)).
  • Nucleic acid fragments were prepared after treating the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t of (7) above with SpeI and XhoI, and the KAR2 overexpressing OLS sequence and PDI1 overexpressing OLS sequence prepared as described above were used. They were mixed and spliced together using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-KAR2OLS-CYC1t and pUC_del_Zeo_Pgap-PDI1OLS-CYC1t.
  • the OLS sequence for overexpression of KAR2 or PDI1 was placed downstream of the GAPDH promoter derived from Komagataera fafi, and it was confirmed by the Sanger method that the nucleotide sequence was appropriate. After linearly converting these plasmids with the restriction enzyme BspQI, they were introduced into Komagataera fafi DNL4 deficient and histidine-requiring strains (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018). It was inserted into the target site (KAR2 gene or PDI1 gene) by homologous recombination.
  • Primer sequence 102 (SEQ ID NO: 202) and primer sequence 103 (SEQ ID NO: 203) that sandwich the KAR2 gene on the genome, and primer sequence 104 (SEQ ID NO: 204) and primer sequence 105 (SEQ ID NO: 205) that sandwich the PDI1 gene. ) was designed, and the insertion of the above-mentioned plasmid into the target site of the Komagataera fafi genome was verified by the colony PCR method. The position of the band of the PCR product on agarose gel electrophoresis showed that each plasmid was inserted into the target site on the genome.
  • KAR2 or PDI1 gene of the inserted strain was overexpressed was evaluated by using the RT-qPCR method for the transcription amount of KAR2 and PDI1.
  • KAR2 overexpressing strain and PDI1 overexpressing strain using RNeasy kit (manufactured by Kiagen), reverse transcription using the reverse transcription kit (ReverTraAce qPCR RT Master Mix, manufactured by TOYOBO).
  • a photoreaction was performed, and the amount of KAR2 and PDI1 transcriptions of each strain was quantified using a quantitative PCR kit (KOD SYBR® qPCR Mix, manufactured by TOYOBO).
  • Primer sequence 106 (SEQ ID NO: 206) and primer sequence 107 (SEQ ID NO: 207) are used to quantify the amount of KAR2 transcription
  • primer sequence 108 (SEQ ID NO: 208) and primer sequence 109 (SEQ ID NO: 209) are used to quantify the amount of PDI1 transcription. It was used.
  • the ACT1 gene of Komagataera fafi was used, and primer sequence 110 (SEQ ID NO: 210) and primer sequence 111 (SEQ ID NO: 211) were used.
  • KAR2 overexpressing strains and PDI1 overexpressing strains as controls having the plasmids pUC_Arg4_Pgap_KAR2_T37552_Zeo and pUC_Arg4_Pgap_PDI1_T37552_Zeo in which the KAR2 and PDI1 genes and their terminator regions were introduced downstream of the GAPDH promoter were prepared and compared, respectively) were prepared and compared.
  • RT-qPCR it was shown that the KAR2 and PDI1 overexpressing strains prepared by the above method overexpressed their respective genes in the same manner as the respective control strains (Fig. 3). The above results indicate that it is possible to prepare a cell library overexpressing the Komagataera fafi gene by this method.
  • Example 2 Preparation of Komagataera fafi gene overexpressing cell library Based on the results of Example 1, the above OLS sequences were designed (Fig. 2) and prepared for all Komagataera fafi genes (5,001 genes) (Agilent). ⁇ Agilent). In order to convert the obtained total OLS sequence into double-stranded DNA, DNA is amplified by PCR using primers 112 (SEQ ID NO: 212) and primer 113 (SEQ ID NO: 213) complementary to both ends of the OLS sequence. I let you.
  • the double-stranded OLS sequence was ligated to the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t described in (7) above using the In fusion method, and the Escherichia coli DH5 ⁇ strain was transformed with the obtained plasmid.
  • the obtained transformant colonies (4x10 5 pieces) were collected from a plurality of plates, and a plasmid was extracted using a Plasmamid Plus Midi kit (Qiagen) to prepare a plasmid library.
  • the 12 transformant colonies obtained here were cultured in LB medium, the plasmid was extracted, and the nucleotide sequence of the OLS sequence contained in the plasmid was confirmed by the Sanger method.
  • the obtained plasmid library was cleaved with the restriction enzyme BspQI, DNA was purified, and then introduced into a Komagataera fafi strain secreting small molecule antibodies (anti-lysothium scFv antibody, tandem scFv226, blinatumomab antibody) using an electroporation method. ..
  • Example 3 High-throughput screening using the amount of small molecule antibody secreted as an index
  • the transformant group (gene overexpressing cell library) obtained by the electroporation method in Example 2 was selected as a colony picker (PM-2, microtechnition).
  • a square plate of YPD agar medium supplemented with Zeocin was arranged in a 96-well format and colonized by culturing at 30 ° C., and this was used as a master plate. From the master plate, 96 strains were simultaneously inoculated using 96 pins into a deep well plate containing 0.5 mL of BMGY medium. After stirring and culturing at 30 ° C.
  • Example 4 Identification of useful factors The genes overexpressed in the obtained screening positive strain were identified by the following methods (Fig. 4) ((a) to (f) below are (a) to (a) to 4 in FIG. 4). Corresponds to (f)).
  • Gen Toru-kun manufactured by Takara Bio Inc.
  • Genomic DNA was fragmented by simultaneous treatment with multiple restriction enzymes (using any of the following (i) to (iii)).
  • i) After treating the genomic DNA with the 5'protruding end-type restriction enzymes EcoRI, SalI, NheI, BamHI and ClaI, the 5'protruding end of the fragmented DNA was blunt-ended with Klenow fragment.
  • Genomic DNA was treated with 5'protruding end-type restriction enzymes BamHI, BclI and BglII (the 5'protruding end has the same base sequence (GATC)).
  • Genomic DNA was treated with blunt-ended restriction enzymes BsaAI, BsaBI, BstZ17I, HpaI, PmlI, SnaBI and StuI.
  • Fragmented DNA was cyclized by self-ligation.
  • Escherichia coli DH5 ⁇ strain was transformed with cyclized DNA on LB agar medium supplemented with ampicillin and zeocin.
  • a plasmid was extracted from the transformant.
  • the overexpression vectors of 36 genes determined by the above method (36 expression vectors from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo) were applied to the Arg4 site of each small molecule antibody-producing strain used in Example 2 ( A strain introduced by the method described in 13) was prepared, and it was examined whether or not the amount of small molecule antibody secreted increased.
  • EF1st-1 has also been reported separately by the inventors. From the above, among the genes listed in Table 4, 32 types of factors excluding EF1st-1, EF2nd-8, EF3rd-2 and EF3rd-4 were newly discovered in this study. Next, it was investigated whether the useful factors obtained in a specific antibody-producing strain also affect the increase in the productivity of other antibodies.
  • Eighteen useful factor expression vectors (18 types from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF1st-18_T37552_Zeo) obtained from anti-lysothium scFv antibody-producing strains were introduced into tandem scFv226 antibody and blinatumomab antibody-producing strains. ..
  • the tandem scFv226 antibody-producing strain was 0.9 to 1.6 times (Table 5), and the blinatumomab antibody-producing strain was 1.0 to 2.3 times as much as the host strain.
  • the anti-lysothium scFv antibody-producing strain was 1.0 to 1.2 times (Table 7), and the blinatumomab antibody-producing strain was 1.0 to 1.8 times as much as the host strain. (Table 8).
  • Example 5 Development of a high-producing small molecule antibody strain by accumulating useful factors
  • the effect of promoting the production of small molecule antibodies by the useful factors obtained in the above screening is relatively low, 1.1 to 1.6 times (in tandem scFv226 production).
  • Met In order to increase the amount of small molecule antibody secreted, the accumulation of useful factors was examined. This time, a total of 8 of the top 4 factors (EF1st-1 to 4, Table 1) obtained from the anti-lysozyme scFv antibody strain and the top 4 factors (EF2nd-1 to 4, Table 2) obtained from the tandem scFv226 antibody strain. Factors were selected. By accumulating these, we aimed to prepare a high-producing strain of tandem scFv226 antibody.
  • each of the eight selected factors was introduced into the tandem scFv226-producing parent strain by the method described in (13) above, and the amount of tandem scFv226 antibody secreted was evaluated.
  • the high stock was used as the next-generation parent stock.
  • one gene-deficient strain (A strain) that produces tandem scFv226 antibody was used as the parent strain, and only the useful factor EF1st-1, which increased the amount of taFv226 antibody secreted most, was introduced into the ARG4 site, and the tandem scFv226 antibody was introduced. The amount of secretion was evaluated. As a result, a strain B in which the amount of tandem scFv226 antibody secreted was increased by about 1.5 times as compared with the strain A was obtained (Table 11). With strain B as the second-generation parent strain, each of the eight factors selected above was introduced into the URA3 site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF2nd-4 introduced strain (C strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain B (Table 12). ).
  • the C strain was used as the third-generation parent strain, and the eight factors selected above were introduced into the GUT1 site, respectively, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF1st-4 introduced strain (D strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain C (Table 13). ).
  • each of the 8 factors selected above was introduced into the AOX1 promoter site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above.
  • the EF2nd-1-introduced strain had the highest amount of secretion, and secreted about 1.3 times as much antibody as the parent strain D (Table 14).
  • This strain was designated as E strain.
  • E strain the E strain into which the four useful factors were introduced secreted tandem scFv226 antibody, which was about 2.9 times higher than that of the A strain before the introduction of the useful factors.
  • Example 6 Exchange of antibody expression cassette from antibody-producing strain If the antibody expression cassette of the antibody-producing strain can be inserted and removed, different antibodies can be produced in the tandem scFv226 high-producing strain prepared in Example 5. Therefore, we examined the exchange of gene expression cassettes using the Cre-loxP system. Specifically, when a loxP sequence (34 base length) is inserted at both ends of the genome transfer sequence of Komagataera fafi of the antibody expression cassette, the expression cassette introduced into Komagataera fafi has loxP sequences at both ends. (Fig. 6). Next, a plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No.
  • Cre recombinase gene regulated by a methanol-induced AOX1 promoter is introduced into an antibody-producing strain using a Zeo marker. ..
  • Cre recombinase expression at the leak level of the AOX1 promoter deletes the nucleotide sequence between loxPs.
  • the deletion of the antibody expression cassette can be evaluated by the colony PCR method using primers designed at both ends of the genome introduction site or the presence or absence of a drug marker in a drug assay.
  • a different antibody expression vector By introducing a different antibody expression vector into the same genome insertion site of the strain from which this antibody cassette has been deleted, expression from the tandem scFv226 antibody of the tandem scFv226 high-producing strain to a different antibody becomes possible (Fig. 6).
  • the protein expression cassette was exchanged using green fluorescent protein (GFP) and red fluorescent protein gene (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein gene
  • a gene encoding EGFP SEQ ID NO: 214
  • E2crimson SEQ ID NO: 215
  • pPGP_GFP As the GFP expression vector, pPGP_GFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was used.
  • This vector is designed for GFP to be expressed under the control of the GAPDH promoter.
  • the RFP gene was prepared by PCR using synthetic DNA as a template and primer 114 (SEQ ID NO: 216) and primer 115 (SEQ ID NO: 217).
  • This nucleic acid fragment is prepared after SpeI and XhoI treatment, mixed with the nucleic acid fragment of the nucleotide sequence encoding RFP prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to obtain pPGP_RFP. It was constructed.
  • This vector is designed so that RFP is expressed under the control of the GAPDH promoter.
  • FIG. 7 shows the process of exchanging the reporter gene from the GFP-expressing strain to the RFP-expressing strain using the Komagataera fafi wild strain.
  • a strain into which a GFP expression cassette (pPGP_GFP fragment linearly converted at the EcoRV site in the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site of Komagataera fafi (Fig. 7 (1)) was introduced into the above (13). It was produced by the method described in (Fig. 7 (2)). This strain was G418 resistant, and GFP fluorescence was confirmed by flow cytometry (FCM).
  • FCM flow cytometry
  • Example 7 Evaluation of antibody high-producing strains with different small molecule antibodies
  • loxP sequences were introduced at both ends of the sequence for introduction of the antibody expression vector into the CCA38473 terminator site. Therefore, loxP sequences were introduced at both ends of the antibody cassette introduced into the yeast genome (Fig. 8). Therefore, if Cre recombinase is expressed in yeast cells, the tandem scFv226 antibody expression cassette can be removed.
  • pPAP_CP Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018
  • E1, E2, E3 strains were prepared by the method described in (13) above.
  • the amount of each antibody secreted was evaluated by the ELISA method and SDS-PAGE, the amount of antibody secreted was 10 times or more in the anti-lysothium scFv antibody-producing strain and 3 times or more in the blinatumomab antibody-producing strain as compared with the host strain. confirmed.
  • the amount of antibody secreted was confirmed to be about twice that of the D3 strain derived from the D strain and about three times that of the E3 strain derived from the E strain (Fig. 9). From the above results, it was shown that the stocks of useful factors obtained by using the productivity of the tandem scFv226 antibody as an index have high productivity not only in the production of the tandem scFv226 antibody but also in different small molecule antibodies.
  • Example 8 Preparation of antibody production promoting protein overexpressing cell library for pair screening and screening of high-producing strains As shown in Example 5, the accumulation experiment conducted using eight kinds of useful factors showed low accumulation of useful factors. It was shown to be effective in promoting the production and secretion of molecular antibodies. Therefore, as a method for efficiently accumulating the obtained useful factors, a yeast library in which two kinds of useful factors were overexpressed at the same time was prepared, and a strain that secretes the most small molecule antibody from the library is shown in Example 3. We devised to find out using a high-throughput screening method (pair screening, FIG. 10).
  • a plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 81).
  • the prepared plasmid library was introduced into the blinatumomab secretory strain prepared in (13) above to obtain about 2,400 transformants.
  • the transformants that appeared were screened using eight 96-well deep-well plates by the high-throughput screening method shown in Example 3.
  • the amount of blinatumomab secreted in the culture supernatant was evaluated by the ELISA method using the His tag as an index.
  • the amount of antibody secretion increased about 5 times as compared with the host strain (Table 15).
  • EF3rd-1 was introduced in both of them.
  • EF3rd-2 and EF3rd-5 were inserted.
  • the blinatumomab hypersecretory strain E2 strain (4 useful factor-introduced strains) obtained in the useful factor accumulation experiment in Example 7 had an antibody secretion amount of about 3.2 times that of the host strain. It was shown that the highly secreted strain (2 useful factor-introduced strain) obtained in 1) secreted a higher amount of blinatumomab antibody. Next, pair screening was performed in the second cycle using these two strains as parent strains. This time, a total of 10 EF1st-1 to 6 and EF2nd-1 to 4 were selected as useful factors for promoting blinatumomab secretion obtained in Example 2, and the combination was optimized.
  • a useful factor cassette (GAPDH promoter-useful factor) using two primer sets (primer 128 (SEQ ID NO: 233) and primer 129 (SEQ ID NO: 234), primer 130 (SEQ ID NO: 235) and primer 131 (SEQ ID NO: 236)). DNA was amplified by PCR using ORF and its terminator sequence). These two types of PCR products and pUC2_Hyg_TARG83 cleaved with BamHI and XhoI are mixed so that they have the same number of DNA molecules, and they are joined using the In-fusion HD Cloning Kit to obtain two useful factor expression cassettes.
  • a plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 100).
  • the prepared plasmid library was introduced into the two types of blinatumomab hypersecretory strains shown above to obtain about 4,400 transformants. From the transformants that appeared, 9 96-well well plates (864 strains) were screened by the above high-throughput screening method.
  • the amount of secretion was 13 to 15 times higher than that of the host strain. Was shown (Table 15).
  • EF1st-1 was introduced into all the strains (Table 15).
  • the other useful factors were EF2nd-4 (same gene as EF3rd-2) (2nd_11-7 strain and 2nd_11-8 strain) and EF1st-3 (2nd_12-2 strain and 2nd_12-7 strain) (Table). 15).
  • the blinatumomab antibody hypersecreting strain contained useful factors in a combination different from the optimal combination in the tandem scFv226-producing strain shown in Example 7. It is considered that there is an optimal combination of useful factors depending on the modality and amino acid sequence of the small molecule antibody.
  • Example 9 Pair screening with different small molecule antibodies
  • the pair screening method was repeated twice to obtain a high-producing strain of blinatumomab antibody.
  • the useful factors selected as the optimal combination in the blinatumomab hypersecretory strain were EF1st-1, EF1st-3, EF2nd-4, EF3rd-1 and EF3rd-5.
  • a pair screening method using the blinatumomab antibody shown in Example 8 was carried out to try to identify useful factors to be selected.
  • Small molecule antibody (tandem scFv226) secretory strain prepared in (13) above using a plasmid library consisting of 9 useful factors (EF3rd-1-9) prepared in the first cycle of pair screening in Example 8. Introduced into, 1,200 transformants were obtained. From the transformants that appeared, screening for 4 96-well deep-well plates (384 strains) was performed by the high-throughput screening method shown in Example 3. The amount of taFv226 secreted in the culture supernatant was evaluated by the ELISA method shown in (15) above.
  • Example 8 In the first generation pair screening in Example 8, the optimal combination of useful factors was EF3rd-1 and EF3rd-2 (EF2nd-4), EF3rd-1 and EF3rd-5, and the tandem scFv226 secretion of this example. It was different from EF3rd-2 (EF2nd-4) and EF3rd-3 obtained by screening using strains. That is, from this result, it was shown that the optimum combination of useful factors for the secretion of each small molecule antibody is different.

Abstract

The present invention provides: a preparation method for genetically modified cells having enhanced endogenous gene expression; a genetically modified cell prepared by said method; and a method for screening for an endogenous gene that enhances the production of a target protein by using an endogenous gene-overexpressing cell library.

Description

内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法Method for producing recombinant cells with enhanced expression of endogenous gene
　本発明は、内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法、該方法によって製造された遺伝子組換え細胞、該細胞を含む内在性遺伝子過剰発現細胞ライブラリを用いた目的タンパク質の生産を増強する内在性遺伝子のスクリーニング方法に関する。 The present invention is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell. The present invention relates to a method for screening an endogenous gene that enhances.
　医療及び診断に活用するための抗体、酵素、及びサイトカイン等の産業上有用なバイオマテリアルの生産には、遺伝子組換え法が広く用いられている。遺伝子組換え法によって目的タンパク質を生産するための宿主としては、ニワトリ等の動物、CHO等の動物細胞、カイコ等の昆虫、sf9等の昆虫細胞、並びに酵母、大腸菌、及び放線菌等の微生物が用いられている。宿主生物の中でも酵母は安価な培地で大規模かつ高密度での培養が可能であるため目的タンパク質を低コストで生産することができること、シグナルペプチド等を利用すれば培養液中へ目的タンパク質の分泌も可能なため目的タンパク質の精製過程も容易となること、及び真核生物であるため糖鎖付加等の翻訳後修飾も可能であること等から、非常に有益であり、様々な研究が行われている。酵母において種々の目的タンパク質に対応できる革新的な生産技術を開発することができれば、生産性の劇的向上によるコスト競争力の強化に加え、幅広い産業展開が期待できる。 The gene recombination method is widely used for the production of industrially useful biomaterials such as antibodies, enzymes, and cytokines for medical and diagnostic purposes. Hosts for producing the target protein by the gene recombination method include animals such as chickens, animal cells such as CHO, insects such as silk moth, insect cells such as sf9, and microorganisms such as yeast, Escherichia coli, and actinomycetes. It is used. Among host organisms, yeast can be cultivated on a large scale and at high density in an inexpensive medium, so that the target protein can be produced at low cost, and if a signal peptide or the like is used, the target protein is secreted into the culture solution. It is very useful because it is possible to facilitate the purification process of the target protein, and because it is a eukaryotic organism, post-translational modification such as glycosylation is possible, and various studies have been conducted. ing. If we can develop innovative production technology that can handle various target proteins in yeast, we can expect a wide range of industrial development in addition to strengthening cost competitiveness by dramatically improving productivity.
　酵母の一種であるコマガタエラ・パストリス（Komagataella pastoris）はタンパク質発現能力に優れ、工業生産上有利である安価な炭素源を活用できるメタノール資化性（Mut+）酵母である。例えば、非特許文献１には、コマガタエラ・パストリスを用いて、緑色蛍光タンパク質、ヒト血清アルブミン、B型肝炎ウイルス表面抗原、ヒトインシュリン、及び一本鎖抗体等の異種タンパク質を生産する方法が報告されている。酵母において異種タンパク質を生産する場合、その生産性の向上のために、シグナル配列の付加、強力なプロモーターの利用、コドン改変、シャペロン遺伝子の共発現、転写因子遺伝子の共発現、宿主酵母由来のプロテアーゼ遺伝子の不活性化、及び培養条件の検討等様々な試みがなされている。 Komagataella pastoris, a type of yeast, is a methanol-utilizing (Mut + ) yeast that has excellent protein expression capacity and can utilize an inexpensive carbon source that is advantageous for industrial production. For example, Non-Patent Document 1 reports a method for producing a heterologous protein such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataera pastris. ing. When heterologous proteins are produced in yeast, in order to improve their productivity, addition of signal sequences, utilization of strong promoters, codon modification, co-expression of chaperon genes, co-expression of transcription factor genes, proteases derived from host yeast Various attempts have been made such as gene inactivation and examination of culture conditions.
　以上の通り、酵母に代表される宿主細胞を用いた異種タンパク質の生産においては、異種タンパク質の生産量を向上させる内在性遺伝子の探索が重要となっている。異種タンパク質の生産量を向上させる宿主細胞の内在性遺伝子の探索を試みる場合、通常、宿主細胞のcDNAライブラリまたはゲノムライブラリを用いて目的の遺伝子を探索してきた。しかし、cDNAライブラリまたはゲノムライブラリを用いたスクリーニングでは、盲目的なライブラリ作製のためにバイアスがかかり、評価すべきサンプル数が膨大となって多大な労力がかかっていた。 As described above, in the production of heterologous proteins using host cells represented by yeast, it is important to search for endogenous genes that improve the production of heterologous proteins. When attempting to search for an endogenous gene in a host cell that improves the production of heterologous proteins, the gene of interest has usually been searched for using the host cell's cDNA library or genomic library. However, screening using a cDNA library or a genomic library is biased due to blind library preparation, and the number of samples to be evaluated becomes enormous, which requires a great deal of labor.
　本発明は、内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法、該方法によって製造された遺伝子組換え細胞、該細胞を含む内在性遺伝子過剰発現細胞ライブラリを用いた目的タンパク質の生産を増強する内在性遺伝子のスクリーニング方法を提供することを課題とする。 The present invention is a method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, the recombinant cell produced by the method, and the production of a target protein using an endogenous gene overexpressing cell library containing the cell. It is an object of the present invention to provide a screening method for an endogenous gene that enhances the protein.
　本発明者らは、相同組換えによってコマガタエラ・ファフィの全内在性遺伝子（5001種類）に高発現性プロモーターが機能可能に連結された、各内在性遺伝子の発現が増強されたコマガタエラ・ファフィ集団を作製することに成功した。さらに発明者らは、低分子抗体発現ベクターを導入されたコマガタエラ・ファフィから各内在性遺伝子の発現が増強されたコマガタエラ・ファフィ集団を作製し、該低分子抗体の生産量を増加させる内在性遺伝子を３５種類特定した。また、発明者らは、これら３５種類の内在性遺伝子うち４種類を組み合わせることによって、さらに低分子抗体の生産量を増加させることに成功した。
　発明者らは、これらの知見に基づいてさらに検討を重ねた結果、本発明を完成するに至った。 We have created a Komagataera fafi population in which the expression of each endogenous gene is enhanced, in which a highly expressive promoter is operably linked to all endogenous genes (5001 types) of Komagataera fafi by homologous recombination. I succeeded in producing it. Furthermore, the inventors created a Komagataera fafi population in which the expression of each endogenous gene was enhanced from the Komagataera fafi into which a small molecule antibody expression vector was introduced, and the endogenous gene that increases the production of the small molecule antibody. 35 types were identified. In addition, the inventors have succeeded in further increasing the production of small molecule antibodies by combining four of these 35 types of endogenous genes.
As a result of further studies based on these findings, the inventors have completed the present invention.
　すなわち、本発明は、以下を提供する。
［１］以下の工程を含む、内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法。
（１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
（２）該直鎖状核酸を宿主細胞に導入する工程、および
（３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程。
［２］宿主細胞および遺伝子組換え細胞が酵母、細菌、真菌、昆虫細胞、動物細胞または植物細胞である、［１］に記載の方法。
［３］酵母がメタノール資化性酵母、分裂酵母または出芽酵母である、［２］に記載の方法。
［４］メタノール資化性酵母がコマガタエラ属酵母またはオガタエア属酵母である、［３］に記載の方法。
［５］内在性遺伝子が以下の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子である、［１］～［４］のいずれか１つに記載の方法。
（１）配列番号４６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２）配列番号４７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３）配列番号４８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（４）配列番号４９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（５）配列番号５０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（６）配列番号５１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（７）配列番号５２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（８）配列番号５３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（９）配列番号５４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１０）配列番号５５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１１）配列番号５６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１２）配列番号５７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１３）配列番号５８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１４）配列番号５９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１５）配列番号６０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１６）配列番号６１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１７）配列番号６２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１８）配列番号６３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１９）配列番号６４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２０）配列番号６５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２１）配列番号６６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２２）配列番号６７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２３）配列番号６８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２４）配列番号６９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２５）配列番号７０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２６）配列番号７１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２７）配列番号７３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２８）配列番号７５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２９）配列番号７６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３０）配列番号７７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３１）配列番号７８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
（３２）配列番号７９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。
［６］核酸断片が、以下の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片である、［５］に記載の方法。
（i）配列番号２３８で表される塩基配列を含む核酸断片、
（ii）配列番号２３９で表される塩基配列を含む核酸断片、
（iii）配列番号２４０で表される塩基配列を含む核酸断片、
（iv）配列番号２４１で表される塩基配列を含む核酸断片、
（v）配列番号２４２で表される塩基配列を含む核酸断片、
（vi）配列番号２４３で表される塩基配列を含む核酸断片、
（vii）配列番号２４４で表される塩基配列を含む核酸断片、
（viii）配列番号２４５で表される塩基配列を含む核酸断片、
（ix）配列番号２４６で表される塩基配列を含む核酸断片、
（x）配列番号２４７で表される塩基配列を含む核酸断片、
（xi）配列番号２４８で表される塩基配列を含む核酸断片、
（xii）配列番号２４９で表される塩基配列を含む核酸断片、
（xiii）配列番号２５０で表される塩基配列を含む核酸断片、
（xiv）配列番号２５１で表される塩基配列を含む核酸断片、
（xv）配列番号２５２で表される塩基配列を含む核酸断片、
（xvi）配列番号２５３で表される塩基配列を含む核酸断片、
（xvii）配列番号２５４で表される塩基配列を含む核酸断片、
（xviii）配列番号２５５で表される塩基配列を含む核酸断片、
（xix）配列番号２５６で表される塩基配列を含む核酸断片、
（xx）配列番号２５７で表される塩基配列を含む核酸断片、
（xxi）配列番号２５８で表される塩基配列を含む核酸断片、
（xxii）配列番号２５９で表される塩基配列を含む核酸断片、
（xxiii）配列番号２６０で表される塩基配列を含む核酸断片、
（xxiv）配列番号２６１で表される塩基配列を含む核酸断片、
（xxv）配列番号２６２で表される塩基配列を含む核酸断片、
（xxvi）配列番号２６３で表される塩基配列を含む核酸断片、
（xxvii）配列番号２６５で表される塩基配列を含む核酸断片、
（xxviii）配列番号２６７で表される塩基配列を含む核酸断片、
（xxix）配列番号２６８で表される塩基配列を含む核酸断片、
（xxx）配列番号２６９で表される塩基配列を含む核酸断片、
（xxxi）配列番号２７０で表される塩基配列を含む核酸断片、および
（xxxii）配列番号２７１で表される塩基配列を含む核酸断片。
［７］内在性遺伝子が核酸断片を含むプラスミドが制限酵素で切断された直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された該内在性遺伝子を含む遺伝子組換え細胞であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、遺伝子組換え細胞。
［８］遺伝子組換え細胞が酵母、細菌、真菌、昆虫細胞、動物細胞または植物細胞である、［７］に記載の遺伝子組換え細胞。
［９］酵母がメタノール資化性酵母、分裂酵母または出芽酵母である、［８］に記載の遺伝子組換え細胞。
［１０］メタノール資化性酵母がコマガタエラ属酵母またはオガタエア属酵母である、［９］に記載の遺伝子組換え細胞。
［１１］内在性遺伝子が以下の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子である、［７］～［１０］のいずれか１つに記載の遺伝子組換え細胞。
（１）配列番号４６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２）配列番号４７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３）配列番号４８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（４）配列番号４９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（５）配列番号５０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（６）配列番号５１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（７）配列番号５２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（８）配列番号５３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（９）配列番号５４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１０）配列番号５５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１１）配列番号５６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１２）配列番号５７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１３）配列番号５８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１４）配列番号５９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１５）配列番号６０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１６）配列番号６１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１７）配列番号６２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１８）配列番号６３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１９）配列番号６４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２０）配列番号６５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２１）配列番号６６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２２）配列番号６７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２３）配列番号６８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２４）配列番号６９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２５）配列番号７０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２６）配列番号７１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２７）配列番号７３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２８）配列番号７５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２９）配列番号７６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３０）配列番号７７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３１）配列番号７８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
（３２）配列番号７９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。
［１２］核酸断片が、以下の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片である、［１１］に記載の遺伝子組換え細胞。
（i）配列番号２３８で表される塩基配列を含む核酸断片、
（ii）配列番号２３９で表される塩基配列を含む核酸断片、
（iii）配列番号２４０で表される塩基配列を含む核酸断片、
（iv）配列番号２４１で表される塩基配列を含む核酸断片、
（v）配列番号２４２で表される塩基配列を含む核酸断片、
（vi）配列番号２４３で表される塩基配列を含む核酸断片、
（vii）配列番号２４４で表される塩基配列を含む核酸断片、
（viii）配列番号２４５で表される塩基配列を含む核酸断片、
（ix）配列番号２４６で表される塩基配列を含む核酸断片、
（x）配列番号２４７で表される塩基配列を含む核酸断片、
（xi）配列番号２４８で表される塩基配列を含む核酸断片、
（xii）配列番号２４９で表される塩基配列を含む核酸断片、
（xiii）配列番号２５０で表される塩基配列を含む核酸断片、
（xiv）配列番号２５１で表される塩基配列を含む核酸断片、
（xv）配列番号２５２で表される塩基配列を含む核酸断片、
（xvi）配列番号２５３で表される塩基配列を含む核酸断片、
（xvii）配列番号２５４で表される塩基配列を含む核酸断片、
（xviii）配列番号２５５で表される塩基配列を含む核酸断片、
（xix）配列番号２５６で表される塩基配列を含む核酸断片、
（xx）配列番号２５７で表される塩基配列を含む核酸断片、
（xxi）配列番号２５８で表される塩基配列を含む核酸断片、
（xxii）配列番号２５９で表される塩基配列を含む核酸断片、
（xxiii）配列番号２６０で表される塩基配列を含む核酸断片、
（xxiv）配列番号２６１で表される塩基配列を含む核酸断片、
（xxv）配列番号２６２で表される塩基配列を含む核酸断片、
（xxvi）配列番号２６３で表される塩基配列を含む核酸断片、
（xxvii）配列番号２６５で表される塩基配列を含む核酸断片、
（xxviii）配列番号２６７で表される塩基配列を含む核酸断片、
（xxix）配列番号２６８で表される塩基配列を含む核酸断片、
（xxx）配列番号２６９で表される塩基配列を含む核酸断片、
（xxxi）配列番号２７０で表される塩基配列を含む核酸断片、および
（xxxii）配列番号２７１で表される塩基配列を含む核酸断片。
［１３］目的タンパク質をコードする塩基配列をゲノムに含む、［７］～［１２］のいずれか１つに記載の遺伝子組換え細胞。
［１４］目的タンパク質が異種タンパク質である、［１３］に記載の遺伝子組換え細胞。
［１５］［１３］または［１４］に記載の遺伝子組換え細胞を培養する工程を含む、目的タンパク質の製造方法。
［１６］［７］～［１０］のいずれか１つに記載の遺伝子組換え細胞を含む、内在性遺伝子過剰発現細胞ライブラリ。
［１７］以下の工程を含む、目的タンパク質の生産を増強する内在性遺伝子をスクリーニングする方法。
（１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
（２）該直鎖状核酸を目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞に導入する工程、
（３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程、
（４）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞を培養する工程、
（５）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞による目的タンパク質の生産量をそれぞれ測定する工程、および
（６）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。
［１８］以下の工程を含む、目的タンパク質の生産を増強する内在性遺伝子をスクリーニングする方法。
（１）［１６］に記載の内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞に目的タンパク質をコードする塩基配列を含む発現ベクターを導入し、培養する工程、
（２）内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞による目的タンパク質の生産量を測定する工程、および
（３）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。 That is, the present invention provides the following.
[1] A method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, which comprises the following steps.
(1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the start codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
(2) The step of introducing the linear nucleic acid into a host cell, and (3) the endogenous gene is homologously recombined by the linear nucleic acid, and a highly expressive promoter is operably linked. A step of selecting transgenic cells containing a sex gene.
[2] The method according to [1], wherein the host cell and the recombinant cell are yeast, bacteria, fungi, insect cells, animal cells or plant cells.
[3] The method according to [2], wherein the yeast is methanol-utilizing yeast, fission yeast or budding yeast.
[4] The method according to [3], wherein the methanol-utilizing yeast is a yeast of the genus Komagataera or a yeast of the genus Ogataea.
[5] Described in any one of [1] to [4], wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). the method of.
(1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46.
(2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47.
(3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48.
(4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
(5) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50.
(6) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51.
(7) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52.
(8) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53.
(9) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54.
(10) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55.
(11) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56.
(12) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57.
(13) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58.
(14) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59.
(15) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60.
(16) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61.
(17) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 62.
(18) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63.
(19) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 64.
(20) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 65.
(21) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66.
(22) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 67.
(23) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 68.
(24) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 69.
(25) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 70.
(26) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71.
(27) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73.
(28) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 75.
(29) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76.
(30) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77.
(31) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78, and (32) the same or substantially the same base as the base sequence represented by SEQ ID NO: 79. An endogenous gene containing a sequence.
[6] The method according to [5], wherein the nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
(I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238,
(Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239,
(Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240,
(Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
(V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242,
(Vi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243,
(Vii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244,
(Viii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 245,
(Ix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 246,
(X) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247,
(Xi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248,
(Xii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249,
(Xiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250,
(Xiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251.
(Xv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252,
(Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253,
(Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254,
(Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255,
(Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256,
(Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
(Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258,
(Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259,
(Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260,
(Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
(Xxv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262,
(Xxvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263,
(Xxvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265,
(Xxviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267,
(Xxix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 268,
(Xxx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269,
(Xxxi) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 270, and (xxxii) a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 271.
[7] A gene set containing an endogenous gene in which a highly expressive promoter is operably linked, in which a plasmid containing a nucleic acid fragment is homologously recombined with a linear nucleic acid in which a nucleic acid fragment is cleaved with a restriction enzyme. In a replacement cell, the nucleic acid fragment was a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, and the partial sequence into which the restriction enzyme recognition site was inserted and the end codon were sequentially linked. A recombinant cell containing a base sequence.
[8] The recombinant cell according to [7], wherein the recombinant cell is a yeast, bacterium, fungus, insect cell, animal cell or plant cell.
[9] The genetically modified cell according to [8], wherein the yeast is a methanol-utilizing yeast, a fission yeast or a budding yeast.
[10] The genetically modified cell according to [9], wherein the methanol-utilizing yeast is a yeast of the genus Komagataera or a yeast of the genus Ogataea.
[11] Described in any one of [7] to [10], wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). Genetically modified cells.
(1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46.
(2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47.
(3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48.
(4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
(5) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50.
(6) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51.
(7) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52.
(8) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53.
(9) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54.
(10) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55.
(11) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56.
(12) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57.
(13) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58.
(14) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59.
(15) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60.
(16) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61.
(17) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 62.
(18) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63.
(19) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 64.
(20) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 65.
(21) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66.
(22) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 67.
(23) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 68.
(24) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 69.
(25) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 70.
(26) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71.
(27) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73.
(28) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 75.
(29) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76.
(30) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77.
(31) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78, and (32) the same or substantially the same base as the base sequence represented by SEQ ID NO: 79. An endogenous gene containing a sequence.
[12] The transgenic cell according to [11], wherein the nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
(I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238,
(Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239,
(Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240,
(Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
(V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242,
(Vi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243,
(Vii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244,
(Viii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 245,
(Ix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 246,
(X) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247,
(Xi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248,
(Xii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249,
(Xiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250,
(Xiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251.
(Xv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252,
(Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253,
(Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254,
(Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255,
(Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256,
(Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
(Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258,
(Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259,
(Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260,
(Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
(Xxv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262,
(Xxvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263,
(Xxvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265,
(Xxviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267,
(Xxix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 268,
(Xxx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269,
(Xxxi) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 270, and (xxxii) a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 271.
[13] The genetically modified cell according to any one of [7] to [12], which comprises a base sequence encoding a target protein in its genome.
[14] The transgenic cell according to [13], wherein the target protein is a heterologous protein.
[15] A method for producing a target protein, which comprises the step of culturing the recombinant cells according to [13] or [14].
[16] An endogenous gene overexpressing cell library containing the recombinant cell according to any one of [7] to [10].
[17] A method for screening an endogenous gene that enhances the production of a target protein, which comprises the following steps.
(1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
(2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding a target protein in the genome.
(3) A step of selecting a recombinant cell containing an endogenous gene in which the endogenous gene is homologously recombined by the linear nucleic acid and to which a highly expressive promoter is operably linked.
(4) A step of culturing a host cell containing the cell obtained in step (3) and the base sequence encoding the target protein in the genome.
(5) The step of measuring the production amount of the target protein by the cells obtained in the step (3) and the host cell containing the base sequence encoding the target protein in the genome, respectively, and (6) increasing the production amount of the target protein. The step of identifying the endogenous gene to be caused.
[18] A method for screening an endogenous gene that enhances the production of a target protein, which comprises the following steps.
(1) A step of introducing an expression vector containing a base sequence encoding a target protein into the endogenous gene overexpressing cell library and the host cell according to [16] and culturing the cells.
(2) A step of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell, and (3) a step of identifying the endogenous gene that increases the production amount of the target protein.
　各内在性遺伝子の発現を増強する方法を提供することにより、簡便に各内在性遺伝子の発現が増強された遺伝子組換え細胞ライブラリを構築することが可能となった。また、該ライブラリを用いることによって目的タンパク質の生産を増加させる内在性遺伝子のスクリーニングが可能となった。 By providing a method for enhancing the expression of each endogenous gene, it has become possible to easily construct a transgenic cell library in which the expression of each endogenous gene is enhanced. In addition, the use of the library has made it possible to screen for endogenous genes that increase the production of the target protein.
コマガタエラ・ファフィ遺伝子過剰発現細胞ライブラリを作製するための工程を示す図である。It is a figure which shows the process for making the Komagataera fafi gene overexpressing cell library. KAR2過剰発現用OLS配列およびPDI1過剰発現用OLS配列の構造を示す図である。It is a figure which shows the structure of the OLS sequence for KAR2 overexpression and the OLS sequence for PDI1 overexpression. KAR2過剰発現株におけるKAR2発現量の増加およびPDI1過剰発現株におけるPDI1発現量の増加を示す図である。It is a figure which shows the increase of the KAR2 expression level in the KAR2 overexpression strain and the increase of the PDI1 expression level in a PDI1 overexpression strain. スクリーニングポジティブ株で過剰発現された遺伝子を同定するための工程を示す図である。It is a figure which shows the process for identifying the gene overexpressed in a screening positive strain. 有用因子EF1st-1、EF2nd-4、EF1st-4およびEF2nd-1を1つのタンデムscFv226抗体生産株に集積することによって抗体生産量が増加することを示す図である。It is a figure which shows that the antibody production amount increases by accumulating useful factors EF1st-1, EF2nd-4, EF1st-4 and EF2nd-1 in one tandem scFv226 antibody production strain. CCA38473ターミネーター配列内に挿入された特定の遺伝子を別の遺伝子へ変換するための工程を示す図である。It is a figure which shows the process for converting a specific gene inserted into a CCA38473 terminator sequence into another gene. コマガタエラ・ファフィのゲノムのCCA38473ターミネーター配列内に挿入されたGFP遺伝子をRFP遺伝子へ変換するための工程を示す図である。It is a figure which shows the process for converting the GFP gene inserted into the CCA38473 terminator sequence of the genome of Komagataera fafi into the RFP gene. タンデムscFv226抗体高生産株に導入されたタンデムscFv226抗体カセットの構造を示す図である。It is a figure which shows the structure of the tandem scFv226 antibody cassette introduced into the tandem scFv226 antibody high production strain. 有用因子EF1st-1、EF2nd-4、EF1st-4およびEF2nd-1が集積されたタンデムscFv226抗体生産株の抗体産生遺伝子を入れ替えた株の抗体生産量を示す図である。It is a figure which shows the antibody production amount of the strain which exchanged the antibody production gene of the tandem scFv226 antibody production strain which accumulated useful factors EF1st-1, EF2nd-4, EF1st-4 and EF2nd-1. ペアスクリーニング用抗体産生促進タンパク質過剰発現細胞ライブラリを示す図である。It is a figure which shows the antibody production promotion protein overexpressing cell library for pair screening.
１．内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法
　本発明は、内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法（以下、本発明の遺伝子組換え細胞の製造方法）を提供する。 1. 1. Method for producing recombinant cell with enhanced expression of endogenous gene The present invention is a method for producing a recombinant cell with enhanced expression of an endogenous gene (hereinafter, the method for producing a recombinant cell of the present invention). I will provide a.
　本発明の遺伝子組換え細胞の製造方法において、遺伝子組換え細胞は、後述する直鎖状核酸が導入され、遺伝子組換えによって内在性遺伝子の発現が増強された細胞をいう。本明細書では直鎖状核酸を導入する前の遺伝子組換え細胞を宿主細胞と呼ぶ場合がある。宿主細胞は直鎖状核酸を導入することの出来る細胞であれば特に限定されない。 In the method for producing a recombinant cell of the present invention, the recombinant cell refers to a cell in which a linear nucleic acid described later is introduced and the expression of an endogenous gene is enhanced by the gene recombination. In the present specification, the recombinant cell before the introduction of the linear nucleic acid may be referred to as a host cell. The host cell is not particularly limited as long as it is a cell into which a linear nucleic acid can be introduced.
　遺伝子組換え細胞および宿主細胞（以下、本発明の細胞）の生物種は特に限定されないが、酵母、細菌、真菌、昆虫細胞、動物細胞および植物細胞等が挙げられ、酵母が好ましく、メタノール資化性酵母、分裂酵母、出芽酵母がより好ましく、メタノール資化性酵母がさらに好ましい。一般に、メタノール資化性酵母は、唯一の炭素源としてメタノールを利用して培養可能な酵母と定義されるが、本来メタノール資化性酵母であったが、人為的な改変あるいは変異によりメタノール資化性能を喪失した酵母も、本発明におけるメタノール資化性酵母に包含される。 The biological species of the recombinant cell and the host cell (hereinafter referred to as the cell of the present invention) are not particularly limited, and examples thereof include yeast, bacteria, fungi, insect cells, animal cells and plant cells, with yeast being preferred and methanol assimilation. Sex yeast, fission yeast, and germination yeast are more preferable, and methanol-utilizing yeast is even more preferable. Generally, methanol-utilizing yeast is defined as yeast that can be cultivated using methanol as the only carbon source. Originally, it was methanol-utilizing yeast, but it was assimilated into methanol by artificial modification or mutation. Yeasts that have lost their performance are also included in the methanol-utilizing yeasts of the present invention.
　メタノール資化性酵母細胞としては、ピキア（Pichia）属、オガタエア（Ogataea）属、コマガタエラ（Komagataella）属等に属する酵母が挙げられ、コマガタエラ属酵母またはオガタエア属酵母が好ましく、コマガタエラ属酵母が特に好ましい。 Examples of the methanol-utilizing yeast cells include yeasts belonging to the genus Pichia, Ogataea, Komagataella, etc., and yeasts of the genus Komagataella or yeasts of the genus Ogataea are preferable, and yeasts of the genus Komagataela are particularly preferable. ..
　ピキア（Pichia）属ではピキア・メタノリカ（Pichia methanolica）、オガタエア（Ogataea）属ではオガタエア・アングスタ（Ogataea angusta）、オガタエア・ポリモルファ（Ogataea polymorpha）、オガタエア・パラポリモルファ（Ogataea parapolymorpha）、オガタエア・ミヌータ(Ogataea minuta)、コマガタエラ（Komagataella）属ではコマガタエラ・パストリス（Komagataella pastoris）、コマガタエラ・ファフィ（Komagataella phaffii）等が好ましい例として挙げられ、その中でもコマガタエラ・ファフィが最も好ましい。なお、コマガタエラ・パストリスおよびコマガタエラ・ファフィはどちらもピキア・パストリス（Pichia pastoris）の別名を有する。 In the genus Pichia, Pichia meethanolica, in the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea parapolymorpha, Ogataea parapolymorpha, Ogataea parapolymorpha ), In the genus Komagataella, Komagataella pastoris, Komagataella phaffii and the like are mentioned as preferable examples, and among them, Komagataella fafi is the most preferable. Both Komagataera pastoris and Komagataera fafi have another name for Pichia pastoris.
　具体的に本発明の細胞として使用できる細胞株として、コマガタエラ・パストリスNRBC0948（CBS704, DSMZ 70382）、コマガタエラ・パストリスX-33、コマガタエラ・ファフィCBS7435（Y-11430）等の株が挙げられる。これらの細胞株は、アメリカン・タイプ・カルチャー・コレクションやサーモフィッシャーサイエンティフィック社等から入手することができる。 Specific examples of cell lines that can be used as the cells of the present invention include strains such as Komagataera pastris NRBC0948 (CBS704, DSMZ70382), Komagataera pastris X-33, and Komagataera fafi CBS7435 (Y-11430). These cell lines can be obtained from the American Type Culture Collection, Thermo Fisher Scientific, and others.
　また、本発明においては、これらコマガタエラ属酵母菌株からの誘導株も使用でき、例えばヒスチジン要求性としてコマガタエラ・パストリスGS115株（サーモフィッシャーサイエンティフィック社より入手可能）、等が挙げられる。また、別の誘導株としては、コマガタエラ・ファフィの非相同組み換え機構破壊株（Δku70, Δdnl4）もまた使用できる。本発明においては、これらの菌株からの誘導株等も使用できる。 Further, in the present invention, a strain derived from these yeast strains of the genus Komagataera can also be used, and examples thereof include Komagataera pastris GS115 strain (available from Thermo Fisher Scientific Co., Ltd.) as a histidine requirement. As another inducible strain, a non-homologous recombination mechanism disrupting strain (Δku70, Δdnl4) of Komagataera fafi can also be used. In the present invention, strains derived from these strains and the like can also be used.
　本発明の遺伝子組換え細胞の製造方法において、内在性遺伝子は、本発明の細胞が有する核酸のうちDNAのみならずそのmRNA及びcDNAも包含するが、典型的にはDNAであることができ、特にゲノムDNAであることができる。また、内在性遺伝子は、機能領域の別を問うものではなく、例えば、エキソンのみ含むのであってもよいし、エキソン及びイントロンを含むものであってもよい。 In the method for producing a recombinant cell of the present invention, the endogenous gene includes not only DNA but also its mRNA and cDNA among the nucleic acids possessed by the cell of the present invention, but can be typically DNA. In particular, it can be genomic DNA. Further, the endogenous gene does not ask the distinction of the functional region, and may contain, for example, only exons, or may contain exons and introns.
　内在性遺伝子は、本発明の細胞が備える遺伝子であれば特に制限されないが、例えば、後述する目的タンパク質の生産を増強させるという目的からは以下の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子が例示される。
（１）配列番号４６（EF1st-2）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２）配列番号４７（EF1st-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３）配列番号４８（EF1st-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（４）配列番号４９（EF1st-5）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（５）配列番号５０（EF1st-6）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（６）配列番号５１（EF1st-7）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（７）配列番号５２（EF1st-8）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（８）配列番号５３（EF1st-9）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（９）配列番号５４（EF1st-10）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１０）配列番号５５（EF1st-11）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１１）配列番号５６（EF1st-12）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１２）配列番号５７（EF1st-13）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１３）配列番号５８（EF1st-14）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１４）配列番号５９（EF1st-15）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１５）配列番号６０（EF1st-16）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１６）配列番号６１（EF1st-17）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１７）配列番号６２（EF1st-18）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１８）配列番号６３（EF2nd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（１９）配列番号６４（EF2nd-2）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２０）配列番号６５（EF2nd-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２１）配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２２）配列番号６７（EF2nd-5）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２３）配列番号６８（EF2nd-6）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２４）配列番号６９（EF2nd-7）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２５）配列番号７０（EF2nd-9）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２６）配列番号７１（EF3rd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２７）配列番号７３（EF3rd-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２８）配列番号７５（EF3rd-5）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（２９）配列番号７６（EF3rd-6）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３０）配列番号７７（EF3rd-7）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（３１）配列番号７８（EF3rd-8）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
（３２）配列番号７９（EF3rd-9）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。 The endogenous gene is not particularly limited as long as it is a gene contained in the cell of the present invention. For example, it is composed of the following endogenous genes (1) to (32) for the purpose of enhancing the production of the target protein described later. At least one endogenous gene selected from the group is exemplified.
(1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46 (EF1st-2).
(2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
(3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48 (EF1st-4).
(4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49 (EF1st-5).
(5) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50 (EF1st-6).
(6) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51 (EF1st-7).
(7) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52 (EF1st-8).
(8) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53 (EF1st-9).
(9) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54 (EF1st-10).
(10) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55 (EF1st-11).
(11) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56 (EF1st-12).
(12) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57 (EF1st-13).
(13) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58 (EF1st-14).
(14) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59 (EF1st-15).
(15) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60 (EF1st-16).
(16) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61 (EF1st-17).
(17) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 62 (EF1st-18).
(18) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
(19) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 64 (EF2nd-2).
(20) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 65 (EF2nd-3).
(21) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4).
(22) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 67 (EF2nd-5).
(23) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 68 (EF2nd-6).
(24) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 69 (EF2nd-7).
(25) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 70 (EF2nd-9).
(26) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71 (EF3rd-1).
(27) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73 (EF3rd-3).
(28) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 75 (EF3rd-5).
(29) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76 (EF3rd-6).
(30) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77 (EF3rd-7).
(31) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78 (EF3rd-8), and (32) the base represented by SEQ ID NO: 79 (EF3rd-9). An endogenous gene containing a base sequence that is the same as or substantially the same as the sequence.
　上記の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子は、好ましくは、以下の（a）～（g）の内在性遺伝子からなる群から選択される少なくとも２つの内在性遺伝子であってよい。
（a）配列番号４７（EF1st-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（b）配列番号４８（EF1st-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（c）配列番号６３（EF2nd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（d）配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（e）配列番号７１（EF3rd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
（f）配列番号７３（EF3rd-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
（g）配列番号７５（EF3rd-5）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。 At least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32) is preferably selected from the group consisting of the following (a) to (g) endogenous genes. It may be at least two endogenous genes.
(A) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
(B) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48 (EF1st-4).
(C) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1).
(D) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4).
(E) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71 (EF3rd-1).
(F) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73 (EF3rd-3), and (g) the base represented by SEQ ID NO: 75 (EF3rd-5). An endogenous gene containing a base sequence that is the same as or substantially the same as the sequence.
　また内在性遺伝子は、上記の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子に加えて、さらに以下の内在性遺伝子を含んでもよい。
（３３）配列番号４５（EF1st-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。 Further, the endogenous gene may further include the following endogenous genes in addition to at least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32).
(33) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
　内在性遺伝子は、例えば、以下の組み合わせが挙げられる。
（A）配列番号４５（EF1st-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号４８（EF1st-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号６３（EF2nd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。
（B）配列番号４５（EF1st-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号７１（EF3rd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
配列番号７５（EF3rd-5）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。
（C）配列番号４５（EF1st-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号４７（EF1st-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
配列番号７１（EF3rd-1）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。
（D）配列番号６６（EF2nd-4）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
配列番号７３（EF3rd-3）で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。 Examples of the endogenous gene include the following combinations.
(A) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48 (EF1st-4).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63 (EF2nd-1), and the same or substantially the same as the base sequence represented by SEQ ID NO: 66 (EF2nd-4). An endogenous gene containing the same base sequence.
(B) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71 (EF3rd-1), and the same or substantially the same as the base sequence represented by SEQ ID NO: 75 (EF3rd-5). An endogenous gene containing the same base sequence.
(C) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 45 (EF1st-1).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47 (EF1st-3).
An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4), and the same or substantially the same as the base sequence represented by SEQ ID NO: 71 (EF3rd-1). An endogenous gene containing the same base sequence.
(D) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66 (EF2nd-4), and the same base sequence represented by SEQ ID NO: 73 (EF3rd-3). Or an endogenous gene containing substantially the same base sequence.
　配列番号４５～７１、７３、７５～７９で表される塩基配列と実質的に同一の塩基配列としては、配列番号４５～７１、７３、７５～７９で表される塩基配列と約85％以上、好ましくは約90％以上、最も好ましくは約95％以上の同一性を有する塩基配列などが挙げられる。ここで「同一性」とは、当該技術分野において公知の数学的アルゴリズムを用いて２つの塩基配列をアラインさせた場合の、最適なアラインメント（好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方もしくは両方へのギャップの導入を考慮し得るものである）における、オーバーラップする全塩基配列に対する同一塩基配列の割合（％）を意味する。
　本明細書における塩基配列の同一性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値=10；ギャップを許す；フィルタリング=ON；マッチスコア=1；ミスマッチスコア=-3）にて計算することができる。。 The base sequence substantially the same as the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79 is about 85% or more of the base sequence represented by SEQ ID NOs: 45 to 71, 73, 75 to 79. , Preferably about 90% or more, most preferably about 95% or more base sequences and the like. Here, "identity" means the optimum alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is used for the optimum alignment of sequences. It means the ratio (%) of the same base sequence to the total base sequence that overlaps in (which can consider the introduction of a gap in one or both).
For the identity of the base sequence in the present specification, the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) is used, and the following conditions (expected value = 10; allow gaps; filtering = ON; match) It can be calculated with score = 1; mismatch score = -3). ..
　本発明の遺伝子組換え細胞の製造方法において、内在性遺伝子の発現が増強されているとは、内在性遺伝子の転写産物であるmRNAまたは翻訳産物であるポリペプチドの発現量が増強されている状態をいう。なお、mRNAの発現量はリアルタイムPCR法、RNA-Seq法、ノーザンハイブリダイゼーション又はDNAアレイを利用したハイブリダイゼーション法等を用いて定量することができ、ポリペプチドの発現量は、ポリペプチドを認識する抗体やポリペプチドと結合性を有する染色化合物等を用いて定量することができる。また、上記に挙げた定量方法以外にも、当業者で用いられている従来法であってもよい。 In the method for producing a recombinant cell of the present invention, enhanced expression of an endogenous gene means that the expression level of mRNA, which is a transcript of the endogenous gene, or polypeptide, which is a translation product, is enhanced. To say. The expression level of mRNA can be quantified by using a real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using a DNA array, or the like, and the expression level of a polypeptide recognizes a polypeptide. It can be quantified using a staining compound or the like having binding property to an antibody or a polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
　本発明の遺伝子組換え細胞の製造方法において、内在性遺伝子の発現の増強の度合は、後述する目的タンパク質の生産量が増強されれば特に限定されないが、内在性遺伝子の転写産物または翻訳産物の発現量が5％以上、10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上または95％以上増強されていることが好ましい。 In the method for producing a recombinant cell of the present invention, the degree of enhancement of the expression of the endogenous gene is not particularly limited as long as the production amount of the target protein described later is enhanced, but the transcript or translation product of the endogenous gene is not particularly limited. The expression level is enhanced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more. Is preferable.
　本発明の遺伝子組換え細胞の製造方法は、以下の工程を含む。
（１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
（２）該直鎖状核酸を宿主細胞に導入する工程、および
（３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程。 The method for producing a recombinant cell of the present invention includes the following steps.
(1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
(2) The step of introducing the linear nucleic acid into a host cell, and (3) the endogenous gene is homologously recombined by the linear nucleic acid, and a highly expressive promoter is operably linked. A step of selecting transgenic cells containing a sex gene.
　本発明の遺伝子組換え細胞の製造方法は、核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程（工程（１））を含む。ここで、核酸断片は、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む核酸断片である。 The method for producing a transgenic cell of the present invention includes a step (step (1)) of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme. Here, the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked. It is a nucleic acid fragment.
　工程（１）において、高発現性プロモーター（以下、本発明の高発現性プロモーター）は、本発明の細胞の内在性遺伝子の発現を増強させるプロモーターであれば特に制限されない。本発明の高発現性プロモーターの種類は、本発明の細胞に対応して適切なプロモーターであればいかなるものでもよい。
　例えば、本発明の細胞が酵母である場合、本発明の高発現性プロモーターは、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーターなどが好ましい。
　本発明の細胞がエシェリヒア属菌である場合、本発明の高発現性プロモーターは、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
　本発明の細胞がバチルス属菌である場合、本発明の高発現性プロモーターは、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。
　本発明の細胞が真菌である場合、本発明の高発現性プロモーターは、ADHプロモーター、CMV（サイトメガロウイルス）プロモーターなどが好ましい。
　本発明の細胞が昆虫細胞である場合、本発明の高発現性プロモーターは、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。
　本発明の細胞が動物細胞である場合、本発明の高発現性プロモーターは、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMVプロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが好ましい。
　本発明の細胞が植物細胞である場合、本発明の高発現性プロモーターは、CaMV（カリフラワーモザイクウイルス）35Sプロモーターなどが好ましい。 In the step (1), the highly expressive promoter (hereinafter, the highly expressive promoter of the present invention) is not particularly limited as long as it is a promoter that enhances the expression of the endogenous gene of the cell of the present invention. The type of the highly expressive promoter of the present invention may be any promoter suitable for the cells of the present invention.
For example, when the cell of the present invention is yeast, the highly expressive promoter of the present invention is preferably the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like.
When the cell of the present invention is a bacterium of the genus Escherichia, high expression promoter of the present invention, trp promoter, lac promoter, recA promoter, .lambda.P L promoter, lpp promoter, T7 promoter and the like are preferable.
When the cell of the present invention is a bacterium belonging to the genus Bacillus, the highly expressive promoter of the present invention is preferably the SPO1 promoter, SPO2 promoter, penP promoter or the like.
When the cell of the present invention is a fungus, the highly expressive promoter of the present invention is preferably an ADH promoter, a CMV (cytomegalovirus) promoter or the like.
When the cell of the present invention is an insect cell, the highly expressive promoter of the present invention is preferably a polyhedrin promoter, a P10 promoter or the like.
When the cells of the present invention are animal cells, the highly expressive promoters of the present invention are SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, HSV. -TK (herpes simplex virus thymidine kinase) promoter and the like are preferred.
When the cell of the present invention is a plant cell, the highly expressive promoter of the present invention is preferably the CaMV (cauliflower mosaic virus) 35S promoter or the like.
　工程（１）において、部分配列（以下、本発明の部分配列）は、本発明の細胞の内在性遺伝子の開始コドンから始まる部分配列であって制限酵素認識部位が挿入された部分配列である。本発明の細胞の内在性遺伝子の開始コドンから始まる部分配列の塩基配列情報は、公知のデータベースに記載の塩基配列情報から取得することができる。例えば、本発明の細胞としてコマガタエラ・ファフィを用いる場合、コマガタエラ・ファフィの全内在性遺伝子の開始コドンから始まる塩基配列情報は、コマガタエラ・ファフィCBS7435株の4本の染色体DNAの塩基配列情報(ACCESSION No. FR839628～FR839631（J. Biotechnol.154 (4), 312-320 (2011)））から取得することができる。また、本発明の細胞としてコマガタエラ・パストリスを用いる場合、コマガタエラ・パストリスの全内在性遺伝子の開始コドンから始まる塩基配列情報は、コマガタエラ・パストリスNBRC 0948株の4本の染色体DNAの塩基配列情報（Mattanovich et al., Microbial Cell Factories 8, 29 (2009)）、及びコマガタエラ・パストリスGS115株の4本の染色体DNAの塩基配列情報（ACCESSION No. FN392319～FN392322（Nat. Biotechnol. 27 (6), 561-566 (2009))）から取得することができる。このようにして得られる塩基配列情報を基にして内在性遺伝子の開始コドンから始まる部分配列を設計することができる。 In step (1), the partial sequence (hereinafter referred to as the partial sequence of the present invention) is a partial sequence starting from the start codon of the endogenous gene of the cell of the present invention and into which a restriction enzyme recognition site is inserted. The nucleotide sequence information of the partial sequence starting from the start codon of the endogenous gene of the cell of the present invention can be obtained from the nucleotide sequence information described in a known database. For example, when Komagataera fafi is used as the cell of the present invention, the nucleotide sequence information starting from the start codon of all endogenous genes of Komagataera fafi is the nucleotide sequence information (ACCESSION No.) of the four chromosomal DNAs of the Komagataera fafi CBS7435 strain. It can be obtained from FR839628 to FR839631 (J. Biotechnol.154 (4), 312-320 (2011)). When Komagataera pastris is used as the cell of the present invention, the nucleotide sequence information starting from the starting codon of the entire endogenous gene of Komagataera pastris is the nucleotide sequence information of the four chromosomal DNAs of the Komagataera pastris NBRC 0948 strain (Mattanovich). Et al., Microbial Cell Factories 8, 29 (2009)), and the nucleotide sequence information of the four chromosomal DNAs of the Komagataera Pastris GS115 strain (ACCESSION No. FN392319 to FN392322 (Nat. Biotechnol. 27 (6), 561- It can be obtained from 566 (2009))). Based on the base sequence information obtained in this way, a partial sequence starting from the start codon of the endogenous gene can be designed.
　本発明の部分配列の長さは、内在性遺伝子と直鎖状核酸の間で相同組換えが生じる長さである限り特に制限されないが、例えば、20塩基長以上、50塩基長以上、100塩基長以上、150塩基長以上である。また、本発明の部分配列の長さは、例えば、1000塩基長以下、750塩基長以下、500塩基長以下、または250塩基長以下である。 The length of the partial sequence of the present invention is not particularly limited as long as it is the length at which homologous recombination occurs between the endogenous gene and the linear nucleic acid, and is, for example, 20 bases or more, 50 bases or more, and 100 bases. Long or longer, 150 bases or longer. The length of the partial sequence of the present invention is, for example, 1000 bases or less, 750 bases or less, 500 bases or less, or 250 bases or less.
　また、本発明の部分配列は制限酵素認識部位が挿入された部分配列である。該制限酵素認識部位は、上記の核酸断片が含まれるプラスミドの全塩基配列中で該部分配列中にのみ存在する制限酵素認識部位であれば特に制限されない。本発明の部分配列に挿入される制限酵素認識部位としては、BspQI、BbsI、BsaI、BsmBIなど、認識部位と切断部位が異なるタイプIIS型の制限酵素によって認識される部位が挙げられる。 Further, the partial sequence of the present invention is a partial sequence in which a restriction enzyme recognition site is inserted. The restriction enzyme recognition site is not particularly limited as long as it is a restriction enzyme recognition site present only in the partial sequence in the entire base sequence of the plasmid containing the above nucleic acid fragment. Examples of restriction enzyme recognition sites inserted into the partial sequences of the present invention include sites recognized by type IIS type restriction enzymes, such as BspQI, BbsI, BsaI, and BsmBI, whose recognition sites and cleavage sites are different.
　上記の制限酵素認識部位は、本発明の部分配列内の任意の部位に挿入されてよいが、内在性遺伝子との間で相同的に組換えが生じる程度に本発明の部分配列が分割される位置に挿入されることが好ましい。そのような部分配列内における制限酵素認識部位の挿入位置としては、通常、本発明の部分配列の中間に挿入される。例えば、本発明の部分配列が内在性遺伝子の開始コドンから始まる部分配列であって、183塩基長である部分配列である場合、制限酵素認識部位は92番目塩基を置換することによって挿入される。 The above restriction enzyme recognition site may be inserted at any site within the partial sequence of the present invention, but the partial sequence of the present invention is divided to such an extent that recombination occurs homologously with the endogenous gene. It is preferably inserted in position. The insertion position of the restriction enzyme recognition site in such a partial sequence is usually inserted in the middle of the partial sequence of the present invention. For example, when the partial sequence of the present invention is a partial sequence starting from the start codon of an endogenous gene and has a length of 183 bases, the restriction enzyme recognition site is inserted by substituting the 92nd base.
　本発明の部分配列中に挿入される上記の制限酵素認識部位の数は、上記の核酸断片が含まれるプラスミドの全塩基配列中で該部分配列中にのみ存在する限り特に制限されないが、通常、１～数個であり、好ましくは、１又は２個である。 The number of the restriction enzyme recognition sites inserted in the partial sequence of the present invention is not particularly limited as long as it is present only in the partial sequence in the entire base sequence of the plasmid containing the nucleic acid fragment, but is usually limited. The number is one to several, preferably one or two.
　工程（１）において、終止コドン（以下、本発明の終止コドン）は、TAA、TAGまたはTGAのいずれかである。 In step (1), the stop codon (hereinafter referred to as the stop codon of the present invention) is either TAA, TAG, or TGA.
　工程（１）において、核酸断片（以下、本発明の核酸断片）は、本発明の高発現性プロモーター、本発明の部分配列、本発明の終止コドンがこの順に連結された塩基配列を含む。即ち、本発明の核酸断片では、本発明の高発現性プロモーターの３’末端の炭素と本発明の部分配列の５’末端の炭素はホスホジエステル結合を形成し、本発明の部分配列の３’末端の炭素と本発明の終止コドンの５’末端の炭素がホスホジエステル結合を形成している。なお、本発明の高発現性プロモーターと本発明の部分配列の間、本発明の部分配列と本発明の終止コドンの間は、スペーサー配列が挿入されてもよい。スペーサー配列の長さは、当業者が適宜決定してよく、例えば、15から25塩基長であってよい。 In step (1), the nucleic acid fragment (hereinafter referred to as the nucleic acid fragment of the present invention) includes a highly expressive promoter of the present invention, a partial sequence of the present invention, and a base sequence in which the stop codon of the present invention is linked in this order. That is, in the nucleic acid fragment of the present invention, the carbon at the 3'end of the highly expressive promoter of the present invention and the carbon at the 5'end of the partial sequence of the present invention form a phosphodiester bond, and the 3'of the partial sequence of the present invention. The terminal carbon and the 5'terminal carbon of the termination codon of the present invention form a phosphodiester bond. A spacer sequence may be inserted between the highly expressive promoter of the present invention and the partial sequence of the present invention, and between the partial sequence of the present invention and the stop codon of the present invention. The length of the spacer sequence may be appropriately determined by those skilled in the art, and may be, for example, 15 to 25 bases in length.
　工程（１）において、本発明の核酸断片は、本発明の細胞より調製したゲノムDNA画分を鋳型として用い、公知のデータベースに記載の高発現性プロモーター、内在性遺伝子の塩基配列情報からプライマーを設計し、Polymerase Chain Reaction（以下、「PCR法」と略称する）によって直接増幅することができる。また、本発明の部分配列の挿入される制限酵素認識部位は、公知の部位特異的変異導入法によって部分配列内に導入することができる。あるいは、後述する実施例の通り、本発明の核酸断片は、アジレント・テクノロジー社に製造を委託することによって取得することも可能である。 In step (1), the nucleic acid fragment of the present invention uses the genomic DNA fraction prepared from the cells of the present invention as a template, and a primer is used from the base sequence information of the highly expressive promoter and the endogenous gene described in a known database. It can be designed and directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method"). In addition, the restriction enzyme recognition site into which the partial sequence of the present invention is inserted can be introduced into the partial sequence by a known site-specific mutagenesis method. Alternatively, as in the examples described later, the nucleic acid fragment of the present invention can be obtained by outsourcing the production to Agilent Technologies.
　本発明の核酸断片の長さは、特に制限されないが、例えば、20塩基長以上、50塩基長以上、100塩基長以上、150塩基長以上、または200塩基長以上である。また、本発明の部分配列の長さは、例えば、1000塩基長以下、500塩基長以下、または400塩基長以下である。 The length of the nucleic acid fragment of the present invention is not particularly limited, but is, for example, 20 bases or more, 50 bases or more, 100 bases or more, 150 bases or more, or 200 bases or more. The length of the partial sequence of the present invention is, for example, 1000 bases or less, 500 bases or less, or 400 bases or less.
　内在性遺伝子が、上記の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子である場合、本発明の核酸断片は、以下の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片である。
（i）配列番号２３８（EF1st-2 OLS）で表される塩基配列を含む核酸断片、
（ii）配列番号２３９（EF1st-3 OLS）で表される塩基配列を含む核酸断片、
（iii）配列番号２４０（EF1st-4 OLS）で表される塩基配列を含む核酸断片、
（iv）配列番号２４１（EF1st-5 OLS）で表される塩基配列を含む核酸断片、
（v）配列番号２４２（EF1st-6 OLS）で表される塩基配列を含む核酸断片、
（vi）配列番号２４３（EF1st-7 OLS）で表される塩基配列を含む核酸断片、
（vii）配列番号２４４（EF1st-8 OLS）で表される塩基配列を含む核酸断片、
（viii）配列番号２４５（EF1st-9 OLS）で表される塩基配列を含む核酸断片、
（ix）配列番号２４６（EF1st-10 OLS）で表される塩基配列を含む核酸断片、
（x）配列番号２４７（EF1st-11 OLS）で表される塩基配列を含む核酸断片、
（xi）配列番号２４８（EF1st-12 OLS）で表される塩基配列を含む核酸断片、
（xii）配列番号２４９（EF1st-13 OLS）で表される塩基配列を含む核酸断片、
（xiii）配列番号２５０（EF1st-14 OLS）で表される塩基配列を含む核酸断片、
（xiv）配列番号２５１（EF1st-15 OLS）で表される塩基配列を含む核酸断片、
（xv）配列番号２５２（EF1st-16 OLS）で表される塩基配列を含む核酸断片、
（xvi）配列番号２５３（EF1st-17 OLS）で表される塩基配列を含む核酸断片、
（xvii）配列番号２５４（EF1st-18 OLS）で表される塩基配列を含む核酸断片、
（xviii）配列番号２５５（EF2nd-1 OLS）で表される塩基配列を含む核酸断片、
（xix）配列番号２５６（EF2nd-2 OLS）で表される塩基配列を含む核酸断片、
（xx）配列番号２５７（EF2nd-3 OLS）で表される塩基配列を含む核酸断片、
（xxi）配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片、
（xxii）配列番号２５９（EF2nd-5 OLS）で表される塩基配列を含む核酸断片、
（xxiii）配列番号２６０（EF2nd-6 OLS）で表される塩基配列を含む核酸断片、
（xxiv）配列番号２６１（EF2nd-7 OLS）で表される塩基配列を含む核酸断片、
（xxv）配列番号２６２（EF2nd-9 OLS）で表される塩基配列を含む核酸断片、
（xxvi）配列番号２６３（EF3rd-1 OLS）で表される塩基配列を含む核酸断片、
（xxvii）配列番号２６５（EF3rd-3 OLS）で表される塩基配列を含む核酸断片、
（xxviii）配列番号２６７（EF3rd-5 OLS）で表される塩基配列を含む核酸断片、
（xxix）配列番号２６８（EF3rd-6 OLS）で表される塩基配列を含む核酸断片、
（xxx）配列番号２６９（EF3rd-7 OLS）で表される塩基配列を含む核酸断片、
（xxxi）配列番号２７０（EF3rd-8 OLS）で表される塩基配列を含む核酸断片、および
（xxxii）配列番号２７１（EF3rd-9 OLS）で表される塩基配列を含む核酸断片。 When the endogenous gene is at least one endogenous gene selected from the group consisting of the above-mentioned endogenous genes (1) to (32), the nucleic acid fragment of the present invention is the following (i) to (xxxii). ) Is at least one nucleic acid fragment selected from the group consisting of nucleic acid fragments.
(I) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238 (EF1st-2 OLS),
(Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS),
(Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS),
(Iv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241 (EF1st-5 OLS),
(V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242 (EF1st-6 OLS),
(Vi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243 (EF1st-7 OLS),
(Vii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244 (EF1st-8 OLS),
(Viii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 245 (EF1st-9 OLS),
(Ix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 246 (EF1st-10 OLS),
(X) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247 (EF1st-11 OLS),
(Xi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248 (EF1st-12 OLS),
(Xii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249 (EF1st-13 OLS),
(Xiii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250 (EF1st-14 OLS),
(Xiv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251 (EF1st-15 OLS),
(Xv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (EF1st-16 OLS),
(Xvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253 (EF1st-17 OLS),
(Xvii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254 (EF1st-18 OLS),
(Xviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS),
(Xix) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256 (EF2nd-2 OLS),
(Xx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257 (EF2nd-3 OLS),
(Xxi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS),
(Xxii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259 (EF2nd-5 OLS),
(Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260 (EF2nd-6 OLS),
(Xxiv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261 (EF2nd-7 OLS),
(Xxv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262 (EF2nd-9 OLS),
(Xxvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS),
(Xxvii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS),
(Xxviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267 (EF3rd-5 OLS),
(Xxix) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 268 (EF3rd-6 OLS),
(Xxx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269 (EF3rd-7 OLS),
A nucleic acid fragment containing the base sequence represented by (xxxi) SEQ ID NO: 270 (EF3rd-8 OLS) and a nucleic acid fragment containing the base sequence represented by (xxxii) SEQ ID NO: 271 (EF3rd-9 OLS).
　上記の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片は、好ましくは、以下の（a’）～（g’）の核酸断片からなる群から選択される少なくとも２つの核酸断片であってよい。
（a’）配列番号２３９（EF1st-3 OLS）で表される塩基配列を含む核酸断片、
（b’）配列番号２４０（EF1st-4 OLS）で表される塩基配列を含む核酸断片、
（c’）配列番号２５５（EF2nd-1 OLS）で表される塩基配列を含む核酸断片、
（d’）配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片、
（e’）配列番号２６３（EF3rd-1 OLS）で表される塩基配列を含む核酸断片、
（f’）配列番号２６５（EF3rd-3 OLS）で表される塩基配列を含む核酸断片、および
（g’）配列番号２６７（EF3rd-5 OLS）で表される塩基配列を含む核酸断片。 At least one nucleic acid fragment selected from the group consisting of the above nucleic acid fragments (i) to (xxxii) is preferably selected from the group consisting of the following nucleic acid fragments (a') to (g'). It may be at least two nucleic acid fragments.
(A') Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS),
(B') Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS),
(C') Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS),
(D') Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS),
(E') Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS),
A nucleic acid fragment containing the base sequence represented by (f') SEQ ID NO: 265 (EF3rd-3 OLS) and a nucleic acid fragment containing the base sequence represented by (g') SEQ ID NO: 267 (EF3rd-5 OLS).
　また核酸断片は、上記の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片に加えて、さらに以下の核酸断片を含んでもよい。
（xxxiii）配列番号２３７（EF1st-1 OLS）で表される塩基配列を含む核酸断片。 Further, the nucleic acid fragment may further contain the following nucleic acid fragments in addition to at least one nucleic acid fragment selected from the group consisting of the above-mentioned nucleic acid fragments (i) to (xxxii).
(Xxxiii) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 237 (EF1st-1 OLS).
　核酸断片は、例えば、以下の組み合わせが挙げられる。
（A）配列番号２３７（EF1st-1 OLS）で表される塩基配列を含む核酸断片、
配列番号２４０（EF1st-4 OLS）で表される塩基配列を含む核酸断片、
配列番号２５５（EF2nd-1 OLS）で表される塩基配列を含む核酸断片、および
配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片。
（B）配列番号２３７（EF1st-1 OLS）で表される塩基配列を含む核酸断片、
配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片、
配列番号２６３（EF3rd-1 OLS）で表される塩基配列を含む核酸断片、および
配列番号２６７（EF3rd-5 OLS）で表される塩基配列を含む核酸断片。
（C）配列番号２３７（EF1st-1 OLS）で表される塩基配列を含む核酸断片、
配列番号２３９（EF1st-3 OLS）で表される塩基配列を含む核酸断片、
配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片、および
配列番号２６３（EF3rd-1 OLS）で表される塩基配列を含む核酸断片。
（D）配列番号２５８（EF2nd-4 OLS）で表される塩基配列を含む核酸断片、および
配列番号２６５（EF3rd-3 OLS）で表される塩基配列を含む核酸断片。 Examples of the nucleic acid fragment include the following combinations.
(A) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS),
Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS),
A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS).
(B) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS),
Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS),
A nucleic acid fragment containing a base sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS) and a nucleic acid fragment containing a base sequence represented by SEQ ID NO: 267 (EF3rd-5 OLS).
(C) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS),
Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS),
A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS).
(D) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS) and a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS).
　以上の通りにして得られる核酸断片は、プラスミドに含まれる。本発明においてプラスミドとは人為的に構築された核酸分子である。プラスミドを構成する核酸分子は、通常DNA、好ましくは二本鎖DNAである。プラスミドの例としては、YEpベクター、YRpベクター、YCpベクター、pPICHOLI、pHIP（Journal of General Microbioiogy (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha）、pHRP（pHIPについて挙げた前記文献参照）、pHARS（Molecular and General Genetics MGG February1986, Volume 202, Issue 2, pp 302-308, Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors）、大腸菌由来プラスミドベクター（pUC18、pUC19、pBR322、pBluescript、pQE）、枯草菌由来プラスミドベクター（pHY300PLK、pMTLBS72）等を使用することができる。 The nucleic acid fragment obtained as described above is contained in the plasmid. In the present invention, a plasmid is an artificially constructed nucleic acid molecule. The nucleic acid molecule that constitutes the plasmid is usually DNA, preferably double-stranded DNA. Examples of plasmids include YEp vector, YRp vector, YCp vector, pPICHOLI, pHIP (Journal of General Microbioiogy (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha), pHRP (pHIP). (Refer to the above-mentioned literature), pHARS (Molecular and General Genetics MGG February1986, Volume 202, Issue 2, pp 302-308, Transformation of the methylotrophic yeast Hansenula polymorpha by automatic replication and integration vector UC), derived from E. coli pBR322, pBluescript, pQE), plasmid vector derived from bacillus (pHY300PLK, pMTLBS72) and the like can be used.
　上記のプラスミドはさらに、本発明の核酸断片に加えて、１つ以上の制限酵素認識部位を含むクローニングサイト、Clontech社のIn-FusionクローニングシステムやNew England Biolabs社のGibson Assemblyシステム等を利用するためのオーバーラップ領域、選択マーカー遺伝子（栄養要求性相補遺伝子、薬剤耐性遺伝子など）の塩基配列等を含むことができる。
　栄養要求性相補遺伝子の例としては、URA3遺伝子、LEU2遺伝子、ADE1遺伝子、HIS4遺伝子、ARG4遺伝子等が挙げられる。
　薬剤耐性遺伝子の例としては、G418耐性遺伝子、Zeocin(商標)耐性遺伝子、ハイグロマイシン耐性遺伝子、Clone NAT耐性遺伝子、ブラストサイジンS耐性遺伝子、ノーセオスリシン耐性遺伝子等が挙げられる。 In addition to the nucleic acid fragments of the present invention, the above plasmids further utilize cloning sites containing one or more restriction enzyme recognition sites, Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, and the like. Can include the overlap region of the above, the base sequence of the selectable marker gene (nutrition-requiring complementary gene, drug resistance gene, etc.) and the like.
Examples of auxotrophic complementary genes include URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene and the like.
Examples of drug resistance genes include G418 resistance gene, Zeocin ™ resistance gene, hyglomycin resistance gene, Clone NAT resistance gene, blastsaidin S resistance gene, noseoslisin resistance gene and the like.
　工程（１）において、上記の通りに得られる核酸断片を含むプラスミドを本発明の部分配列に含まれる制限酵素認識部位を切断できる制限酵素で切断して直鎖状の核酸（以下、本発明の直鎖状核酸）を調製する。本発明の核酸断片を含むプラスミドを制限酵素で切断することによって、５’末端に終止コドンに連結された本発明の部分配列の後半部（相同配列１）が配置され、３’末端に高発現性プロモーターに連結された本発明の部分配列の前半部（相同配列２）が配置された直鎖状核酸を調製することができる。 In step (1), the plasmid containing the nucleic acid fragment obtained as described above is cleaved with a restriction enzyme capable of cleaving the restriction enzyme recognition site contained in the partial sequence of the present invention to form a linear nucleic acid (hereinafter referred to as the present invention). Linear nucleic acid) is prepared. By cleaving the plasmid containing the nucleic acid fragment of the present invention with a restriction enzyme, the latter half of the partial sequence of the present invention (homologous sequence 1) linked to the stop codon is placed at the 5'end and highly expressed at the 3'end. A linear nucleic acid can be prepared in which the first half (homologous sequence 2) of the partial sequence of the present invention linked to the sex promoter is arranged.
　本発明の遺伝子組換え細胞の製造方法は、本発明の直鎖状核酸を宿主細胞に導入する工程（工程（２））を含む。本発明の直鎖状核酸を宿主細胞へ導入する方法、すなわち形質転換法は公知の方法を適宜用いることができ、例えば宿主細胞として酵母細胞を用いる場合、エレクトロポレーション法、酢酸リチウム法、スフェロプラスト法等が挙げられるが特にこれらに限定されるものではない。例えば、コマガタエラ・ファフィの形質転換法としては、High efficiency transformation by electroporation of Pichia pastoris pretreated with lithiumacetate and dithiothreitol（Biotechniques. 2004 Jan;36(1):152-4.）に記載されているエレクトロポレーション法が一般的である。 The method for producing a recombinant cell of the present invention includes a step (step (2)) of introducing the linear nucleic acid of the present invention into a host cell. As a method for introducing the linear nucleic acid of the present invention into a host cell, that is, a transformation method, a known method can be appropriately used. For example, when yeast cells are used as host cells, an electroporation method, a lithium acetate method, or a spheroplast method can be used. Examples thereof include the spheroplast method, but the method is not particularly limited thereto. For example, as a transformation method for Komagataera fafi, the electroporation method described in High efficiency transformation by electroporation of Pichia pastoris treated with lithium acetate and dithiothreitol (Biotechniques. 2004 Jan; 36 (1): 152-4.) Is common.
　本発明の遺伝子組換え細胞の製造方法は、内在性遺伝子が本発明の直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程（工程（３））を含む。 The method for producing a recombinant cell of the present invention is a gene recombination containing an endogenous gene in which an endogenous gene is homologously recombined by the linear nucleic acid of the present invention and a highly expressive promoter is operably linked. The step of selecting cells (step (3)) is included.
　工程（３）において、宿主細胞に導入された本発明の直鎖状核酸は、５’末端に配置された終止コドンに連結された本発明の部分配列の後半部（相同配列１）と３’末端に配置された高発現性プロモーターに連結された本発明の部分配列の前半部（相同配列２）を内在性遺伝子に対する相同性領域として、相同組換え（シングルクロスオーバー組換え）が生じる。相同組換えの結果、本発明の直鎖状核酸は内在性遺伝子に含まれる相同配列１と相同配列２の間に挿入され、宿主細胞に元々備わっていた内在性遺伝子は本発明の直鎖状核酸に含まれる終止コドンによって機能を喪失する。その代わりに、機能を喪失した該遺伝子の下流では、本発明の直鎖状核酸に含まれる高発現性プロモーターに機能可能に連結された新たな内在性遺伝子が生じる。結果的に、本発明の遺伝子組換え細胞では、高発現性プロモーターによって内在性遺伝子の発現は増強される。 In step (3), the linear nucleic acid of the present invention introduced into the host cell is the latter half (homologous sequence 1) and 3'of the partial sequence of the present invention linked to the stop codon located at the 5'end. Homologous recombination (single crossover recombination) occurs with the first half (homologous sequence 2) of the partial sequence of the present invention linked to the highly expressive promoter located at the end as a homologous region for the endogenous gene. As a result of homologous recombination, the linear nucleic acid of the present invention is inserted between the homologous sequence 1 and the homologous sequence 2 contained in the endogenous gene, and the endogenous gene originally provided in the host cell is the linear of the present invention. It loses its function due to the stop codon contained in the nucleic acid. Instead, downstream of the dysfunctional gene results in a new endogenous gene operably linked to the highly expressive promoter contained in the linear nucleic acid of the invention. As a result, in the recombinant cells of the present invention, the expression of the endogenous gene is enhanced by the highly expressive promoter.
　工程（３）において、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞（以下、本発明の遺伝子組換え細胞）を選抜する場合、栄養要求性相補遺伝子または薬剤耐性遺伝子などの選択マーカー遺伝子を用いることが好ましい。選択マーカーは特に限定されないが、宿主細胞がコマガタエラ属酵母であれば、URA3遺伝子、LEU2遺伝子、ADE1遺伝子、HIS4遺伝子、ARG4遺伝子などの栄養要求性相補遺伝子であれば、それぞれウラシル、ロイシン、アデニン、ヒスチジン、アルギニンの栄養要求性株において原栄養株表現型の回復により本発明の遺伝子組換え細胞を選抜することができる。また、G418耐性遺伝子、Zeocin(商標)耐性遺伝子、ハイグロマイシン耐性遺伝子、Clone NAT耐性遺伝子、ブラストサイジンS耐性遺伝子などの薬剤耐性遺伝子であれば、それぞれG418、Zeocin(商標)、ハイグロマイシン、Clone NAT、ブラストサイジンSを含む培地上における耐性により本発明の遺伝子組換え細胞を選抜することができる。なお、遺伝子組換え酵母を作製するときに用いる栄養要求性選択マーカーは、宿主酵母において該選択マーカーが破壊されていない場合は、用いることができない。この場合、宿主酵母において該選択マーカーを破壊すればよく、方法は当業者には公知の方法を用いることができる。 In step (3), when a transgenic cell containing an endogenous gene operably linked to a highly expressive promoter (hereinafter referred to as the recombinant cell of the present invention) is selected, a auxotrophic complementary gene or drug resistance is selected. It is preferable to use a selectable marker gene such as a gene. The selection marker is not particularly limited, but if the host cell is yeast of the genus Komagataera, if it is an auxotrophic complementary gene such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, uracil, leucine, adenin, respectively. The genetically modified cells of the present invention can be selected by restoring the auxotrophic strain phenotype in the auxotrophic strains of yeast and arginine. In addition, if it is a drug resistance gene such as G418 resistance gene, Zeocin ™ resistance gene, Hyglomycin resistance gene, Clone NAT resistance gene, Blasticidin S resistance gene, G418, Zeocin ™, Hyglomycin, Clone, respectively. The recombinant cells of the present invention can be selected by resistance on a medium containing NAT and Blasticidin S. The auxotrophic selectable marker used when producing the recombinant yeast cannot be used if the selectable marker is not destroyed in the host yeast. In this case, the selectable marker may be destroyed in the host yeast, and a method known to those skilled in the art can be used as the method.
２．内在性遺伝子の発現が増強された遺伝子組換え細胞
　本発明は、内在性遺伝子の発現が増強された遺伝子組換え細胞（以下、本発明の遺伝子組換え細胞）を提供する。 2. Recombinant cells with enhanced expression of endogenous genes The present invention provides transgenic cells with enhanced expression of endogenous genes (hereinafter, the recombinant cells of the present invention).
　本発明の遺伝子組換え細胞は、本発明の遺伝子組換え細胞の製造方法によって調製することができる。具体的には、本発明の遺伝子組換え細胞は、内在性遺伝子が核酸断片を含むプラスミドが制限酵素で切断された直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された該内在性遺伝子を含む遺伝子組換え細胞であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、遺伝子組換え細胞である。 The genetically modified cell of the present invention can be prepared by the method for producing a genetically modified cell of the present invention. Specifically, the recombinant cell of the present invention can function as a highly expressive promoter in which an endogenous gene is homologously recombined with a linear nucleic acid in which a plasmid containing a nucleic acid fragment is cleaved with a limiting enzyme. In a recombinant cell containing the ligated endogenous gene, the nucleic acid fragment is a highly expressive promoter, a partial sequence starting from the starting codon of the endogenous gene, and the restriction enzyme recognition site is inserted. A recombinant cell containing a base sequence in which a partial sequence and a termination codon are sequentially linked.
　本発明の遺伝子組換え細胞における、遺伝子組換え細胞、内在性遺伝子、核酸断片、プラスミド、制限酵素、高発現性プロモーター、部分配列などは本発明の遺伝子組換え細胞の製造方法で記載されたものと同一であってよい。 The gene-recombinant cell, endogenous gene, nucleic acid fragment, plasmid, restriction enzyme, highly expressive promoter, partial sequence, etc. in the gene-recombinant cell of the present invention are described in the method for producing a gene-recombinant cell of the present invention. May be the same as.
　本発明の遺伝子組換え細胞は、目的タンパク質をコードする塩基配列をゲノムに含んでもよい。本発明において目的タンパク質とは、目的タンパク質をコードする塩基配列をゲノムに含む細胞が生産するタンパク質であって、その細胞の内在性タンパク質であってもよいし、異種タンパク質であってもよい。目的タンパク質の例として、微生物由来の酵素類、多細胞生物である動物や植物が産生するタンパク質等が挙げられる。例えば、フィターゼ、プロテインA、プロテインG、プロテインL、アミラーゼ、グルコシダーゼ、セルラーゼ、リパーゼ、プロテアーゼ、グルタミナーゼ、ペプチダーゼ、ヌクレアーゼ、オキシダーゼ、ラクターゼ、キシラナーゼ、トリプシン、ペクチナーゼ、イソメラーゼ、フィブロイン、及び蛍光タンパク質等が挙げられるが、これらに限定はされない。特に、ヒト及び/又は動物治療用タンパク質が好ましい。ヒト及び/又は動物治療用タンパク質として、具体的には、B型肝炎ウイルス表面抗原、ヒルジン、抗体、ヒト抗体、部分抗体、ヒト部分抗体、血清アルブミン、ヒト血清アルブミン、上皮成長因子、ヒト上皮成長因子、インシュリン、成長ホルモン、エリスロポエチン、インターフェロン、血液凝固第VIII因子、顆粒球コロニー刺激因子（G-CSF）、顆粒球マクロファージコロニー刺激因子（GM-CSF）、トロンボポエチン、IL-1、IL-6、組織プラスミノーゲン活性化因子（TPA）、ウロキナーゼ、レプチン、及び幹細胞成長因子（SCF）等が挙げられる。 The recombinant cell of the present invention may contain a base sequence encoding a target protein in its genome. In the present invention, the target protein is a protein produced by a cell whose genome contains a base sequence encoding the target protein, and may be an endogenous protein of the cell or a heterologous protein. Examples of the target protein include enzymes derived from microorganisms, proteins produced by animals and plants that are multicellular organisms, and the like. Examples thereof include phytase, protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, fluorescent protein and the like. However, it is not limited to these. In particular, human and / or animal therapeutic proteins are preferred. As proteins for human and / or animal treatment, specifically, hepatitis B virus surface antigen, hirudin, antibody, human antibody, partial antibody, human partial antibody, serum albumin, human serum albumin, epithelial growth factor, human epithelial growth. Factors, insulin, growth hormone, erythropoetin, interferon, blood coagulation factor VIII, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), thrombopoetin, IL-1, IL-6, Tissue plasminogen activator (TPA), urokinase, leptin, stem cell growth factor (SCF) and the like.
　ここで抗体とは、L鎖とH鎖の各2本のポリペプチド鎖がジスルフィド結合して構成されるヘテロテトラマータンパク質のことを指し、特定の抗原に結合する能力を有していれば、特に限定されない。 Here, the antibody refers to a heterotetramer protein composed of two polypeptide chains, an L chain and an H chain, formed by disulfide bonds, especially if it has the ability to bind to a specific antigen. Not limited.
　ここで部分抗体とは、Fab抗体、（Fab）2抗体、scFv抗体、diabody抗体、ラクダVHH抗体や、これらの誘導体等のことを指し、特定の抗原に結合する能力を有していれば、特に限定されない。Fab抗体とは、抗体のL鎖とFd鎖がS-S結合で結合したヘテロマータンパク質、又は抗体のL鎖とFd鎖がS-S結合を含まず会合したヘテロマータンパク質のことを指し、特定の抗原に結合する能力を有していれば、特に限定されない。 Here, the partial antibody refers to a Fab antibody, (Fab) 2 antibody, scFv antibody, diabody antibody, camel VHH antibody, derivatives thereof, etc., as long as it has the ability to bind to a specific antigen. There is no particular limitation. Fab antibody refers to a heteromer protein in which the L chain and Fd chain of an antibody are bound by an SS bond, or a heteromer protein in which the L chain and Fd chain of an antibody are associated without an SS bond. It is not particularly limited as long as it has the ability to combine.
　上記の目的タンパク質を構成するアミノ酸は天然のものでもよいし、非天然のものでもよいし、修飾を受けていてもよい。また、タンパク質のアミノ酸配列は、人為的な改変が施されていてもよいし、de-novoで設計されたものでもよい。 The amino acids constituting the above-mentioned target protein may be natural, non-natural, or modified. In addition, the amino acid sequence of the protein may be artificially modified or may be designed by de-novo.
　上記の目的タンパク質をコードする塩基配列は発現ベクターに含まれ、本発明の遺伝子組換え細胞のゲノムの任意の部位に相同組換えによって組み込まれる。該発現ベクターは、例えば、上記の目的タンパク質をコードする塩基配列を含むDNA断片を切り出し、該DNA断片を適当な発現ベクター中のプロモーターの下流に連結することにより製造することができる。
　発現ベクターとしては、大腸菌由来のプラスミド（例、pBR322，pBR325，pUC12，pUC13）；枯草菌由来のプラスミド（例、pUB110，pTP5，pC194）；酵母由来プラスミド（例、pSH19，pSH15）；昆虫細胞発現プラスミド（例：pFast-Bac）；動物細胞発現プラスミド（例：pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo）；λファージなどのバクテリオファージ；バキュロウイルスなどの昆虫ウイルスベクター（例：BmNPV、AcNPV）；レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。
　プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよく、例えば、本発明の高発現性プロモーターなどが好ましく挙げられる。 The base sequence encoding the above-mentioned target protein is contained in an expression vector, and is integrated by homologous recombination into an arbitrary site in the genome of the recombinant cell of the present invention. The expression vector can be produced, for example, by cutting out a DNA fragment containing a base sequence encoding the above-mentioned target protein and linking the DNA fragment downstream of a promoter in an appropriate expression vector.
Expression vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacteriophage-derived plasmids (eg, pUB110, pTP5, pC194); Yeast-derived plasmids (eg, pSH19, pSH15); Insect cell expression. Plasmid (eg pFast-Bac); animal cell expression plasmid (eg pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophage such as λ phage; insect virus vector such as baculovirus (eg) Example: BmNPV, AcNPV); Animal virus vectors such as retrovirus, vaccinia virus, and adenovirus are used.
The promoter may be any promoter as long as it is suitable for the host used for gene expression, and for example, the highly expressive promoter of the present invention is preferably mentioned.
　発現ベクターとしては、上記の他に、所望によりエンハンサー、スプライシングシグナル、ポリA付加シグナル、選択マーカー、SV40複製起点などを含有しているものを用いることができる。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素（dhfr）遺伝子、アンピシリン耐性遺伝子、ネオマイシン耐性遺伝子等が挙げられる。特に、dhfr遺伝子欠損チャイニーズハムスター細胞を用い、dhfr遺伝子を選択マーカーとして使用する場合、チミジンを含まない培地によって目的遺伝子を選択することもできる。
　また、必要に応じて、宿主細胞に合ったシグナル配列をコードする塩基配列（シグナルコドン）を、目的タンパク質をコードする塩基配列の5’末端側に付加（またはネイティブなシグナルコドンと置換）してもよい。例えば、宿主細胞がエシェリヒア属菌である場合、PhoAシグナル配列、OmpAシグナル配列などが；宿主細胞がバチルス属菌である場合、α-アミラーゼシグナル配列、サブチリシンシグナル配列などが；宿主細胞が酵母である場合、MFαシグナル配列、SUC2シグナル配列などが；宿主細胞が動物細胞である場合、インスリンシグナル配列、α-インターフェロンシグナル配列、抗体分子シグナル配列などがそれぞれ用いられる。
　また、発現ベクターは、本発明の遺伝子組換え細胞のゲノムの部分配列であって、制限酵素認識部位が挿入された部分配列を含む。制限酵素によって切断された直鎖状の発現ベクターは、本発明の遺伝子組換え細胞に導入され、５’末端に配置された部分配列の後半部（相同配列１）と３’末端に配置された部分配列の前半部（相同配列２）を本発明の遺伝子組換え細胞のゲノムの部分配列に対する相同性領域として、相同組換え（シングルクロスオーバー組換え）が生じる。相同組換えの結果、本発明の目的タンパク質をコードする塩基配列は本発明の遺伝子組換え細胞のゲノムに挿入される。 As the expression vector, in addition to the above, a vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication, or the like can be used, if desired. Examples of the selectable marker include a dihydrofolate reductase (dhfr) gene, an ampicillin resistance gene, a neomycin resistance gene and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selectable marker, the target gene can also be selected using a thymidine-free medium.
If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host cell is added (or replaced with a native signal codon) to the 5'end side of the base sequence encoding the target protein. May be good. For example, if the host cell is a bacterium of the genus Escherichia, the PhoA signal sequence, the OmpA signal sequence, etc .; if the host cell is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc.; If the host cell is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. are used, respectively.
In addition, the expression vector is a partial sequence of the genome of the recombinant cell of the present invention, and contains a partial sequence in which a restriction enzyme recognition site is inserted. The linear expression vector cleaved by the restriction enzyme was introduced into the recombinant cell of the present invention and placed at the latter half (homologous sequence 1) and 3'end of the partial sequence arranged at the 5'end. Homologous recombination (single crossover recombination) occurs with the first half of the partial sequence (homologous sequence 2) as a region of homologousness to the partial sequence of the genome of the recombinant cell of the present invention. As a result of homologous recombination, the base sequence encoding the target protein of the present invention is inserted into the genome of the recombinant cell of the present invention.
３．目的タンパク質をコードする塩基配列をゲノムに含む本発明の遺伝子組換え細胞を培養する工程を含む、目的タンパク質の製造方法（以下、本発明の目的タンパク質の製造方法）を提供する。 3. 3. Provided is a method for producing a target protein (hereinafter, a method for producing a target protein of the present invention), which comprises a step of culturing a recombinant cell of the present invention containing a base sequence encoding the target protein in the genome.
　細胞の培養条件は特に限定されず、細胞に応じて適宜選択すればよい。該培養においては、細胞が資化しうる栄養源を含む培地であれば何でも使用できる。該栄養源としては、グルコース、シュークロース、マルトース等の糖類、乳酸、酢酸、クエン酸、プロピオン酸等の有機酸類、メタノール、エタノール、グリセロール等のアルコール類、パラフィン等の炭化水素類、大豆油、菜種油等の油類、又はこれらの混合物等の炭素源、硫酸アンモニウム、リン酸アンモニウム、尿素、酵母エキス、肉エキス、ペプトン、コーンスチープリカー等の窒素源、更に、その他の無機塩、ビタミン類等の栄養源を適宜混合した通常の培地を用いることができる。また、培養はバッチ培養や連続培養のいずれでも可能である。 The cell culture conditions are not particularly limited and may be appropriately selected according to the cells. In the culture, any medium containing a nutrient source capable of assimilating cells can be used. Examples of the nutrient source include sugars such as glucose, shoe cloth and maltose, organic acids such as lactic acid, acetic acid, citric acid and propionic acid, alcohols such as methanol, ethanol and glycerol, hydrocarbons such as paraffin and soybean oil. Oils such as rapeseed oil, carbon sources such as mixtures thereof, nitrogen sources such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, and other inorganic salts, vitamins, etc. A normal medium in which nutrient sources are appropriately mixed can be used. In addition, the culture can be either batch culture or continuous culture.
　本発明の好ましい態様として、酵母にコマガタエラ属酵母又はオガタエア属酵母を用いた場合、上記炭素源は、グルコース、グリセロール、メタノールの1種でもよく、又は2種以上であってもよい。また、これらの炭素源は培養初期から存在していてもよいし、培養途中に添加してもよい。 As a preferred embodiment of the present invention, when Komagataera yeast or Ogataea yeast is used as the yeast, the carbon source may be one kind of glucose, glycerol, and methanol, or two or more kinds. Further, these carbon sources may be present from the initial stage of culturing, or may be added during culturing.
　目的タンパク質をコードする塩基配列をゲノムに含む本発明の遺伝子組換え細胞を培養することで細胞中又は培養液中に目的タンパク質を蓄積させ、回収することができる。目的タンパク質の回収方法については、公知の精製法を適当に組み合わせて用いることができる。例えば、まず、目的タンパク質をコードする塩基配列をゲノムに含む本発明の遺伝子組換え細胞を適当な培地で培養し、培養液の遠心分離、あるいは、濾過処理により培養上清から細胞を除く。得られた培養上清を、塩析（硫酸アンモニウム沈殿、リン酸ナトリウム沈殿等）、溶媒沈殿（アセトン又はエタノール等によるタンパク質分画沈殿法）、透析、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、アフィニティークロマトグラフィー、逆相クロマトグラフィー、限外濾過等の手法を単独で、又は組み合わせて用いることにより、該培養上清から目的タンパク質を回収する。 By culturing the recombinant cell of the present invention containing the base sequence encoding the target protein in the genome, the target protein can be accumulated and recovered in the cell or in the culture medium. As a method for recovering the target protein, a known purification method can be used in an appropriate combination. For example, first, the recombinant cells of the present invention containing the nucleotide sequence encoding the target protein in the genome are cultured in an appropriate medium, and the cells are removed from the culture supernatant by centrifugation or filtration of the culture medium. The obtained culture supernatant is subjected to salting (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone, ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography. The target protein is recovered from the culture supernatant by using techniques such as imaging, affinity chromatography, reverse phase chromatography, and ultrafiltration alone or in combination.
　細胞の培養は通常一般の条件により行なうことができ、例えば、酵母の場合、pH2．5～10．0、温度範囲10℃～48℃の範囲で、好気的に10時間～10日間培養することにより行うことができる。 The cells can usually be cultured under general conditions. For example, in the case of yeast, the cells are aerobically cultured for 10 hours to 10 days in a pH range of 2.5 to 10.0 and a temperature range of 10 ° C to 48 ° C. It can be done by.
　回収された目的タンパク質は、そのまま使用することもできるが、その後PEG化等の薬理学的な変化をもたらす修飾、酵素やアイソトープ等の機能を付加する修飾を加えて使用することもできる。また、各種の製剤化処理を使用してもよい。 The recovered target protein can be used as it is, but it can also be used afterwards with modifications that bring about pharmacological changes such as PEGylation and modifications that add functions such as enzymes and isotopes. Moreover, various formulation treatments may be used.
４．本発明の遺伝子組換え細胞を含む、各内在遺伝子の発現が増強された細胞ライブラリ（以下、本発明の内在性遺伝子過剰発現細胞ライブラリ）および本発明の内在性遺伝子過剰発現細胞ライブラリを用いた目的タンパク質の生産を増強する内在性遺伝子をスクリーニングする方法（以下、本発明のスクリーニング方法１および２）を提供する。 4. Purpose of using the cell library in which the expression of each endogenous gene is enhanced, including the recombinant cell of the present invention (hereinafter referred to as the endogenous gene overexpressing cell library of the present invention), and the endogenous gene overexpressing cell library of the present invention. Provided are methods for screening endogenous genes that enhance protein production (hereinafter, screening methods 1 and 2 of the present invention).
　本発明の遺伝子組換え細胞では、宿主細胞に元々備わっていた内在性遺伝子は本発明の直鎖状核酸に含まれる終止コドンによって機能を喪失する。その代わりに、機能を喪失した該遺伝子の下流では、本発明の直鎖状核酸に含まれる高発現性プロモーターに機能可能に連結された新たな遺伝子（内在性遺伝子と同一遺伝子）が生じる。結果的に、本発明の遺伝子組換え細胞では、高発現性プロモーターによって内在性遺伝子の発現は増強される。従って、宿主細胞の全内在性遺伝子の各遺伝子について発現が増強された本発明の遺伝子組換え細胞集団は、各内在遺伝子の発現が増強された細胞ライブラリとして使用することができる。 In the recombinant cell of the present invention, the endogenous gene originally provided in the host cell loses its function due to the stop codon contained in the linear nucleic acid of the present invention. Instead, downstream of the dysfunctional gene results in a new gene (the same gene as the endogenous gene) operably linked to the highly expressive promoter contained in the linear nucleic acid of the invention. As a result, in the recombinant cells of the present invention, the expression of the endogenous gene is enhanced by the highly expressive promoter. Therefore, the recombinant cell population of the present invention in which the expression of each gene of all endogenous genes of the host cell is enhanced can be used as a cell library in which the expression of each endogenous gene is enhanced.
　本発明のスクリーニング方法１は、以下の工程を含む。
（１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
（２）該直鎖状核酸を目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞に導入する工程、
（３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程、
（４）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞を培養する工程、
（５）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞による目的タンパク質の生産量をそれぞれ測定する工程、および
（６）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。 The screening method 1 of the present invention includes the following steps.
(1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
(2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding a target protein in the genome.
(3) A step of selecting a recombinant cell containing an endogenous gene in which the endogenous gene is homologously recombined by the linear nucleic acid and to which a highly expressive promoter is operably linked.
(4) A step of culturing a host cell containing the cell obtained in step (3) and the base sequence encoding the target protein in the genome.
(5) The step of measuring the production amount of the target protein by the cells obtained in the step (3) and the host cell containing the base sequence encoding the target protein in the genome, respectively, and (6) increasing the production amount of the target protein. The step of identifying the endogenous gene to be caused.
　本発明のスクリーニング方法１は、核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程（工程（１））を含む。
　本発明のスクリーニング方法１における工程（１）は、本発明の遺伝子組換え細胞の製造方法における工程（１）と同一であってよい。 The screening method 1 of the present invention is a step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a stop codon of the endogenous gene. The step (step (1)) is included, which comprises a partial sequence starting from, a partial sequence into which the restriction enzyme recognition site is inserted, and a base sequence in which stop codons are sequentially linked.
The step (1) in the screening method 1 of the present invention may be the same as the step (1) in the method for producing a recombinant cell of the present invention.
　本発明のスクリーニング方法１は、本発明の直鎖状核酸を目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞（以下、本発明の目的タンパク質発現細胞）に導入する工程（工程（２））を含む。
　本発明の直鎖状核酸を本発明の目的タンパク質発現細胞へ導入する方法は、本発明の遺伝子組換え細胞の製造方法における工程（２）に記載のものと同一であってよい。目的タンパク質は、本発明の遺伝子組換え細胞のゲノムに含まれる目的タンパク質と同一であってよい。また、宿主細胞は、本発明の遺伝子組換え細胞の製造方法で用いられる宿主細胞と同一であってよい。 The screening method 1 of the present invention is a step of introducing the linear nucleic acid of the present invention into a host cell (hereinafter, a cell expressing the target protein of the present invention) containing a base sequence encoding the target protein in the genome (step (2)). )including.
The method for introducing the linear nucleic acid of the present invention into the target protein-expressing cell of the present invention may be the same as that described in step (2) in the method for producing a recombinant cell of the present invention. The target protein may be the same as the target protein contained in the genome of the recombinant cell of the present invention. Further, the host cell may be the same as the host cell used in the method for producing a recombinant cell of the present invention.
　本発明のスクリーニング方法１は、内在性遺伝子が本発明の直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程（工程（３））を含む。
　本発明のスクリーニング方法１における工程（３）は、本発明の遺伝子組換え細胞の製造方法における工程（３）と同一であってよい。 The screening method 1 of the present invention selects transgenic cells containing an endogenous gene operably linked with a highly expressive promoter, in which the endogenous gene is homologously recombined by the linear nucleic acid of the present invention. Includes step (step (3)).
The step (3) in the screening method 1 of the present invention may be the same as the step (3) in the method for producing a recombinant cell of the present invention.
　本発明のスクリーニング方法１は、工程（３）で得られた細胞および本発明の目的タンパク質発現細胞を培養する工程（工程（４））を含む。
　工程（３）で得られた細胞および本発明の目的タンパク質発現細胞を培養する方法は、本発明の遺伝子組換え細胞を培養する方法に記載の培養方法と同一であってよい。 The screening method 1 of the present invention includes a step (step (4)) of culturing the cells obtained in the step (3) and the target protein-expressing cells of the present invention.
The method for culturing the cells obtained in step (3) and the target protein-expressing cells of the present invention may be the same as the culturing method described in the method for culturing the recombinant cells of the present invention.
　本発明のスクリーニング方法１は、工程（３）で得られた細胞および目的タンパク質発現細胞による目的タンパク質の生産量をそれぞれ測定する工程（工程（５））を含む。 The screening method 1 of the present invention includes a step (step (5)) of measuring the production amount of the target protein by the cells obtained in the step (3) and the target protein-expressing cells, respectively.
　工程（３）で得られた細胞および本発明の目的タンパク質発現細胞による目的タンパク質の生産量の測定は、目的タンパク質の転写産物であるmRNAまたは翻訳産物であるポリペプチドの発現量を測定することによって行うことができる。mRNAの発現量はリアルタイムPCR法、RNA-Seq法、ノーザンハイブリダイゼーション又はDNAアレイを利用したハイブリダイゼーション法等を用いて定量することができ、ポリペプチドの発現量は、ポリペプチドを認識する抗体やポリペプチドと結合性を有する染色化合物等を用いて定量することができる。また、上記に挙げた定量方法以外にも、当業者で用いられている従来法であってもよい。 The amount of target protein produced by the cells obtained in step (3) and the target protein-expressing cells of the present invention is measured by measuring the expression level of mRNA, which is a transcript of the target protein, or polypeptide, which is a translation product. It can be carried out. The expression level of mRNA can be quantified by using real-time PCR method, RNA-Seq method, Northern hybridization, hybridization method using DNA array, etc., and the expression level of polypeptide can be determined by an antibody that recognizes a polypeptide or It can be quantified using a staining compound or the like having binding property to the polypeptide. Further, in addition to the quantification method described above, a conventional method used by those skilled in the art may be used.
　本発明のスクリーニング方法１は、目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程（工程（６））を含む。 The screening method 1 of the present invention includes a step (step (6)) of identifying an endogenous gene that increases the production amount of the target protein.
　工程（６）において、工程（３）で得られた細胞における目的タンパク質の生産量が本発明の目的タンパク質発現細胞における目的タンパク質の生産量より高ければ、該細胞は目的タンパク質の生産量を増加させる内在性遺伝子を含む細胞であると判断することができる。工程（３）で得られた細胞における目的タンパク質の生産量の増加は、本発明の目的タンパク質発現細胞における目的タンパク質の生産量に対して、例えば1.01倍、1.02倍、1.03倍、1.04倍、1.05倍、1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、1.6倍、1.7倍、1.8倍、1.9倍、2倍、2.5倍、3倍、3.5倍、4倍、4.5倍、又は5倍以上、また100倍、90倍、80倍、70倍、60倍、50倍、40倍、30倍、20倍、10倍、9倍、8倍、7倍、6倍、又は5倍以下であればよい。目的タンパク質を分泌生産する場合、細胞からの全タンパク質の分泌生産量は、細胞の培養上清等を用いて、当業者に公知の方法、例えばBladford法、Lowry法、及びBCA法等により容易に決定することができる。細胞からの特定の目的タンパク質の分泌生産量は、細胞の培養上清等を用いて、ELISA法等により容易に決定することができる。 In step (6), if the production amount of the target protein in the cells obtained in step (3) is higher than the production amount of the target protein in the target protein-expressing cells of the present invention, the cells increase the production amount of the target protein. It can be determined that the cell contains an endogenous gene. The increase in the production amount of the target protein in the cells obtained in step (3) is, for example, 1.01 times, 1.02 times, 1.03 times, 1.04 times, 1.05 times the production amount of the target protein in the target protein-expressing cells of the present invention. Double, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times Above, 100 times, 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, or 5 times or less All you need is. When the target protein is secreted and produced, the amount of total protein secreted and produced from the cells can be easily determined by a method known to those skilled in the art, such as the Bladeford method, the Lowry method, the BCA method, etc., using the cell culture supernatant or the like. Can be decided. The amount of secreted production of a specific target protein from cells can be easily determined by an ELISA method or the like using a cell culture supernatant or the like.
　上記の通りに工程（３）で得られた細胞から目的タンパク質の発現を増加させる遺伝子組換え細胞を特定した後、公知の手段で発現が増強されている内在性遺伝子を特定することができる。例えば、細胞からゲノムを抽出し、制限酵素で断片化した後、断片化したゲノムをセルフライゲーションさせる。セルフライゲーションにより環状化した目的の内在性遺伝子を含むゲノム断片は、薬剤耐性遺伝子によりスクリーニングすることができる。スクリーニングされた目的の内在性遺伝子を含むゲノム断片をサンガー法により塩基配列を特定することによって、該内在性遺伝子を具体的に特定することができる。 As described above, after identifying the recombinant cell that increases the expression of the target protein from the cells obtained in the step (3), it is possible to identify the endogenous gene whose expression is enhanced by a known means. For example, the genome is extracted from cells, fragmented with a restriction enzyme, and then the fragmented genome is self-ligated. Genome fragments containing the endogenous gene of interest cyclized by self-ligation can be screened by drug resistance genes. The endogenous gene can be specifically identified by specifying the nucleotide sequence of the screened genomic fragment containing the target endogenous gene by the Sanger method.
　また別の実施態様として、本発明のスクリーニング方法２は、以下の工程を含む。
（１）本発明の内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞に目的タンパク質をコードする塩基配列を含む発現ベクターを導入し、培養する工程、
（２）内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞による目的タンパク質の生産量を測定する工程、および
（３）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。 As another embodiment, the screening method 2 of the present invention includes the following steps.
(1) A step of introducing an expression vector containing a base sequence encoding a target protein into a cell library overexpressing an endogenous gene of the present invention and a host cell and culturing the cells.
(2) A step of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell, and (3) a step of identifying the endogenous gene that increases the production amount of the target protein.
　本発明のスクリーニング方法２は、本発明の内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞に目的タンパク質をコードする塩基配列を含む発現ベクターを導入し、培養する工程（工程（１））を含む。
　本発明の内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞に目的タンパク質をコードする塩基配列を含む発現ベクターを導入し、培養する方法は、本発明の直鎖状核酸を宿主細胞に導入する方法および本発明の遺伝子組換え細胞を培養する方法に記載のもの同一であってよい。 The screening method 2 of the present invention includes a step (step (1)) of introducing an expression vector containing a base sequence encoding a target protein into a host cell and an endogenous gene overexpressing cell library of the present invention and culturing the cells.
The method of introducing the endogenous gene overexpressing cell library of the present invention and the expression vector containing the base sequence encoding the target protein into the host cell and culturing the method is the method of introducing the linear nucleic acid of the present invention into the host cell and the present invention. It may be the same as that described in the method for culturing transgenic cells of the present invention.
　本発明のスクリーニング方法２は、内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞による目的タンパク質の生産量を測定する工程（工程（２））を含む。
　内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞による目的タンパク質の生産量の測定方法は、本発明のスクリーニング方法１における目的タンパク質の生産量の測定方法と同一であってよい。 The screening method 2 of the present invention includes a step (step (2)) of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell.
The method for measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell may be the same as the method for measuring the production amount of the target protein in the screening method 1 of the present invention.
　本発明のスクリーニング方法２は、目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程（工程（３））を含む。
　目的タンパク質の生産量を増加させる内在性遺伝子の特定方法は、本発明のスクリーニング方法１における内在性遺伝子の特定方法と同一であってよい。 The screening method 2 of the present invention includes a step (step (3)) of identifying an endogenous gene that increases the production of the target protein.
The method for identifying the endogenous gene that increases the production of the target protein may be the same as the method for identifying the endogenous gene in the screening method 1 of the present invention.
　以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されない。 The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto.
材料と方法
（１）ベクターの調製に用いた各種遺伝子の調製
　以下の実施例において用いた組換えDNA技術に関する詳細な操作方法等は、次の成書に記載されている：Molecular Cloning 2nd Edition（Cold Spring Harbor Laboratory Press,1989）、Current Protocols in Molecular Biology（Greene Publishing Associates and Wiley-Interscience）。
　また、以下の実施例において、酵母の形質転換に用いるプラスミドは、構築したベクターを大腸菌E.coli DH5αコンピテントセル（タカラバイオ社製）に導入し、得られた形質転換体を培養して増幅することによって調製した。プラスミド保持株からのプラスミドの調製は、Wizard（登録商標）Plus SV Minipreps DNA Purification Systems（プロメガ社製）を用いて行った。
　ベクターの構築において利用したGAPプロモーター（配列番号1）、AOX1プロモーター（配列番号2）、AOX1ターミネーター（配列番号3）、CCA38473ターミネーター（配列番号4）、ARG4遺伝子（配列番号5）、URA3遺伝子及びその下流配列（配列番号6）、GUT1遺伝子（配列番号7）、ターミネーターが連結されたコマガタエラ・ファフィ由来のKAR2をコードする遺伝子（配列番号8）及びPDI1をコードする遺伝子（配列番号9）はコマガタエラ・ファフィCBS7435株の染色体DNA（塩基配列はEMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628～FR839631に記載)混合物をテンプレートにしてPCRで調製した。ターミネーターが連結されたコマガタエラ・ファフィ由来の抗体発現促進タンパク質群（配列番号10～44で示されるアミノ酸配列）をコードする遺伝子（配列番号45～79）はスクリーニングにより得られたコマガタエラ・ファフィのゲノムをテンプレートとしてPCRで調製した。GAPプロモーターはプライマー1（配列番号80）及びプライマー2（配列番号81）、AOX1プロモーターはプライマー3（配列番号82）及びプライマー4（配列番号83）、AOX1ターミネーターはプライマー5（配列番号84）及びプライマー6（配列番号85）、CCA38473ターミネーターはプライマー7（配列番号86）及びプライマー8（配列番号87）、プロモーターで制御されたARG4遺伝子はプライマー9（配列番号88）及びプライマー10（配列番号89）、開始コドンを欠損し、かつ内部にAscI-PmeI認識部位を付加したURA3遺伝子はプライマー11（配列番号90）及び変異導入用のプライマー12（配列番号91）、変異導入用のプライマー13（配列番号92）及びプライマー14（配列番号93）、開始コドンを欠損し、かつ内部にAscI-PmeI認識部位を付加したGUT1遺伝子はプライマー15（配列番号94）及び変異導入用のプライマー16（配列番号95）、変異導入用のプライマー17（配列番号96）及びプライマー18（配列番号97）、ターミネーターが連結された抗体発現促進タンパク質（配列番号10～44で示されるアミノ酸配列）をコードする遺伝子はフォワード用のプライマー19（配列番号98）とそれぞれのリバース用のプライマー20～55（配列番号99～134）を用いてPCRで調製した。
　ベクターの構築において利用した分泌シグナルMFα遺伝子（配列番号135）はサッカロマイセス・セレビシエBY4741株の染色体DNA（塩基配列はACCESSION No. BK006934～BK006949に記載)混合物をテンプレートにしてプライマー56（配列番号136）及びプライマー57（配列番号137）を用いてPCRで調製した。また分泌シグナルMFα遺伝子の２アミノ酸置換体（L42S/V50A）（配列番号138）は合成DNAをテンプレートとして、プライマー56（配列番号136）及びプライマー57（配列番号137）を用いてPCRで調製した。
　ベクターの構築において利用した、プロモーターで制御されたZeocin(商標)耐性遺伝子（配列番号139）は合成DNAをテンプレートにしてPCRで調製した。ベクターの構築において利用した、プロモーターで制御されたG418耐性遺伝子（配列番号140）は合成DNAをテンプレートにしてPCRで調製した。ベクターの構築において利用した、プロモーターで制御されたハイグロマイシン耐性遺伝子（配列番号141）は合成DNAをテンプレートにしてPCRで調製した。ベクターの構築において利用した、プロモーターで制御されたノーセオスリシン耐性遺伝子（配列番号142）は合成DNAをテンプレートにしてPCRで調製した。ベクターの構築において利用した、プロモーターで制御されたブラストサイジン耐性遺伝子（配列番号143）は合成DNAをテンプレートにしてPCRで調製した。
　ベクターの構築において利用した、抗リゾチウム一本鎖抗体遺伝子（配列番号144）、タンデムscFv226抗体遺伝子（配列番号145）、ブリナツモマブ抗体遺伝子（配列番号146）及びミニボディー抗体（配列番号147）は合成DNAをテンプレートにしてPCRで調製した。またベクターの構築において利用した、KAR2遺伝子（配列番号8）及びPDI1遺伝子（配列番号9）はコマガタエラ・ファフィCBS7435株のゲノムをテンプレートにしてPCRで調製した。
　PCRにはPrime STAR HS DNA Polymerase（タカラバイオ社製）等を用い、反応条件は添付のマニュアルに記載の方法で行った。染色体DNAの調製は、コマガタエラ・パストリスATCC76273株又はサッカロマイセス・セレビシエBY4741株からカネカ 簡易DNA抽出キット version 2（カネカ社製）等を用いて、これに記載の条件で実施した。 Materials and Methods (1) Preparation of Various Genes Used for Vector Preparation Detailed operating methods related to the recombinant DNA technology used in the following examples are described in the following textbook: Molecular Cloning 2nd Edition (Molecular Cloning 2nd Edition ( Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).
Further, in the following examples, for the plasmid used for yeast transformation, the constructed vector was introduced into E. coli DH5α competent cells (manufactured by Takara Bio Inc.), and the obtained transformant was cultured and amplified. Prepared by Preparation of the plasmid from the plasmid-carrying strain was performed using Wizard® Plus SV Minipreps DNA Purification Systems (manufactured by Promega).
GAP promoter (SEQ ID NO: 1), AOX1 promoter (SEQ ID NO: 2), AOX1 terminator (SEQ ID NO: 3), CCA38473 terminator (SEQ ID NO: 4), ARG4 gene (SEQ ID NO: 5), URA3 gene and their components used in the construction of the vector. The downstream sequence (SEQ ID NO: 6), GUT1 gene (SEQ ID NO: 7), the gene encoding KAR2 (SEQ ID NO: 8) derived from Komagataera fafi to which the terminator is linked, and the gene encoding PDI1 (SEQ ID NO: 9) are Komagataera. The chromosomal DNA of the Faffy CBS7435 strain (the base sequence is described in EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) was prepared by PCR using a mixture as a template. The gene (SEQ ID NO: 45-79) encoding the antibody expression-promoting protein group (amino acid sequence shown by SEQ ID NOs: 10 to 44) derived from Komagataera fafi to which the terminator is linked is the genome of Komagataera fafi obtained by screening. Prepared by PCR as a template. The GAP promoter is Primer 1 (SEQ ID NO: 80) and Primer 2 (SEQ ID NO: 81), the AOX1 promoter is Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83), and the AOX1 terminator is Primer 5 (SEQ ID NO: 84) and Primer. 6 (SEQ ID NO: 85), CCA38473 terminator is primer 7 (SEQ ID NO: 86) and primer 8 (SEQ ID NO: 87), promoter-regulated ARG4 gene is primer 9 (SEQ ID NO: 88) and primer 10 (SEQ ID NO: 89), The URA3 gene lacking the starting codon and having the AscI-PmeI recognition site added inside is Primer 11 (SEQ ID NO: 90), Primer 12 for mutagenesis (SEQ ID NO: 91), and Primer 13 for mutagenesis (SEQ ID NO: 92). ) And primer 14 (SEQ ID NO: 93), the GUT1 gene lacking the starting codon and having the AscI-PmeI recognition site added internally is primer 15 (SEQ ID NO: 94) and primer 16 for mutagenesis (SEQ ID NO: 95), The gene encoding the mutation-introducing primer 17 (SEQ ID NO: 96) and primer 18 (SEQ ID NO: 97), and the antibody expression-promoting protein (amino acid sequence shown by SEQ ID NOs: 10 to 44) to which the terminator is linked is a primer for forward. It was prepared by PCR using 19 (SEQ ID NO: 98) and primers 20 to 55 (SEQ ID NO: 99 to 134) for each reverse.
The secretory signal MFα gene (SEQ ID NO: 135) used in the construction of the vector is the chromosomal DNA of the Saccharomyces cerevisiae BY4741 strain (the base sequence is described in ACCESSION No. BK006934 to BK006949). It was prepared by PCR using Primer 57 (SEQ ID NO: 137). The diamino acid substitution (L42S / V50A) (SEQ ID NO: 138) of the secretory signal MFα gene was prepared by PCR using synthetic DNA as a template and primers 56 (SEQ ID NO: 136) and 57 (SEQ ID NO: 137).
The promoter-controlled Zeocin ™ resistance gene (SEQ ID NO: 139) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. The promoter-regulated G418 resistance gene (SEQ ID NO: 140) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. The promoter-controlled hygromycin resistance gene (SEQ ID NO: 141) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. The promoter-regulated noseoslisin resistance gene (SEQ ID NO: 142) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. The promoter-controlled blastidin resistance gene (SEQ ID NO: 143) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.
The anti-lysothium single-chain antibody gene (SEQ ID NO: 144), tandem scFv226 antibody gene (SEQ ID NO: 145), blinatumomab antibody gene (SEQ ID NO: 146) and minibody antibody (SEQ ID NO: 147) used in the construction of the vector are synthetic DNAs. Was used as a template and prepared by PCR. The KAR2 gene (SEQ ID NO: 8) and PDI1 gene (SEQ ID NO: 9) used in the construction of the vector were prepared by PCR using the genome of the Komagataera fafi CBS7435 strain as a template.
Prime STAR HS DNA Polymerase (manufactured by Takara Bio Inc.) was used for PCR, and the reaction conditions were as described in the attached manual. The chromosomal DNA was prepared from the Komagataera pastris ATCC76273 strain or the Saccharomyces cerevisiae BY4741 strain using Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka Corporation) under the conditions described therein.
（２）抗体発現用基本ベクターの構築
　HindIII-NotI-BamHI-BglII-XbaI-EcoRIのマルチクローニングサイトもつ遺伝子断片（配列番号148）を全合成し、これをpUC19（タカラバイオ社製、Code No. 3219）のHindIII-EcoRIサイト間に挿入して、pUC-1を調製した。プロモーターで制御されたG418耐性遺伝子（配列番号140）の両端にEcoRI認識配列を付加した核酸断片を、プライマー58（配列番号149）及びプライマー59（配列番号150）を用いたPCRにより調製し、EcoRI処理後にpUC-1のEcoRIサイトに挿入して、pUC_G418を構築した。
　次に、CCA38473ターミネーター（配列番号4）の核酸断片をプライマー7（配列番号86）及びプライマー8（配列番号87）を用いてPCRで調製し、上記pUC_G418をXbaI処理した核酸断片と混合し、In-fusion HD Cloning Kit（クロンテック社製）を用いて繋ぎ合わせて、pUC_T38473_G418を構築した。
　次に、AOX1プロモーター（配列番号2）の末端にBamHI認識配列及びSpeI認識配列を付加した核酸断片を、プライマー3（配列番号82）及びプライマー4（配列番号83）を用いてPCRで調製し、BamHI及びSpeI処理、pUC_T38473_G418のBamHI-BglIIサイト間に挿入して、pUC_Paox1_T38473_G418を構築した。
　次に、AOX1ターミネーター（配列番号3）の末端にMluI認識配列及びBglII認識配列を付加した核酸断片を、プライマー5（配列番号84）及びプライマー6（配列番号85）を用いてPCRで調製し、MluI及びBglII処理後にpUC_Paox1_T38473_G418のMluI-BglIIサイト間に挿入して、pUC_Paox1_Taox1_T38473_G418を構築した。
　次に、分泌シグナルMFα遺伝子（配列番号135）及びその2アミノ酸置換体（L42S/V50A）(配列番号138）の末端にSpeI認識配列及びBglII認識配列を付加した核酸断片をプライマー56（配列番号136）及びプライマー57（配列番号137）を用いてそれぞれPCRで調製し、SpeI及びBglII処理後、それぞれをpUC_Paox1_Taox1_T38473_G418のMluI-BglIIサイト間に挿入して、pUC_Paox1_MFα_Taox1_T38473_G418及びpUC_Paox1_MFα(mut)_Taox1_T38473_G418を構築した。 (2) Construction of basic vector for antibody expression A gene fragment (SEQ ID NO: 148) having a multi-cloning site of HindIII-NotI-BamHI-BglII-XbaI-EcoRI was completely synthesized, and pUC19 (manufactured by Takara Bio Inc., Code No. 3219) was inserted between the HindIII-EcoRI sites to prepare pUC-1. A nucleic acid fragment in which an EcoRI recognition sequence was added to both ends of a promoter-controlled G418 resistance gene (SEQ ID NO: 140) was prepared by PCR using primer 58 (SEQ ID NO: 149) and primer 59 (SEQ ID NO: 150), and EcoRI was prepared. After processing, it was inserted into the EcoRI site of pUC-1 to construct pUC_G418.
Next, a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using Primer 7 (SEQ ID NO: 86) and Primer 8 (SEQ ID NO: 87), and the above pUC_G418 was mixed with the XbaI-treated nucleic acid fragment and In. -pUC_T38473_G418 was constructed by connecting using the fusion HD Cloning Kit (manufactured by Clontech).
Next, a nucleic acid fragment having a BamHI recognition sequence and a SpeI recognition sequence added to the end of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 3 (SEQ ID NO: 82) and Primer 4 (SEQ ID NO: 83). BamHI and SpeI treatment, pUC_T38473_G418 was inserted between the BamHI-BglII sites to construct pUC_Paox1_T38473_G418.
Next, a nucleic acid fragment having the MluI recognition sequence and the BglII recognition sequence added to the end of the AOX1 terminator (SEQ ID NO: 3) was prepared by PCR using Primer 5 (SEQ ID NO: 84) and Primer 6 (SEQ ID NO: 85). After MluI and BglII treatment, pUC_Paox1_T38473_G418 was inserted between the MluI-BglII sites to construct pUC_Paox1_Taox1_T38473_G418.
Next, a nucleic acid fragment having a SpeI recognition sequence and a BglII recognition sequence added to the ends of the secretory signal MFα gene (SEQ ID NO: 135) and its two amino acid substitutions (L42S / V50A) (SEQ ID NO: 138) was added to Primer 56 (SEQ ID NO: 136). ) And primer 57 (SEQ ID NO: 137), respectively, after treatment with SpeI and BglII, each was inserted between the MluI-BglII sites of pUC_Paox1_Taox1_T38473_G418 to construct pUC_Paox1_MFα_Taox1_T38473_G418 and pUC_Paox1_MFα (mut) _43.
（３）抗リゾチウム一本鎖抗体発現ベクターの構築
　抗リゾチウム一本鎖抗体遺伝子（配列番号144）を、合成DNAをテンプレートにしてプライマー60（配列番号151）及びプライマー61（配列番号152）を用いてPCRで調製した。この核酸断片は抗リゾチウム一本鎖抗体をコードする塩基配列の上流にオーバーラップ領域として分泌シグナルMFα遺伝子配列の下流末端領域、下流にオーバーラップ領域としてAOX1ターミネーター配列の上流末端領域が付加されている。また、抗リゾチウム抗体をコードする塩基配列とAOX1ターミネーター配列上流末端領域との間には、Hisタグ（配列番号153）をコードする塩基配列が付加されている。
　上記（２）で構築したpUC_Pgap_MFα_Taox1_T38473_G418をXhoI及びMluI処理後に核酸断片を調製し、上記PCRにより調製された抗リゾチウム一本鎖抗体をコードする塩基配列の核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Paox1_MFα_scFv_Taox1_T38473_G418を構築した。このベクターは、抗リゾチウム一本鎖抗体がAOX1プロモーター制御下で発現するように設計されている。 (3) Construction of anti-lysothium single-chain antibody expression vector Using the anti-lysothium single-chain antibody gene (SEQ ID NO: 144) as a template, primer 60 (SEQ ID NO: 151) and primer 61 (SEQ ID NO: 152) were used. Prepared by PCR. In this nucleic acid fragment, the downstream terminal region of the secretory signal MFα gene sequence is added as an overlapping region upstream of the nucleotide sequence encoding the anti-lysothymic single-chain antibody, and the upstream terminal region of the AOX1 terminator sequence is added downstream as an overlapping region. .. In addition, a nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the anti-lysozyme antibody and the upstream terminal region of the AOX1 terminator sequence.
The pUC_Pgap_MFα_Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the anti-lysodium single-chain antibody prepared by the above PCR, and the In-fusion HD Cloning Kit was used. We constructed pUC_Paox1_MFα_scFv_Taox1_T38473_G418 by connecting them using. This vector is designed to express anti-lysozyme single chain antibodies under the control of the AOX1 promoter.
（４）二重特異性抗体（タンデムsc Fv226）発現ベクターの構築
　抗CD3一本鎖抗体と抗EGFR一本鎖抗体とを融合させた二重特異性抗体（タンデムscFv226; taFv226）遺伝子（配列番号145）を、合成DNAをテンプレートにしてプライマー62（配列番号154）及びプライマー63（配列番号155）を用いてPCRで調製した。この核酸断片はtaFv226抗体をコードする塩基配列の上流にオーバーラップ領域として分泌シグナルMFα遺伝子配列（2アミノ酸置換体（L42S/V50A））の下流末端領域、下流にオーバーラップ領域としてAOX1ターミネーター配列の上流末端領域が付加されている。また、taFv226抗体をコードする塩基配列とAOX1ターミネーター配列上流末端領域との間には、c-Mycタグ（配列番号156）及びHisタグ（配列番号153）をコードする塩基配列が付加されている。
　上記（２）で構築したpUC_Paox1_MFα(mut)_Taox1_T38473_G418をXhoI及びMluI処理後に核酸断片を調製し、上記PCRにより調製された二重特異性抗体（taFv226）をコードする塩基配列の核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Paox1_MFα(mut)_taFv226_Taox1_T38473_G418を構築した。
　次に、CCA38473ターミネーター（配列番号4）の核酸断片をプライマー64（配列番号157）及びプライマー65（配列番号158）を用いてPCRで調製し、上記pUC_Paox1_MFα(mut)_taFv226_Taox1_T38473_G418をXbaI処理した核酸断片と混合し、In-fusion HD Cloning Kit（クロンテック社製）を用いて繋ぎ合わせて、pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418を構築した。このベクターは、二重特異性抗体（taFv226）がAOX1プロモーター制御下で発現するように設計されている。 (4) Construction of bispecific antibody (tandem sc Fv226) expression vector Bispecific antibody (tandem scFv226; taFv226) gene (SEQ ID NO:) in which an anti-CD3 single-chain antibody and an anti-EGFR single-chain antibody are fused. 145) was prepared by PCR using synthetic DNA as a template and Primer 62 (SEQ ID NO: 154) and Primer 63 (SEQ ID NO: 155). This nucleic acid fragment is the downstream terminal region of the secretory signal MFα gene sequence (2-amino acid substitution (L42S / V50A)) as an overlapping region upstream of the base sequence encoding the taFv226 antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream. The terminal region is added. In addition, a nucleotide sequence encoding a c-Myc tag (SEQ ID NO: 156) and a His tag (SEQ ID NO: 153) are added between the nucleotide sequence encoding the taFv226 antibody and the upstream terminal region of the AOX1 terminator sequence.
The pUC_Paox1_MFα (mut) _Taox1_T38473_G418 constructed in (2) above was treated with XhoI and MluI to prepare a nucleic acid fragment, which was mixed with the nucleic acid fragment having the nucleotide sequence encoding the bispecific antibody (taFv226) prepared by PCR above. We constructed pUC_Paox1_MFα (mut) _taFv226_Taox1_T38473_G418 by connecting them using the In-fusion HD Cloning Kit.
Next, a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using primer 64 (SEQ ID NO: 157) and primer 65 (SEQ ID NO: 158), and the above pUC_Paox1_MFα (mut) _taFv226_Taox1_T38473_G418 was treated with the nucleic acid fragment treated with XbaI. The mixture was mixed and spliced using an In-fusion HD Cloning Kit (manufactured by Clontech) to construct pUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T38473_loxP_G418. This vector is designed for bispecific antibody (taFv226) to be expressed under AOX1 promoter control.
（５）ブリナツモマブ発現ベクターの構築
　現時点にて唯一上市されている、二重特異性低分子抗体であるブリナツモマブ遺伝子（配列番号146）を、合成DNAをテンプレートにしてプライマー66（配列番号159）及びプライマー67（配列番号160）を用いてPCRで調製した。
　この核酸断片はブリナツモマブ抗体をコードする塩基配列の上流にオーバーラップ領域として分泌シグナルMFα遺伝子配列（2アミノ酸置換体（L42S/V50A））の下流末端領域、下流にオーバーラップ領域としてAOX1ターミネーター配列の上流末端領域が付加されている。また、ブリナツモマブ抗体をコードする塩基配列とAOX1ターミネーター配列上流末端領域との間には、Hisタグ（配列番号153）をコードする塩基配列が付加されている。
　上記（４）で構築したpUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418をXhoI及びMluI処理後に核酸断片を調製し、上記PCRにより調製されたブリナツモマブ抗体をコードする塩基配列の核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418を構築した。このベクターは、ブリナツモマブ抗体がAOX1プロモーター制御下で発現するように設計されている。 (5) Construction of Blinatumomab Expression Vector The blinatumomab gene (SEQ ID NO: 146), which is the only bispecific low molecular weight antibody currently on the market, is used as a template for primer 66 (SEQ ID NO: 159) and primer. Prepared by PCR using 67 (SEQ ID NO: 160).
This nucleic acid fragment is the downstream terminal region of the secretory signal MFα gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the brinatsumomab antibody, and upstream of the AOX1 terminator sequence as an overlapping region downstream. The terminal region is added. In addition, a nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the blinatumomab antibody and the upstream terminal region of the AOX1 terminator sequence.
The pUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above was treated with XhoI and MluI to prepare a nucleic acid fragment, mixed with the nucleic acid fragment having the nucleotide sequence encoding the blinatumomab antibody prepared by the above PCR, and the In-fusion HD Cloning Kit. We constructed pUC_Paox1_MFα (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 by connecting them using. This vector is designed for blinatumomab antibody expression under AOX1 promoter control.
（６）ミニボディー抗体発現ベクターの構築
　抗EGFR一本鎖抗体遺伝子とFc領域の一部を融合させた低分子抗体であるミニボディー遺伝子（配列番号147）を、合成DNAをテンプレートにしてプライマー68（配列番号161）及びプライマー69（配列番号162）を用いてPCRで調製した。
　この核酸断片はミニボディー抗体をコードする塩基配列の上流にオーバーラップ領域として分泌シグナルMFα遺伝子配列（2アミノ酸置換体（L42S/V50A））の下流末端領域、下流にオーバーラップ領域としてAOX1ターミネーター配列の上流末端領域が付加されている。また、ミニボディー抗体をコードする塩基配列とAOX1ターミネーター配列上流末端領域との間には、Hisタグ（配列番号153）をコードする塩基配列が付加されている。
　上記（４）で構築したpUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418をXhoI及びMluI処理後に核酸断片を調製し、上記PCRにより調製されたミニボディー抗体をコードする塩基配列の核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418を構築した。このベクターは、ミニボディー抗体がAOX1プロモーター制御下で発現するように設計されている。 (6) Construction of minibody antibody expression vector Primer 68 of the minibody gene (SEQ ID NO: 147), which is a small antibody fused with an anti-EGFR single-chain antibody gene and a part of the Fc region, using synthetic DNA as a template. It was prepared by PCR using (SEQ ID NO: 161) and primer 69 (SEQ ID NO: 162).
This nucleic acid fragment is the downstream terminal region of the secretory signal MFα gene sequence (2-amino acid substituent (L42S / V50A)) as an overlapping region upstream of the nucleotide sequence encoding the minibody antibody, and the AOX1 terminator sequence downstream as an overlapping region. The upstream end region is added. In addition, a nucleotide sequence encoding a His tag (SEQ ID NO: 153) is added between the nucleotide sequence encoding the minibody antibody and the upstream terminal region of the AOX1 terminator sequence.
PUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above was treated with XhoI and MluI to prepare a nucleic acid fragment, mixed with a nucleic acid fragment having a nucleotide sequence encoding the minibody antibody prepared by PCR above, and in-fusion HD Cloning. We constructed pUC_Paox1_MFα (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 by connecting them using Kit. This vector is designed for minibody antibodies to be expressed under the control of the AOX1 promoter.
（７）遺伝子過剰発現細胞ライブラリ作製用ベクターの構築
　プラスミドpUC19のXbaI-BspQIサイト間を欠損させるため、XbaI及びBspQI処理後、Klenowフラグメント（タカラバイオ社製）処理にて末端を平滑にした後、ライゲーションキット（DNA Ligation kit <Mighty Mix>）を用いて、セルフライゲーションを行い、pUC_delを調製した。
　次に、プロモーターで制御されたZeocin(商標)耐性遺伝子（配列番号139）の両端にEcoRI認識配列を付加した核酸断片を、プライマー70（配列番号163）及びプライマー71（配列番号164）を用いてPCRで調製し、これをpUC_delのEcoRIサイト間にIn-fusion HD Cloning Kitを用いて挿入して、pUC_del_Zeoを構築した。
　GFP恒常発現プラスミドpPGP_EGFP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）上のGAPDHプロモーター内のBspQI部位を破壊するため、GAPDHプロモーターの前半部分と後半部分に当たる2つの核酸断片を、プライマー72（配列番号165）及び変異導入用のプライマー73（配列番号166）、変異導入用のプライマー74（配列番号167）及びプライマー75（配列番号168）を用いてそれぞれPCRにて調製した。
　pPGP_EGFPをBamHI及びSpeI処理後に核酸断片を調製し、上記PCRにより調製された2つの核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pPGPdel_EGFPを調製した。
　次に、BspQI部位を破壊したGAPDHプロモーター及びEGFP遺伝子断片を調製するため、pPGPdel_EGFPをテンプレートにして、プライマー76（配列番号169）及びプライマー77（配列番号170）を用いてPCRにより核酸断片を調製した。またサッカロマイセス・セレビシエ由来のCYC1ターミネーター（配列番号171）の核酸断片を調製するため、サッカロマイセス・セレビシエBY4741株をテンプレートとして、プライマー78（配列番号172）及びプライマー79（配列番号173）を用いてPCRで調製した。pUC_del_ZeoをSacI及びBamHI処理後に核酸断片を調製し、上記PCRにより調製された2つの核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_del_Zeo_Pgap-EGFP-CYC1tを調製した。このベクターは、SpeI及びXhoIに挟まれたEGFP部分をコマガタエラ・ファフィのゲノム配列情報から設計した下記OLS配列に置き換えることで、コマガタエラ・ファフィを用いた遺伝子過剰発現細胞ライブラリ作製用のベクターとなるように設計されている。 (7) Construction of vector for preparing gene overexpressing cell library In order to delete the XbaI-BspQI site of plasmid pUC19, after treatment with XbaI and BspQI, smoothing the ends with Klenow fragment (manufactured by Takara Bio Inc.), Self-ligation was performed using a ligation kit (DNA Ligation kit <Mighty Mix>) to prepare pUC_del.
Next, a nucleic acid fragment in which an EcoRI recognition sequence was added to both ends of a promoter-controlled Zeocin ™ resistance gene (SEQ ID NO: 139) was prepared using primer 70 (SEQ ID NO: 163) and primer 71 (SEQ ID NO: 164). It was prepared by PCR and inserted between the EcoRI sites of pUC_del using the In-fusion HD Cloning Kit to construct pUC_del_Zeo.
Two nucleic acids in the first and second half of the GAPDH promoter to disrupt the BspQI site within the GAPDH promoter on the GFP constitutive expression plasmid pPGP_EGFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) Fragments were prepared by PCR using Primer 72 (SEQ ID NO: 165), Primer 73 for mutagenesis (SEQ ID NO: 166), Primer 74 for mutagenesis (SEQ ID NO: 167) and Primer 75 (SEQ ID NO: 168), respectively. bottom.
Nucleic acid fragments were prepared by treating pPGP_EGFP with BamHI and SpeI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pPGPdel_EGFP.
Next, in order to prepare the GAPDH promoter and the EGFP gene fragment in which the BspQI site was disrupted, a nucleic acid fragment was prepared by PCR using primer 76 (SEQ ID NO: 169) and primer 77 (SEQ ID NO: 170) using pPGPdel_EGFP as a template. .. In addition, in order to prepare a nucleic acid fragment of CYC1 terminator (SEQ ID NO: 171) derived from Saccharomyces cerevisiae, PCR using Saccharomyces cerevisiae BY4741 strain as a template and Primer 78 (SEQ ID NO: 172) and Primer 79 (SEQ ID NO: 173) was performed. Prepared. Nucleic acid fragments were prepared after treating pUC_del_Zeo with SacI and BamHI, mixed with the two nucleic acid fragments prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-EGFP-CYC1t. By replacing the EGFP portion sandwiched between SpeI and XhoI with the following OLS sequence designed from the genome sequence information of Komagataera fafi, this vector can be used as a vector for producing a gene overexpressing cell library using Komagataera fafi. Is designed for.
（８）KAR2及びPDI1遺伝子過剰発現ベクターの構築
　プロモーターで制御されたZeocin(商標)耐性遺伝子（配列番号139）の両端にEcoRI認識配列を付加した核酸断片を、プライマー70（配列番号163）及びプライマー71（配列番号164）を用いてPCRで調製し、これをpUC19のEcoRIサイト間に挿入して、pUC_Zeoを構築した。
　次に、プロモーターで制御されたARG4遺伝子（配列番号5）の前半部分及び後半部分の核酸断片をプライマー9（配列番号88）及び変異挿入用のプライマー80（配列番号174）、プライマー81（配列番号175）及びプライマー10（配列番号89）を用いたPCRで調製し、HindIII及びPstI処理したpUC_Zeoと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、Arg4遺伝子の中央にAscI-FseI-PmeI制限酵素部位（配列番号176）を導入した配列を有するpUC_Arg4_Zeoを構築した。
　次に、EGFPの発現カセット（GAPDHプロモーター-EGFP遺伝子-AOX1t）の片端にHindIII認識配列を付加した核酸断片を、pPGP_EGFP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）をテンプレートとして、プライマー82（配列番号177）及び83（配列番号178）を用いたPCRにより調製し、BamHI処理させたpUC_Arg4_Zeoと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Arg4_Pgap_EGFP_Taox1_Zeoを構築した。
　次に、ターミネーターが連結されたKAR2及びPDI1タンパク質をコードする遺伝子（それぞれ配列番号179、180）の両末端にSpeI認識配列及びHindIII認識配列を付加した核酸断片を、プライマー84（配列番号181）及びプライマー85（配列番号182）、プライマー86（配列番号183）及びプライマー87（配列番号184）を用いたPCRで調製し、SpeI及びHindIII処理させたpUC_Arg4_Pgap_EGFP_Taox1_Zeoと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、それぞれpUC_Arg4_Pgap_KAR2_T37552_Zeo及びpUC_Arg4_Pgap_PDI1_T37552_Zeoを構築した。このベクターは、モデルタンパク質としてKAR2またはPDI1がGAPDHプロモーター制御下で過剰発現するように設計されている。 (8) Construction of KAR2 and PDI1 gene overexpression vector Primer 70 (SEQ ID NO: 163) and primer were added to the nucleic acid fragment in which the EcoRI recognition sequence was added to both ends of the Zeocin ™ resistance gene (SEQ ID NO: 139) controlled by the promoter. It was prepared by PCR using 71 (SEQ ID NO: 164) and inserted between the EcoRI sites of pUC19 to construct pUC_Zeo.
Next, the nucleic acid fragments of the first half and the second half of the ARG4 gene (SEQ ID NO: 5) regulated by the promoter are subjected to Primer 9 (SEQ ID NO: 88), Primer 80 for mutation insertion (SEQ ID NO: 174), and Primer 81 (SEQ ID NO: 81). Prepared by PCR using 175) and primer 10 (SEQ ID NO: 89), mixed with HindIII and PstI-treated pUC_Zeo, and spliced using the In-fusion HD Cloning Kit, AscI-FseI- in the center of the Arg4 gene. A pUC_Arg4_Zeo having a sequence into which a PmeI restriction enzyme site (SEQ ID NO: 176) was introduced was constructed.
Next, a nucleic acid fragment having a HindIII recognition sequence added to one end of the EGFP expression cassette (GAPDH promoter-EGFP gene-AOX1t) was added to pPGP_EGFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018). As a template, prepared by PCR using primers 82 (SEQ ID NO: 177) and 83 (SEQ ID NO: 178), mixed with BamHI-treated pUC_Arg4_Zeo, and spliced using the In-fusion HD Cloning Kit to obtain pUC_Arg4_Pgap_EGFP_Taox1_Zeo. It was constructed.
Next, a nucleic acid fragment in which the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene encoding the KAR2 and PDI1 proteins to which the terminator was ligated (SEQ ID NOs: 179 and 180, respectively) was added to Primer 84 (SEQ ID NO: 181) and Prepared by PCR using Primer 85 (SEQ ID NO: 182), Primer 86 (SEQ ID NO: 183) and Primer 87 (SEQ ID NO: 184), mixed with SpeI and HindIII treated pUC_Arg4_Pgap_EGFP_Taox1_Zeo, and used with In-fusion HD Cloning Kit. They were joined together to construct pUC_Arg4_Pgap_KAR2_T37552_Zeo and pUC_Arg4_Pgap_PDI1_T37552_Zeo, respectively. This vector is designed to overexpress KAR2 or PDI1 as a model protein under the control of the GAPDH promoter.
（９）抗体産生促進タンパク質過剰発現ベクター１の構築
　ターミネーターが連結された抗体産生促進タンパク質群（配列番号10～44で示されるアミノ酸配列）をコードする遺伝子（配列番号45～79）の両端にSpeI認識配列及びHindIII認識配列を付加した核酸断片を、プライマー19（配列番号98）及びプライマー20～55（配列番号99～134）を用いたPCRでそれぞれ調製し、SpeI及びHindIII処理させたpUC_Arg4_Pgap_EGFP_Taox1_Zeoと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Arg4_Pgap_EF1st-1_T37552_ZeoからpUC_Arg4_Pgap_EF3rd-9_T37552_Zeoの36種類を構築した。
　このベクターは、抗体産生促進タンパク質（配列番号10～44で示されるアミノ酸配列）がそれぞれGAPDHプロモーター制御下で発現するように設計されている。 (9) Construction of antibody production promoting protein overexpression vector 1 SpeI at both ends of a gene (SEQ ID NO: 45 to 79) encoding an antibody production promoting protein group (amino acid sequence shown by SEQ ID NOs: 10 to 44) to which a terminator is linked. Nucleic acid fragments to which the recognition sequence and HindIII recognition sequence were added were prepared by PCR using primers 19 (SEQ ID NO: 98) and primers 20 to 55 (SEQ ID NOs: 99 to 134), respectively, and mixed with pUC_Arg4_Pgap_EGFP_Taox1_Zeo treated with SpeI and HindIII. Then, 36 types of pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo were constructed from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo by connecting them using the In-fusion HD Cloning Kit.
This vector is designed so that antibody production promoting proteins (amino acid sequences shown in SEQ ID NOs: 10 to 44) are each expressed under the control of the GAPDH promoter.
（１０）抗体産生促進タンパク質過剰発現ベクター２の構築
　プロモーターで制御されたハイグロマイシン耐性遺伝子（配列番号141）の両端にEcoRI認識配列を付加した核酸断片を、プライマー88（配列番号185）及びプライマー89（配列番号186）を用いてPCRで調製し、これをpUC_Arg4_Pgap_EGFP_Taox1_ZeoのEcoRIサイト間に挿入して、pUC_Arg4_Pgap_EGFP_Taox1_Hygを構築した。
　次に、開始コドンを削除したURA3遺伝子（配列番号6）の前半部分及び後半部分の核酸断片をそれぞれ、プライマー90（配列番号187）及び変異導入用のプライマー91（配列番号188）、変異導入用のプライマー92（配列番号189）及びプライマー93（配列番号190）を用いたPCRで調製し、NheI及びPstI処理させたpUC_Arg4_Pgap_EGFP_Taox1_Hygと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、URA3遺伝子の中央にAscI-PmeI制限酵素部位（配列番号191）を導入した配列を有するpUC_URA3_Pgap_EGFP_Taox1_Hygを構築した。
　次にターミネーターが連結された抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）をコードする遺伝子（配列番号45～52）の両端にSpeI認識配列及びHindIII認識配列を付加した核酸断片を、フォワード用のプライマー19（配列番号98）及びそれぞれのリバース用のプライマー20～27（配列番号99～106）を用いたPCRで調製し、SpeI及びHindIII処理させたpUC_URA3_Pgap_EGFP_Taox1_Hygと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_URA3_Pgap_EF1st-1_HygからpUC_URA3_Pgap_EF2nd-4_Hygの8種類を構築した。
　このベクターは、抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）がGAPDHプロモーター制御下で発現するように設計されている。 (10) Construction of antibody production promoting protein overexpression vector 2 Nucleic acid fragments in which EcoRI recognition sequences are added to both ends of a hyglomycin resistance gene (SEQ ID NO: 141) controlled by a promoter are added to Primer 88 (SEQ ID NO: 185) and Primer 89. It was prepared by PCR using (SEQ ID NO: 186) and inserted between the EcoRI sites of pUC_Arg4_Pgap_EGFP_Taox1_Zeo to construct pUC_Arg4_Pgap_EGFP_Taox1_Hyg.
Next, the nucleic acid fragments of the first half and the second half of the URA3 gene (SEQ ID NO: 6) from which the starting codon was deleted were subjected to primer 90 (SEQ ID NO: 187), primer 91 for mutagenesis (SEQ ID NO: 188), and mutagenesis, respectively. Prepared by PCR using Primer 92 (SEQ ID NO: 189) and Primer 93 (SEQ ID NO: 190) of No. A pUC_URA3_Pgap_EGFP_Taox1_Hyg having a sequence into which an AscI-PmeI restriction enzyme site (SEQ ID NO: 191) was introduced was constructed in the center of the gene.
Next, the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked. Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_URA3_Pgap_EGFP_Taox1_Hyg and mixed. Eight types of pUC_URA3_Pgap_EF2nd-4_Hyg were constructed from pUC_URA3_Pgap_EF1st-1_Hyg by connecting them using the In-fusion HD Cloning Kit.
This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
（１１）抗体産生促進タンパク質過剰発現ベクター３の構築
　プロモーターで制御されたノーセオスリシン（Nourseothricin）耐性遺伝子（配列番号142）の両端にEcoRI認識配列を付加した核酸断片を、プライマー88（配列番号185）及びプライマー94（配列番号192）を用いてPCRで調製し、これをpUC_Arg4_Pgap_EGFP_Taox1_ZeoのEcoRIサイト間に挿入して、pUC_Arg4_Pgap_EGFP_Taox1_NATを構築した。
　次に、開始コドンを削除したGUT1遺伝子（配列番号7）の前半部分及び後半部分の核酸断片をそれぞれ、プライマー95（配列番号193）及び変異導入用のプライマー96（配列番号194）、変異導入用のプライマー97（配列番号195）及びプライマー98（配列番号196）を用いたPCRで調製し、NheI及びPstI処理させたpUC_Arg4_Pgap_EGFP_Taox1_NATと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、Gut1遺伝子とその下流配列（0.7Kb）の中央にAscI-PmeI制限酵素部位（配列番号191）を導入した配列を有するpUC_Gut1_Pgap_EGFP_Taox1_NATを構築した。
　次にターミネーターが連結された抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）をコードする遺伝子（配列番号45～52）の両端にSpeI認識配列及びHindIII認識配列を付加した核酸断片を、フォワード用のプライマー19（配列番号98）及びそれぞれのリバース用のプライマー20～27（配列番号99～106）を用いたPCRで調製し、SpeI及びHindIII処理させたpUC_Gut1_Pgap_EGFP_Taox1_NATと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_GUT1_Pgap_EF1st-1_NATからpUC_GUT1_Pgap_EF2nd-4_NATの8種類を構築した。
　このベクターは、抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）がGAPDHプロモーター制御下で発現するように設計されている。 (11) Construction of antibody production promoting protein overexpression vector 3 Nucleic acid fragments having EcoRI recognition sequences added to both ends of a promoter-controlled Nourseothricin resistance gene (SEQ ID NO: 142) were added to Primer 88 (SEQ ID NO: 185) and It was prepared by PCR using Primer 94 (SEQ ID NO: 192) and inserted between the EcoRI sites of pUC_Arg4_Pgap_EGFP_Taox1_Zeo to construct pUC_Arg4_Pgap_EGFP_Taox1_NAT.
Next, the nucleic acid fragments of the first half and the second half of the GUT1 gene (SEQ ID NO: 7) from which the starting codon was deleted were used as primer 95 (SEQ ID NO: 193), primer 96 for mutagenesis (SEQ ID NO: 194), and mutagenesis, respectively. Prepared by PCR using Primer 97 (SEQ ID NO: 195) and Primer 98 (SEQ ID NO: 196) of No. We constructed pUC_Gut1_Pgap_EGFP_Taox1_NAT having a sequence in which the AscI-PmeI restriction enzyme site (SEQ ID NO: 191) was introduced in the center of the downstream sequence (0.7Kb).
Next, the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked. Nucleic acid fragments were prepared by PCR using forward primer 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NO: 99-106), mixed with SpeI and HindIII treated pUC_Gut1_Pgap_EGFP_Taox1_NAT and mixed. Eight types of pUC_GUT1_Pgap_EF2nd-4_NAT were constructed from pUC_GUT1_Pgap_EF1st-1_NAT by connecting them using the In-fusion HD Cloning Kit.
This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
（１２）抗体産生促進タンパク質過剰発現ベクター４の構築
　プロモーターで制御されたブラストサイジン耐性遺伝子（配列番号143）の両端にEcoRI認識配列を付加した核酸断片を、プライマー88（配列番号185）及びプライマー99（配列番号197）を用いてPCRで調製し、これをpUC_Arg4_Pgap_EGFP_Taox1_ZeoのEcoRIサイト間に挿入して、pUC_Arg4_Pgap_EGFP_Taox1_bsdを構築した。
　次に、AOX1プロモーター（配列番号2）の核酸断片をプライマー100（配列番号198）及びプライマー101（配列番号199）を用いたPCRで調製し、NheI及びPstI処理させたpUC_Arg4_Pgap_EGFP_Taox1_bsdと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、ゲノム導入部位としてのAOX1プロモーター配列を有するpUC_Paox1_Pgap_EGFP_Taox1_bsdを構築した。
　次にターミネーターが連結された抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）をコードする遺伝子（配列番号45～52）の両端にSpeI認識配列及びHindIII認識配列を付加した核酸断片を、フォワード用のプライマー19（配列番号98）及びそれぞれのリバース用のプライマー20～27（配列番号99～106）を用いたPCRで調製し、SpeI及びHindIII処理させたpUC_Paox1_Pgap_EGFP_Taox1_bsdと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_Paox1_Pgap_EF1st-1_bsdからpUC_Paox1_Pgap_EF2nd-4_bsdの8種類を構築した。
　このベクターは、抗体産生促進タンパク質（配列番号10～17で示される8種類のアミノ酸配列）がGAPDHプロモーター制御下で発現するように設計されている。 (12) Construction of antibody production-promoting protein overexpression vector 4 Nucleic acid fragments in which EcoRI recognition sequences are added to both ends of a blastsaidin resistance gene (SEQ ID NO: 143) controlled by a promoter are added to Primer 88 (SEQ ID NO: 185) and Primer. It was prepared by PCR using 99 (SEQ ID NO: 197) and inserted between the EcoRI sites of pUC_Arg4_Pgap_EGFP_Taox1_Zeo to construct pUC_Arg4_Pgap_EGFP_Taox1_bsd.
Next, a nucleic acid fragment of the AOX1 promoter (SEQ ID NO: 2) was prepared by PCR using Primer 100 (SEQ ID NO: 198) and Primer 101 (SEQ ID NO: 199), mixed with NheI and PstI-treated pUC_Arg4_Pgap_EGFP_Taox1_bsd, and In- By connecting using the fusion HD Cloning Kit, pUC_Paox1_Pgap_EGFP_Taox1_bsd having the AOX1 promoter sequence as the genome transfer site was constructed.
Next, the SpeI recognition sequence and the HindIII recognition sequence were added to both ends of the gene (SEQ ID NO: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) to which the terminator was linked. Nucleic acid fragments were prepared by PCR using forward primers 19 (SEQ ID NO: 98) and their respective reverse primers 20-27 (SEQ ID NOs: 99-106) and mixed with SpeI and HindIII treated pUC_Paox1_Pgap_EGFP_Taox1_bsd. Eight types of pUC_Paox1_Pgap_EF2nd-4_bsd were constructed from pUC_Paox1_Pgap_EF1st-1_bsd by connecting them using the In-fusion HD Cloning Kit.
This vector is designed so that antibody production promoting proteins (8 kinds of amino acid sequences shown in SEQ ID NOs: 10 to 17) are expressed under the control of the GAPDH promoter.
（１３）形質転換酵母の取得
　上記（３）で構築した抗リゾチウム一本鎖抗体発現ベクターpUC_Paox1_MFα_scFv_Taox1_T38473_G418、上記（４）で構築したタンデムscFv226抗体発現ベクターpUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418、上記（５）で構築したブリナツモマブ抗体発現ベクターpUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418及び上記（６）で構築したミニボディー抗体ベクターpUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418を用いて、以下のようにコマガタエラ・ファフィを形質転換した。
　コマガタエラ・ファフィDnl4欠損・ヒスチジン要求性株（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）をYPD培地（1% 乾燥酵母エキス（ナカライテスク社製）、2% Bacto Pepton（Becton Dickinson社製）、2% glucose）2mlにて、30℃で16時間振盪培養後、フレッシュなYPD培地に10倍希釈で植え継ぎ、さらに30℃にて4時間振盪培養した。培養後の酵母細胞を遠心にて回収し、酵母細胞の洗浄（滅菌水6mlを加えて懸濁、遠心での酵母細胞の回収）後、試験管壁に残った滅菌水で酵母細胞を再懸濁し、これをコンピテントセル溶液とした。
　上記（３）で構築した抗リゾチウム一本鎖抗体発現ベクターpUC_Paox1_MFα_scFv_Taox1_T38473_G418、上記（４）で構築したタンデムscFv226抗体発現ベクターpUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418、上記（５）で構築したブリナツモマブ抗体発現ベクターpUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418及び上記（６）で構築したミニボディー抗体ベクターpUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418を用いて大腸菌を形質転換し、得られた形質転換体を5mlのアンピシリン含有LB培地（1% トリプトン（ナカライテスク社製）、0.5% 乾燥酵母エキス（ナカライテスク社製）、1% 塩化ナトリウム、0.01%アンピシリンナトリウム（ナカライテスク社製））で培養し、得られた菌体からPlasmid Plus Midi kit（キアゲン社製）を用いて、プラスミドを取得した。本プラスミドをCCA38473ターミネーター内のEcoRV認識配列を利用して、EcoRV処理により直鎖状にした。
　前記コンピテントセル溶液42μlと直鎖状のpUC_Paox1_MFα_scFv_Taox1_T38473_G418、pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418、pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418及びpUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418を各20μg、10 mg/mlキャリアDNA（タカラバイオ社製）溶液8μl、1M DTT溶液8μl、4M酢酸リチウム溶液4μl、60％ポリエチレングリコール溶液100μlを混合し、42℃で20分間静置した。20分間静置後、酵母細胞を回収し、500μlのYPD培地（1% 乾燥酵母エキス（ナカライテスク社製）、2% Bacto Pepton（Becton Dickinson社製）、2% glucose）に懸濁後、30℃で2時間静置した。2時間静置後、酵母細胞をYPDG418選択寒天プレート（1% 乾燥酵母エキス（ナカライテスク社製）、2% Bacto Pepton（Becton Dickinson社製）、2% glucose、2% アガロース、0.05% G418二硫酸塩（ナカライテスク社製））に塗布し、30℃、3日間の静置培養で生育する株を選択し、抗リゾチウム一本鎖抗体発現酵母、タンデムscFv226抗体発現酵母、ブリナツモマブ抗体発現酵母、およびミニボディー抗体発現酵母を取得した。
　遺伝子活性化の確認は形質転換酵母の染色体DNAをテンプレートにしたPCRによる増幅核酸断片の断片長や内部配列の配列解析、遺伝子発現解析等にて行った。その結果、実施例７で取得した形質転換酵母において、所望の遺伝子が活性化されていることを確認した。 (13) Acquisition of Transformed Yeast The anti-lysothium single-chain antibody expression vector pUC_Paox1_MFα_scFv_Taox1_T38473_G418 constructed in (3) above, and the tandem scFv226 antibody expression vector pUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T438 constructed in (4) above. Blinatumomab antibody expression vector pUC_Paox1_MFα (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and the minibody antibody vector pUC_Paox1_MFα (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above.
Komagataera fafi Dnl4 deficient / histidine auxotrophic strain (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) in YPD medium (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (Manufactured by Becton Dickinson), 2% glucose) 2 ml, shake-cultured at 30 ° C. for 16 hours, subcultured in fresh YPD medium at 10-fold dilution, and further shake-cultured at 30 ° C. for 4 hours. After culturing yeast cells are collected by centrifugation, the yeast cells are washed (suspended by adding 6 ml of sterilized water, and the yeast cells are collected by centrifugation), and then the yeast cells are re-suspended with the sterilized water remaining on the test tube wall. It became turbid and was used as a competent cell solution.
Anti-lysothium single-stranded antibody expression vector constructed in (3) above pUC_Paox1_MFα_scFv_Taox1_T38473_G418, tandem scFv226 antibody expression vector constructed in (4) above pUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T38473_loxP_G418 Escherichia coli was transformed using _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and the minibody antibody vector pUC_Paox1_MFα (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above. , 0.5% dried yeast extract (manufactured by Nakaraitesk), 1% sodium chloride, 0.01% sodium ampicillin (manufactured by Nakaraitesk)), and from the obtained cells, use the Plasmamid Plus Midi kit (manufactured by Kiagen). And obtained a plasmid. This plasmid was linearized by EcoRV treatment using the EcoRV recognition sequence in the CCA38473 terminator.
The competent cell solution 42μl and linear pUC_Paox1_MFα_scFv_Taox1_T38473_G418, pUC_Paox1_MFα (mut) _taFv226_Taox1_loxP_T38473_loxP_G418, pUC_Paox1_MFα (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and pUC_Paox1_MFα (mut) the _Minibody_Taox1_loxP_T38473_loxP_G418 each 20μg, 10 mg / ml carrier DNA (Takara Bio) was 8 [mu] l, 1M 8 μl of DTT solution, 4 μl of 4M lithium acetate solution, and 100 μl of 60% polyethylene glycol solution were mixed and allowed to stand at 42 ° C. for 20 minutes. After standing for 20 minutes, yeast cells are collected, suspended in 500 μl of YPD medium (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose), and then 30 The mixture was allowed to stand at ° C for 2 hours. After standing for 2 hours, yeast cells were subjected to YPDG418 selection agar plate (1% dried yeast extract (manufactured by Nakaraitesk), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate. A strain that is applied to salt (manufactured by Nakaraitesk Co., Ltd.) and grows in a static culture at 30 ° C. for 3 days is selected, and anti-lysodium single-stranded antibody-expressing yeast, tandem scFv226 antibody-expressing yeast, brinatsumomab antibody-expressing yeast, and Minibody antibody-expressing yeast was obtained.
Gene activation was confirmed by PCR using the chromosomal DNA of transformed yeast as a template for fragment length and internal sequence sequence analysis of amplified nucleic acid fragments, gene expression analysis, and the like. As a result, it was confirmed that the desired gene was activated in the transformed yeast obtained in Example 7.
（１４）形質転換酵母の培養
　上記（１３）にて得られた各低分子抗体発現酵母及び実施例４、５、７で作製された低分子抗体発現酵母を2mlのBMGY培地（1% yeast extract bacto（Becton Dickinson社製）、2% polypeptone（日本製薬社製）、0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate（Becton Dickinson社製）、1% Ammonium Sulfate、0.4mg/l Biotin、100mM リン酸カリウム（pH6.0）、2% Glycerol）に接種し、これを30℃、170rpm、24時間振盪培養後、前培養液を得た。2mlのBMMY培地（1% yeast extract bacto（Becton Dickinson社製）、2% polypeptone（日本製薬社製）、0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate（Becton Dickinson社製）、1% Ammonium Sulfate、0.4mg/l Biotin、100mM リン酸カリウム（pH6.0）、2% Methanol）に前培養液を200μl植継ぎ、これを30℃、170rpm、48時間振盪培養後、遠心分離（12,000rpm、5分、4℃）により培養上清を回収した。 (14) Culturing of transformed yeast 2 ml of BMGY medium (1% yeast extract) was prepared by adding each of the low-molecular-weight antibody-expressing yeasts obtained in (13) above and the low-molecular-weight antibody-expressing yeasts prepared in Examples 4, 5 and 7 to 2 ml of BMGY medium (1% yeast extract). bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 0.4 mg / l Biotin, 100 mM phosphate Potassium (pH 6.0), 2% Glycerol) was inoculated, and the mixture was cultured with shaking at 30 ° C., 170 rpm for 24 hours to obtain a preculture solution. 2 ml of BMMY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 200 μl of preculture solution was subcultured in 0.4 mg / l Biotin, 100 mM potassium phosphate (pH 6.0), 2% Methanol), and this was cultured with shaking at 30 ° C., 170 rpm, 48 hours, and then centrifuged (12,000 rpm, 5 minutes). , 4 ° C) to collect the culture supernatant.
（１５）ELISA法による各低分子抗体の分泌量の測定
　上記（１４）で得られた培養上清への抗リゾチウム一本鎖抗体、タンデムscFv226抗体、ブリナツモマブ抗体またはミニボディー抗体の分泌発現量の測定は、サンドイッチELISA（Enzyme-Linked Immunosorbent Assay）法により以下に示す方法で行った。
　ELISA用プレート（Coaster Assay Plate, 96well Clear, EasywashTM（コーニング社製））に固定化バッファー（8g/L 塩化ナトリウム、0.2g/L 塩化カリウム、1.15g/L リン酸一水素ナトリウム（無水）、0.2g/L リン酸二水素カリウム（無水）、1mM 塩化マグネシウム）にて1μM/mLとなるように溶解したリゾチウム（和光純薬社製、抗リゾチウム抗体に対して）または2μg/mLとなるように溶解したプロテインL（レイ・バイオテック社、タンデムscFv226抗体、ブリナツモマブ抗体、ミニボディー抗体に対して）を各ウェル50μlずつ添加し、終夜4℃でインキュベートした。インキュベート後、ウェル中の溶液を除去し、イムノブロック（大日本住友製薬社製）200μlでブロッキングし、室温で1時間静置した。PBSTバッファー（8g/L 塩化ナトリウム、0.2g/L 塩化カリウム、1.15g/L リン酸一水素ナトリウム（無水）、0.2g/L リン酸二水素カリウム（無水）、0.1% Tween20）で3回洗浄後、系列希釈した各標準抗体と希釈した培養上清を各ウェル50μlずつ添加し、室温で1時間反応した。PBSTバッファーで3回洗浄後、PBSTバッファーにて120,000倍希釈した二次抗体溶液（二次抗体：Anti-6X His tag antibody (HRP)（アブカム社製））を各ウェル50μlずつ添加し、室温で1時間反応させた。PBSTバッファーで3回洗浄後、50μlのTMB-1 Component Microwell Peroxidase Substrate SureBlue（KPL社製）を添加し、室温で3分静置した。50μlの1M 塩酸溶液（ナカライテスク社製）を添加して反応を停止させた後、マイクロプレートリーダー（Envison；パーキンエルマー社製）で450nmの吸光度を測定した。培養上清中の各低分子抗体の定量は、各標準抗体の検量線を用いて行った。 (15) Measurement of the amount of each small molecule antibody secreted by the ELISA method. The measurement was performed by the method shown below by the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method.
Immobilized buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.15 g / L sodium monohydrogen phosphate (anhydrous), on an ELISA plate (Coaster Assay Plate, 96well Clear, Easywash TM (manufactured by Corning)), Lyzotium dissolved in 0.2 g / L potassium dihydrogen dihydrogen (anhydrous), 1 mM magnesium chloride) to 1 μM / mL (for anti-lysodium antibody manufactured by Wako Pure Chemical Industries, Ltd.) or 2 μg / mL. Protein L dissolved in (for Ray Biotech, tandem scFv226 antibody, brinattumomab antibody, minibody antibody) was added in 50 μl of each well and incubated overnight at 4 ° C. After incubation, the solution in the well was removed, blocked with 200 μl of immunoblock (manufactured by Sumitomo Dainippon Pharma), and allowed to stand at room temperature for 1 hour. Washed 3 times with PBST buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.15 g / L sodium monohydrogen phosphate (anhydrous), 0.2 g / L potassium dihydrogen phosphate (anhydrous), 0.1% Tween20) Then, 50 μl of each well was added with each serially diluted standard antibody and diluted culture supernatant, and the mixture was reacted at room temperature for 1 hour. After washing 3 times with PBST buffer, add 50 μl of each well of secondary antibody solution (secondary antibody: Anti-6X His tag antibody (HRP) (manufactured by Abcam)) diluted 120,000 times with PBST buffer at room temperature. It was allowed to react for 1 hour. After washing 3 times with PBST buffer, 50 μl of TMB-1 Component Microwell Peroxidase Substrate SureBlue (manufactured by KPL) was added, and the mixture was allowed to stand at room temperature for 3 minutes. After stopping the reaction by adding 50 μl of 1M hydrochloric acid solution (manufactured by Nacalai Tesque), the absorbance at 450 nm was measured with a microplate reader (Envison; manufactured by PerkinElmer). The quantification of each small molecule antibody in the culture supernatant was performed using the calibration curve of each standard antibody.
（１６）SDS-PAGEによる各低分子抗体の分泌量の評価
　上記（１４）で得られた培養上清への抗リゾチウム一本鎖抗体、タンデムscFv226抗体、ブリナツモマブ抗体またはミニボディー抗体の分泌量の評価は、SDS-PAGEにより以下に示す方法で行った。
　各培養上清溶液を2xサンプルバッファーに混合し、100℃にて10分間処理後、ゲル濃度15％のSDS-PAGE用ゲル（e・パジェル、アトー社製）を用いてゲル電気泳動を行った。泳動後のゲルは、CBB Stain One（ナカライテスク社製）にて染色し、目的位置にバンドを確認した。 (16) Evaluation of the amount of each small molecule antibody secreted by SDS-PAGE The amount of anti-lysothium single-chain antibody, tandem scFv226 antibody, blinatumomab antibody or minibody antibody secreted into the culture supernatant obtained in (14) above. The evaluation was performed by the method shown below by SDS-PAGE.
Each culture supernatant solution was mixed with 2x sample buffer, treated at 100 ° C. for 10 minutes, and then gel electrophoresis was performed using an SDS-PAGE gel (e. Pagel, manufactured by Atto) with a gel concentration of 15%. .. The gel after electrophoresis was stained with CBB Stain One (manufactured by Nacalai Tesque), and a band was confirmed at the target position.
（１７）ペアスクリーニング用抗体産生促進タンパク質過剰発現ベクターの構築
　HindIII-NotI-BamHI-SpeI-XhoI-BglII-XbaI-EcoRIのマルチクローニングサイトを有する遺伝子断片（配列番号218）を全合成し、これをpUC19（タカラバイオ社製、Code No. 3219）のHindIII-EcoRIサイト間に挿入して、pUC-2を調製した。プロモーターで制御されたノーセオスリシン耐性遺伝子（配列番号142）及びハイグロマイシン耐性遺伝子（配列番号141）の両端にEcoRI認識配列を付加した核酸断片を、それぞれプライマー88（配列番号185）及びプライマー94（配列番号192）及びプライマー88（配列番号185）及びプライマー89（配列番号186）を用いたPCRにより調製し、EcoRI処理後にpUC-2のEcoRIサイトに挿入して、pUC2_NAT及びpUC2_Hygを構築した。
　次にMRP40ターミネーター（配列番号219）の前半部分及び後半部分の核酸断片をそれぞれ、プライマー116（配列番号220）及び変異導入用のプライマー117（配列番号221）、変異導入用のプライマー118（配列番号222）及びプライマー119（配列番号223）を用いたPCRで調製し、BglII及びXbaI処理させたpUC2_NATと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、MRP40ターミネーターの中央にAscI制限酵素部位（GGCGCGCC）を導入した配列を有するpUC2_NAT_TMRP40を構築した。次にARG83ターミネーター（配列番号224）の前半部分及び後半部分の核酸断片をそれぞれ、プライマー120（配列番号225）及び変異導入用のプライマー121（配列番号226）、変異導入用のプライマー122（配列番号227）及びプライマー123（配列番号228）を用いたPCRで調製し、BglII及びXbaI処理させたpUC2_Hygと混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、MRP40ターミネーターの中央にAscI制限酵素部位（GGCGCGCC）を導入した配列を有するpUC2_Hyg_TARG83を構築した。 (17) Construction of antibody production promoting protein overexpression vector for pair screening A gene fragment (SEQ ID NO: 218) having a multicloning site of HindIII-NotI-BamHI-SpeI-XhoI-BglII-XbaI-EcoRI was totally synthesized and used. pUC-2 was prepared by inserting it between the HindIII-EcoRI sites of pUC19 (manufactured by Takara Bio, Code No. 3219). Nucleic acid fragments with EcoRI recognition sequences added to both ends of the promoter-controlled noseoslisin resistance gene (SEQ ID NO: 142) and hyglomycin resistance gene (SEQ ID NO: 141) were added to Primer 88 (SEQ ID NO: 185) and Primer 94 (SEQ ID NO: 141), respectively. 192) and Primer 88 (SEQ ID NO: 185) and Primer 89 (SEQ ID NO: 186) were prepared by PCR and inserted into the EcoRI site of pUC-2 after EcoRI treatment to construct pUC2_NAT and pUC2_Hyg.
Next, the nucleic acid fragments of the first half and the second half of the MRP40 terminator (SEQ ID NO: 219) were subjected to primer 116 (SEQ ID NO: 220), primer 117 for mutagenesis (SEQ ID NO: 221), and primer 118 for mutagenesis (SEQ ID NO: 221), respectively. Prepared by PCR using 222) and primer 119 (SEQ ID NO: 223), mixed with BglII and XbaI-treated pUC2_NAT, ligated using the In-fusion HD Cloning Kit, and ascI restriction enzyme in the center of the MRP40 terminator. We constructed pUC2_NAT_TMRP40 with a sequence into which the site (GGCGCGCC) was introduced. Next, the nucleic acid fragments of the first half and the second half of the ARG83 terminator (SEQ ID NO: 224) were subjected to primer 120 (SEQ ID NO: 225), primer 121 for mutagenesis (SEQ ID NO: 226), and primer 122 for mutagenesis (SEQ ID NO: 226), respectively. Prepared by PCR using 227) and primer 123 (SEQ ID NO: 228), mixed with BglII and XbaI-treated pUC2_Hyg, ligated using the In-fusion HD Cloning Kit, and ascI restriction enzyme in the center of the MRP40 terminator. We constructed pUC2_Hyg_TARG83 with a sequence into which the site (GGCGCGCC) was introduced.
実施例１　コマガタエラ・ファフィ遺伝子過剰発現細胞ライブラリの作製のための予備検討
　従来のcDNAライブラリ、ゲノムライブラリに代わる遺伝子過剰発現細胞ライブラリとして、全ての酵母遺伝子の内在性プロモーターを高発現型（GAPDHプロモーター）に置き換えることを特徴とする、酵母遺伝子過剰発現細胞ライブラリの作製手法を開発した（図1）。以下に具体的なライブラリの作製方法を示す（(i)～(iv)は図１の(i)～(iv)に対応）。
(i)コマガタエラ・ファフィのすべての遺伝子（GeneX、5,001種類）配列情報を用いて、5,001種類の一本鎖DNA配列（229塩基長）（Oligonucleotide Library Synthesis(OLS)配列）を設計し、作製する。なお、各OLS配列内には2つのBspQI認識部位をBspQI切断部位によって挟まれるように並べて配置する（BspQIは認識部位と切断部位が異なるタイプIIS型の制限酵素である。）。また、各OLS配列は、塩基番号1～91内にBspQI認識部位が存在する場合、アミノ酸置換が生じないように塩基を置換し、当該BspQI認識部位は予め破壊されている。
(ii)次に、上記OLS配列をPCR法により二本鎖DNAにする。上記（７）で作製したpUC_del_Zeo_Pgap-EGFP-CYC1tをSpeI及びXhoIで処理し、上記二本鎖OLS配列と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせることによって、プラスミドライブラリを作製する。
(iii)上記ライブラリのプラスミドを制限酵素BspQIで直鎖状に変換し、シングルクロスオーバーインテグレーションにより上記直鎖状プラスミドをコマガタエラ・ファフィのゲノム上の目的位置（各GeneX）へ相同組換えによって挿入する。
(iv)その結果、相同組換えが生じた細胞のゲノム上では、各GeneXの内因性プロモーターが高発現型（Pgap）に置き換わり、コマガタエラ・ファフィの遺伝子過剰発現細胞ライブラリ（5,001種類）を取得できる。
　上記手法にて遺伝子過剰発現コマガタエラ・ファフィ株を実際に作製できるかどうか、コマガタエラ・ファフィ由来の遺伝子KAR2及びPDI1をモデルタンパク質として用いて検討した。コマガタエラ・ファフィ遺伝子KAR2またはPDI1の開始コドンから91番目までの塩基配列及び93番目から183番目までの塩基配列を取り出し、GAPDHプロモーターの3’末端配列、制限酵素SpeI認識部位、開始コドンから91番目までの塩基配列、制限酵素BspQI認識部位2つ（並んだ2つのBspQI認識部位は2つのBspQI切断部位に挟まれるように連結される）、93番目から183番目までの塩基配列、終止コドン（TGA）、制限酵素認識切断部位XhoI、サッカロマイセス・セレビシエ由来CYC1ターミネーターの5’末端配列の順に連結されたKAR2過剰発現用OLS配列およびPDI1過剰発現用OLS配列を調製した（図２、配列番号200（KAR2）、配列番号201（PDI1））。
　上記（７）の過剰発現細胞ライブラリ作製用ベクターpUC_del_Zeo_Pgap-EGFP-CYC1tをSpeI及びXhoI処理後に核酸断片を調製し、上記のように調製されたKAR2過剰発現用OLS配列およびPDI1過剰発現用OLS配列とそれぞれ混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pUC_del_Zeo_Pgap-KAR2OLS-CYC1t及びpUC_del_Zeo_Pgap-PDI1OLS-CYC1tを調製した。このプラスミドでは、コマガタエラ・ファフィ由来GAPDHプロモーターの下流にKAR2またはPDI1過剰発現用OLS配列が配置されており、その塩基配列が適切であることをサンガー法で確認した。
　これらのプラスミドを制限酵素BspQIで直鎖状に変換後、コマガタエラ・ファフィDNL4欠損・ヒスチジン要求性株（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）にそれぞれ導入し、相同組み換えによって目的部位（KAR2遺伝子またはPDI1遺伝子）へ挿入した。ゲノム上のKAR2遺伝子を挟むようなプライマー配列102（配列番号202）及びプライマー配列103（配列番号203）、及びPDI1遺伝子を挟むようなプライマー配列104（配列番号204）及びプライマー配列105（配列番号205）を設計し、コロニーPCR法により上記プラスミドのコマガタエラ・ファフィゲノムの目的部位への挿入を検証した。アガロースゲル電気泳動でのPCR産物のバンドの位置から、それぞれのプラスミドがゲノム上の目的部位へ挿入されていることが示された。
　また挿入株のKAR2またはPDI1遺伝子がそれぞれ過剰発現されているかどうかをKAR2及びPDI1の転写量をRT-qPCR法を用いて評価した。コマガタエラ・ファフィ野生株、KAR2過剰発現株及びPDI1過剰発現株のトータルRNAをRNeasy kit（キアゲン社製）を用いて取得後、逆転写キット（ReverTraAce qPCR RT Master Mix、TOYOBO社製）を用いて逆転写反応を行い、定量PCRキット（KOD SYBR（登録商標） qPCR Mix、TOYOBO社製）を用いてそれぞれの株のKAR2及びPDI1転写量を定量した。KAR2転写量の定量にはプライマー配列106（配列番号206）及びプライマー配列107（配列番号207）を、PDI1転写量の定量にはプライマー配列108（配列番号208）及びプライマー配列109（配列番号209）を使用した。qPCRのリファレンスとして、コマガタエラ・ファフィのACT1遺伝子を用い、プライマー配列110（配列番号210）及びプライマー配列111（配列番号211）を使用した。コントロールとしてのKAR2過剰発現株及びPDI1過剰発現株（KAR2及びPDI1遺伝子とそのターミネーター領域がGAPDHプロモーターの下流に導入されたプラスミドpUC_Arg4_Pgap_KAR2_T37552_Zeo及びpUC_Arg4_Pgap_PDI1_T37552_Zeoをそれぞれ有する）を作製し、比較対象とした。RT-qPCRを行った結果、上記手法により作製したKAR2及びPDI1過剰発現株は、それぞれのコントロール株と同様に、それぞれの遺伝子を過剰に発現していることが示された（図３）。以上の結果は、本手法によりコマガタエラ・ファフィ遺伝子過剰発現細胞ライブラリの作製が可能であることを示した。 Example 1 Preliminary study for preparation of Komagataera fafi gene overexpressing cell library As a gene overexpressing cell library that replaces the conventional cDNA library and genome library, the endogenous promoters of all yeast genes are highly expressed (GAPDH promoter). We have developed a method for producing a yeast gene overexpressing cell library, which is characterized by replacing it with (Fig. 1). A specific method for producing the library is shown below ((i) to (iv) correspond to (i) to (iv) in FIG. 1).
(i) Design and prepare 5,001 single-stranded DNA sequences (229 base lengths) (Oligonucleotide Library Synthesis (OLS) sequences) using all gene (GeneX, 5,001 types) sequence information of Komagataera fafi. .. Two BspQI recognition sites are arranged side by side in each OLS sequence so as to be sandwiched between the BspQI cleavage sites (BspQI is a type IIS type restriction enzyme having different recognition sites and cleavage sites). Further, in each OLS sequence, when a BspQI recognition site exists in the base numbers 1 to 91, the base is substituted so that amino acid substitution does not occur, and the BspQI recognition site is destroyed in advance.
(ii) Next, the above OLS sequence is converted into double-stranded DNA by the PCR method. A plasmid library is prepared by treating pUC_del_Zeo_Pgap-EGFP-CYC1t prepared in (7) above with SpeI and XhoI, mixing with the above double-stranded OLS sequence, and connecting them using the In-fusion HD Cloning Kit.
(iii) The plasmid of the above library is linearly converted with the restriction enzyme BspQI, and the linear plasmid is inserted into the target position (each GeneX) on the genome of Komagataera fafi by homologous recombination by single crossover integration. ..
(iv) As a result, on the genome of the cells in which homologous recombination has occurred, the endogenous promoter of each GeneX is replaced with the highly expressed type (Pgap), and the gene overexpressing cell library (5,001 types) of Komagataera fafi can be obtained. ..
Whether or not the gene overexpressing Komagataera fafi strain could be actually produced by the above method was examined using the genes KAR2 and PDI1 derived from Komagataera fafi as model proteins. The base sequence from the start codon to the 91st and the base sequence from the 93rd to the 183rd of the Komagataera fafi gene KAR2 or PDI1 are extracted, and the 3'end sequence of the GAPDH promoter, the restriction enzyme SpeI recognition site, and the start codon to the 91st. Nucleotide sequence, two restriction enzyme BspQI recognition sites (two side-by-side BspQI recognition sites are linked so as to be sandwiched between two BspQI cleavage sites), base sequence from 93rd to 183rd, termination codon (TGA) , The KAR2 overexpression OLS sequence and the PDI1 overexpression OLS sequence linked in the order of the restriction enzyme recognition cleavage site XhoI and the CYC1 terminator derived from Saccharomyces cerevisiae were prepared (Fig. 2, SEQ ID NO: 200 (KAR2)). , SEQ ID NO: 201 (PDI1)).
Nucleic acid fragments were prepared after treating the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t of (7) above with SpeI and XhoI, and the KAR2 overexpressing OLS sequence and PDI1 overexpressing OLS sequence prepared as described above were used. They were mixed and spliced together using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-KAR2OLS-CYC1t and pUC_del_Zeo_Pgap-PDI1OLS-CYC1t. In this plasmid, the OLS sequence for overexpression of KAR2 or PDI1 was placed downstream of the GAPDH promoter derived from Komagataera fafi, and it was confirmed by the Sanger method that the nucleotide sequence was appropriate.
After linearly converting these plasmids with the restriction enzyme BspQI, they were introduced into Komagataera fafi DNL4 deficient and histidine-requiring strains (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018). It was inserted into the target site (KAR2 gene or PDI1 gene) by homologous recombination. Primer sequence 102 (SEQ ID NO: 202) and primer sequence 103 (SEQ ID NO: 203) that sandwich the KAR2 gene on the genome, and primer sequence 104 (SEQ ID NO: 204) and primer sequence 105 (SEQ ID NO: 205) that sandwich the PDI1 gene. ) Was designed, and the insertion of the above-mentioned plasmid into the target site of the Komagataera fafi genome was verified by the colony PCR method. The position of the band of the PCR product on agarose gel electrophoresis showed that each plasmid was inserted into the target site on the genome.
In addition, whether or not the KAR2 or PDI1 gene of the inserted strain was overexpressed was evaluated by using the RT-qPCR method for the transcription amount of KAR2 and PDI1. After obtaining the total RNA of Komagataera fafi wild strain, KAR2 overexpressing strain and PDI1 overexpressing strain using RNeasy kit (manufactured by Kiagen), reverse transcription using the reverse transcription kit (ReverTraAce qPCR RT Master Mix, manufactured by TOYOBO). A photoreaction was performed, and the amount of KAR2 and PDI1 transcriptions of each strain was quantified using a quantitative PCR kit (KOD SYBR® qPCR Mix, manufactured by TOYOBO). Primer sequence 106 (SEQ ID NO: 206) and primer sequence 107 (SEQ ID NO: 207) are used to quantify the amount of KAR2 transcription, and primer sequence 108 (SEQ ID NO: 208) and primer sequence 109 (SEQ ID NO: 209) are used to quantify the amount of PDI1 transcription. It was used. As a reference for qPCR, the ACT1 gene of Komagataera fafi was used, and primer sequence 110 (SEQ ID NO: 210) and primer sequence 111 (SEQ ID NO: 211) were used. KAR2 overexpressing strains and PDI1 overexpressing strains as controls (having the plasmids pUC_Arg4_Pgap_KAR2_T37552_Zeo and pUC_Arg4_Pgap_PDI1_T37552_Zeo in which the KAR2 and PDI1 genes and their terminator regions were introduced downstream of the GAPDH promoter were prepared and compared, respectively) were prepared and compared. As a result of RT-qPCR, it was shown that the KAR2 and PDI1 overexpressing strains prepared by the above method overexpressed their respective genes in the same manner as the respective control strains (Fig. 3). The above results indicate that it is possible to prepare a cell library overexpressing the Komagataera fafi gene by this method.
実施例２　コマガタエラ・ファフィ遺伝子過剰発現細胞ライブラリの作製
　実施例１の結果を受けて、すべてのコマガタエラ・ファフィ遺伝子（5,001遺伝子）に対して上記OLS配列を設計し（図２）、作製した（アジレント・テクノロジー社）。得られた全OLS配列を二本鎖DNAに変換するため、OLS配列の両末端と相補的なプライマー112（配列番号212）及びプライマー113（配列番号213）を用いてPCR法にてDNAを増幅させた。二本鎖OLS配列を上記（７）の過剰発現細胞ライブラリ作製用ベクターpUC_del_Zeo_Pgap-EGFP-CYC1tにIn fusion法を用いて連結し、得られたプラスミドで大腸菌DH5α株を形質転換した。得られた形質転換体コロニー（4x105個）を複数のプレートから集め、Plasmid Plus Midi kit（キアゲン社）を用いてプラスミドを抽出し、プラスミドライブラリとした。またここで得られた12株の形質転換体コロニーをLB培地にて培養、プラスミドを抽出し、サンガー法にてプラスミドに含まれるOLS配列の塩基配列を確認した結果、すべてが異なる塩基配列であった。設計したOLS配列をもとに解析したところ、12株中の6株がOLS設計通りの配列であった。しかし残りの6株のうち、2株に1塩基置換（G→T）、1株に1塩基挿入、1株に2塩基置換（共にG→T）及び1塩基欠損、1株に1塩基置換（G→T）及び2塩基欠損が見られ、1株は解析不能であった。
　得られたプラスミドライブラリを制限酵素BspQIにて切断、DNA精製後、低分子抗体（抗リゾチウムscFv抗体、タンデムscFv226、ブリナツモマブ抗体）を分泌するコマガタエラ・ファフィ株に、エレクトロポレーション法を用いて導入した。 Example 2 Preparation of Komagataera fafi gene overexpressing cell library Based on the results of Example 1, the above OLS sequences were designed (Fig. 2) and prepared for all Komagataera fafi genes (5,001 genes) (Agilent).・ Agilent). In order to convert the obtained total OLS sequence into double-stranded DNA, DNA is amplified by PCR using primers 112 (SEQ ID NO: 212) and primer 113 (SEQ ID NO: 213) complementary to both ends of the OLS sequence. I let you. The double-stranded OLS sequence was ligated to the overexpressing cell library preparation vector pUC_del_Zeo_Pgap-EGFP-CYC1t described in (7) above using the In fusion method, and the Escherichia coli DH5α strain was transformed with the obtained plasmid. The obtained transformant colonies (4x10 5 pieces) were collected from a plurality of plates, and a plasmid was extracted using a Plasmamid Plus Midi kit (Qiagen) to prepare a plasmid library. In addition, the 12 transformant colonies obtained here were cultured in LB medium, the plasmid was extracted, and the nucleotide sequence of the OLS sequence contained in the plasmid was confirmed by the Sanger method. As a result, all of them had different nucleotide sequences. rice field. Analysis based on the designed OLS sequence revealed that 6 of the 12 strains had the sequence as designed by OLS. However, of the remaining 6 strains, 1 base was substituted in 2 strains (G → T), 1 base was inserted in 1 strain, 2 bases were substituted in 1 strain (both G → T) and 1 base was deleted, and 1 base was substituted in 1 strain. (G → T) and 2 base deficiencies were observed, and one strain could not be analyzed.
The obtained plasmid library was cleaved with the restriction enzyme BspQI, DNA was purified, and then introduced into a Komagataera fafi strain secreting small molecule antibodies (anti-lysothium scFv antibody, tandem scFv226, blinatumomab antibody) using an electroporation method. ..
実施例３　低分子抗体の分泌量を指標としたハイスループットスクリーニング
　実施例２においてエレクトロポレーション法により得られた形質転換体群（遺伝子過剰発現細胞ライブラリ）をコロニーピッカー（PM-2、マイクロテックニチオン社製）を用いて、Zeocinを添加されたYPD寒天培地の角形プレートに96ウェルフォーマットに整列させ、30℃で培養することでコロニー化させ、これをマスタープレートとした。
　マスタープレートから、BMGY培地0.5 mLが入ったディープウェルプレートに、96ピンを用いて96株を同時に植菌した。30℃にて24時間攪拌培養後、その培養液50μLを、BMMY培地0.5 mLが入った96ディープウェルプレートに加え、さらに30℃にて48時間攪拌培養を行った。培養後、ディープウェルプレートを遠心し、その上清をPBSにて希釈し、Hisタグまたはc-mycタグを指標としたELISA法にて低分子抗体量を評価した。
　上記の方法にて、抗リゾチウムscFv抗体、タンデムscFv226抗体及びブリナツモマブ抗体を生産する各株で遺伝子過剰発現細胞ライブラリを作製し、各株にてディープウェルプレート200枚分の遺伝子過剰発現株（約19,200株）から低分子抗体量が上昇した株（スクリーニングポジティブ株）をスクリーニングした。 Example 3 High-throughput screening using the amount of small molecule antibody secreted as an index The transformant group (gene overexpressing cell library) obtained by the electroporation method in Example 2 was selected as a colony picker (PM-2, microtechnition). A square plate of YPD agar medium supplemented with Zeocin was arranged in a 96-well format and colonized by culturing at 30 ° C., and this was used as a master plate.
From the master plate, 96 strains were simultaneously inoculated using 96 pins into a deep well plate containing 0.5 mL of BMGY medium. After stirring and culturing at 30 ° C. for 24 hours, 50 μL of the culture solution was added to a 96 deep well plate containing 0.5 mL of BMWY medium, and further stirring and culturing was carried out at 30 ° C. for 48 hours. After culturing, the deep well plate was centrifuged, the supernatant was diluted with PBS, and the amount of small molecule antibody was evaluated by the ELISA method using the His tag or c-myc tag as an index.
By the above method, a gene overexpressing cell library was prepared in each strain producing anti-lysothium scFv antibody, tandem scFv226 antibody and blinatumomab antibody, and each strain produced a gene overexpressing strain for 200 deep well plates (about 19,200). A strain (screening positive strain) having an increased amount of small antibody was screened from the strain).
実施例４　有用因子の同定
　得られたスクリーニングポジティブ株で過剰発現された遺伝子の同定を下記の方法にて行った（図４）（下記(a)～(f)は図４の(a)～(f)に対応）。
(a)スクリーニングポジティブ株からGenとるくん（タカラバイオ社製）を用いて、ゲノムDNAを抽出した。
(b)複数の制限酵素を用いた同時処理によってゲノムDNAを断片化した（下記(i)～(iii)のいずれかを使用した）。
(i)5’突出末端型制限酵素EcoRI、SalI、NheI、BamHIおよびClaIでゲノムDNAを処理後、Klenow fragmentを用いて断片化DNAの5’突出末端を平滑末端にした。
(ii)5’突出末端型制限酵素BamHI、BclI及びBglIIでゲノムDNAを処理した（5’突出末端は同じ塩基配列（GATC）となる）。
(iii)平滑末端型制限酵素BsaAI、BsaBI、BstZ17I、HpaI、PmlI、SnaBI及びStuIでゲノムDNAを処理した。
(c)断片化したDNAをセルフライゲーションによって環状化した。
(d)環状化したDNAで大腸菌DH5α株をアンピシリン及びゼオシン添加LB寒天培地上で形質転換した。
(e)形質転換体からプラスミドを抽出した。
(f)サンガー法によって抽出されたプラスミドからコマガタエラ・ファフィのゲノムに挿入されたプラスミドのOLS配列の塩基配列を決定した。
　上記方法にて、スクリーニングポジティブ株から過剰発現した36の遺伝子を決定した。しかし、これらの遺伝子の過剰発現が低分子抗体量の増加に効果があるとは限らない。そこで、上記方法にて決定された36の遺伝子の過剰発現ベクター（pUC_Arg4_Pgap_EF1st-1_T37552_ZeoからpUC_Arg4_Pgap_EF3rd-9_T37552_Zeoの36の発現ベクター）を、実施例２で使用した各低分子抗体生産株のArg4部位に上記（１３）で記した手法にて導入した株を作製し、低分子抗体分泌量が増加するかどうかを調べた。その結果、各過剰発現遺伝子を導入した抗リゾチウムscFv抗体生産株では、宿主株と比較して1.1倍～1.7倍に抗体の分泌量が増加した株が18株得られた（表１）。また各過剰発現遺伝子を導入したタンデムscFv226生産株では、1.1倍～1.6倍に抗体の分泌量が増加した株が９株得られた（表２）。さらに各過剰発現因子を導入したブリナツモマブ生産株では、1.2倍～2.3倍に抗体の分泌量が増加した株が9株得られた（表３）。これらの結果より、それぞれの上記36遺伝子の過剰発現は低分子抗体の産生促進に効果があり、36遺伝子は抗体産生促進のための有用因子であることが確認できた（表４）。今回のスクリーニングで得られた有用因子の中では、2つのみ（EF2nd-8(KAR2)及びEF3rd-4 (KIN2)）がコマガタエラ・ファフィ酵母で先行研究がなされている因子であった（Damasceno et al., Appl. Microbiol. Biotechnol. Vol.74, 2007 (KAR2)、Gasser et al. Appl. Environ. Microbiol. Vol. 73, No. 20 2007 (KIN2)）。また、EF2nd-4とEF3rd-2は同一遺伝子(YCK)であった。EF1st-1もまた本発明者らによって別途報告されている。以上より表４に記載された遺伝子のうち、EF1st-1、EF2nd-8、EF3rd-2およびEF3rd-4を除いた32種類の因子が、本研究にて新規に見つかったものである。
　次に、特定の抗体生産株で得られた有用因子が、他の抗体生産性の増加にも影響するかどうかを調べた。抗リゾチウムscFv抗体生産株から得られた1８個の有用因子発現ベクター（pUC_Arg4_Pgap_EF1st-1_T37552_ZeoからpUC_Arg4_Pgap_EF1st-18_T37552_Zeoの18種類）を、タンデムscFv226抗体及びブリナツモマブ抗体生産株のARG4部位にそれぞれ導入した株を作製した。それぞれの株の抗体分泌量をELISA法にて評価した結果、宿主株と比較して、タンデムscFv226抗体生産株では0.9倍～1.6倍（表５）、ブリナツモマブ抗体生産株では1.0倍～2.3倍に抗体の分泌量になった（表６）。
　同様に、タンデムscFv抗体生産株から得られた９個の有用因子発現ベクター（pUC_Arg4_Pgap_EF2nd-1_T37552_Zeo1～pUC_Arg4_Pgap_EF2nd-9_T37552_Zeoの9種類）を、抗リゾチウムscFv抗体及びブリナツモマブ抗体生産株のARG4部位にそれぞれ導入した株を作製した。それぞれの株の抗体分泌量をELISA法にて評価した結果、宿主株と比較して、抗リゾチウムscFv抗体生産株では1.0倍～1.2倍（表７）、ブリナツモマブ抗体生産株では1.0倍～1.8倍になった（表８）。
　また同様に、ブリナツモマブ抗体生産株から得られた９個の有用因子発現ベクター（pUC_Arg4_Pgap_EF3rd-1_T37552_ZeoからpUC_Arg4_Pgap_EF3rd-9_T37552_Zeo）を、抗リゾチウムscFv抗体及びタンデムscFv226抗体生産株のARG4部位にそれぞれ導入した株を作製した。それぞれの株の抗体分泌量をELISA法にて評価した結果、宿主株と比較して、抗リゾチウムscFv抗体生産株では1.0倍～1.7倍（表９）、タンデムscFv226抗体生産株では0.8倍～1.5倍になった（表１０）。
　タンデムscFv抗体生産株で得られた有用因子群は抗リゾチウムscFv抗体生産の増加に効果は見られなかった。一方、抗リゾチウムscFv抗体及びブリナツモマブ抗体生産株にて得られた有用因子の多くは、異なる低分子抗体の産生促進にも効果が確認できた。 Example 4 Identification of useful factors The genes overexpressed in the obtained screening positive strain were identified by the following methods (Fig. 4) ((a) to (f) below are (a) to (a) to 4 in FIG. 4). Corresponds to (f)).
(a) Genomic DNA was extracted from the screening positive strain using Gen Toru-kun (manufactured by Takara Bio Inc.).
(b) Genomic DNA was fragmented by simultaneous treatment with multiple restriction enzymes (using any of the following (i) to (iii)).
(i) After treating the genomic DNA with the 5'protruding end-type restriction enzymes EcoRI, SalI, NheI, BamHI and ClaI, the 5'protruding end of the fragmented DNA was blunt-ended with Klenow fragment.
(ii) Genomic DNA was treated with 5'protruding end-type restriction enzymes BamHI, BclI and BglII (the 5'protruding end has the same base sequence (GATC)).
(iii) Genomic DNA was treated with blunt-ended restriction enzymes BsaAI, BsaBI, BstZ17I, HpaI, PmlI, SnaBI and StuI.
(c) Fragmented DNA was cyclized by self-ligation.
(d) Escherichia coli DH5α strain was transformed with cyclized DNA on LB agar medium supplemented with ampicillin and zeocin.
(e) A plasmid was extracted from the transformant.
(f) From the plasmid extracted by the Sanger method, the nucleotide sequence of the OLS sequence of the plasmid inserted into the genome of Komagataera fafi was determined.
By the above method, 36 genes overexpressed from the screening positive strain were determined. However, overexpression of these genes is not always effective in increasing the amount of small molecule antibodies. Therefore, the overexpression vectors of 36 genes determined by the above method (36 expression vectors from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo) were applied to the Arg4 site of each small molecule antibody-producing strain used in Example 2 ( A strain introduced by the method described in 13) was prepared, and it was examined whether or not the amount of small molecule antibody secreted increased. As a result, among the anti-lysothium scFv antibody-producing strains into which each overexpressing gene was introduced, 18 strains were obtained in which the amount of antibody secreted increased 1.1- to 1.7-fold as compared with the host strain (Table 1). In addition, among the tandem scFv226-producing strains into which each overexpressing gene was introduced, 9 strains were obtained in which the amount of antibody secreted increased 1.1 to 1.6 times (Table 2). Furthermore, among the blinatumomab-producing strains into which each overexpressing factor was introduced, 9 strains were obtained in which the amount of antibody secreted increased 1.2 to 2.3 times (Table 3). From these results, it was confirmed that the overexpression of each of the above 36 genes was effective in promoting the production of small molecule antibodies, and that the 36 genes were useful factors for promoting antibody production (Table 4). Of the useful factors obtained in this screening, only two (EF2nd-8 (KAR2) and EF3rd-4 (KIN2)) were previously studied in Komagataera fafi yeast (Damasceno et. al., Appl. Microbiol. Biotechnol. Vol.74, 2007 (KAR2), Gasser et al. Appl. Environ. Microbiol. Vol. 73, No. 20 2007 (KIN2)). In addition, EF2nd-4 and EF3rd-2 were the same gene (YCK). EF1st-1 has also been reported separately by the inventors. From the above, among the genes listed in Table 4, 32 types of factors excluding EF1st-1, EF2nd-8, EF3rd-2 and EF3rd-4 were newly discovered in this study.
Next, it was investigated whether the useful factors obtained in a specific antibody-producing strain also affect the increase in the productivity of other antibodies. Eighteen useful factor expression vectors (18 types from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF1st-18_T37552_Zeo) obtained from anti-lysothium scFv antibody-producing strains were introduced into tandem scFv226 antibody and blinatumomab antibody-producing strains. .. As a result of evaluating the antibody secretion amount of each strain by the ELISA method, the tandem scFv226 antibody-producing strain was 0.9 to 1.6 times (Table 5), and the blinatumomab antibody-producing strain was 1.0 to 2.3 times as much as the host strain. It became the amount of antibody secreted (Table 6).
Similarly, 9 useful factor expression vectors (9 types of pUC_Arg4_Pgap_EF2nd-1_T37552_Zeo1 to pUC_Arg4_Pgap_EF2nd-9_T37552_Zeo) obtained from the tandem scFv antibody-producing strain were introduced into the anti-lysothium scFv antibody and blinatumomab antibody-producing strains, respectively. Was produced. As a result of evaluating the antibody secretion amount of each strain by the ELISA method, the anti-lysothium scFv antibody-producing strain was 1.0 to 1.2 times (Table 7), and the blinatumomab antibody-producing strain was 1.0 to 1.8 times as much as the host strain. (Table 8).
Similarly, nine useful factor expression vectors (pUC_Arg4_Pgap_EF3rd-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo) obtained from the blinatumomab antibody-producing strain were introduced into the anti-lysothium scFv antibody and tandem scFv226 antibody-producing strains, respectively. bottom. As a result of evaluating the antibody secretion amount of each strain by the ELISA method, the anti-lysothium scFv antibody-producing strain was 1.0 to 1.7 times (Table 9), and the tandem scFv226 antibody-producing strain was 0.8 to 1.5 times as compared with the host strain. It has doubled (Table 10).
The useful factor group obtained from the tandem scFv antibody-producing strain had no effect on the increase in anti-lysothium scFv antibody production. On the other hand, many of the useful factors obtained from the anti-lysothium scFv antibody and blinatumomab antibody-producing strains were confirmed to be effective in promoting the production of different small molecule antibodies.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
実施例５　有用因子の集積による低分子抗体高生産株の開発
　上記スクリーニングで得られた有用因子による低分子抗体の産生促進効果は、（タンデムscFv226生産では）1.1倍～1.6倍と比較的低いものであった。より低分子抗体の分泌量を増加させるために、有用因子の集積を検討した。今回、抗リゾチウムscFv抗体株から得られた上位４因子（EF1st-1～4、表１）、およびタンデムscFv226抗体株から得られた上位4因子（EF2nd-1～4、表２）の計8因子を選択した。これらを集積させることで、タンデムscFv226抗体の高生産株の作製を目指した。具体的には、選んだ8因子をそれぞれ一つずつ、タンデムscFv226生産親株に上記（１３）で記した手法にて導入し、タンデムscFv226抗体の分泌量を評価し、その中で最も分泌量が高かった株を次世代の親株とした。この有用因子の導入とタンデムscFv226抗体の分泌量評価を4回繰り返すことで、タンデムscFv226抗体の高生産株の取得を目指した。1世代目では、タンデムscFv226抗体を生産する1遺伝子欠損株（A株）を親株として、最もtaFv226抗体の分泌量を増加させた有用因子EF1st-1のみをARG4部位へ導入し、タンデムscFv226抗体の分泌量を評価した。その結果、A株よりもタンデムscFv226抗体の分泌量が約1.5倍増加したB株が得られた（表１１）。B株を2世代目の親株として、上記で選んだ8因子をそれぞれURA3部位へ導入し、それぞれの因子が過剰発現する株を上記（１３）で記した手法にて作製した。作製した株のタンデムscFv226抗体の分泌量を評価したところ、EF2nd-4導入株（C株）が最も分泌量が高く、親株であるB株と比較して約1.2倍抗体を分泌した（表１２）。C株を3世代目の親株として、上記で選んだ8因子をそれぞれGUT1部位へ導入し、それぞれの因子が過剰発現する株を上記（１３）で記した手法にて作製した。作製した株のタンデムscFv226抗体の分泌量を評価したところ、EF1st-4導入株（D株）が最も分泌量が高く、親株であるC株と比較して約1.2倍抗体を分泌した（表１３）。D株を4世代目の親株として、上記で選んだ8因子をそれぞれAOX1プロモーター部位へ導入し、それぞれの因子が過剰発現する株を上記（１３）で記した手法にて作製した。作製した株のタンデムscFv226抗体の分泌量を評価したところ、EF2nd-1導入株が最も分泌量が高く、親株であるD株と比較して約1.3倍抗体を分泌した（表１４）。この株をE株とした。このように4つの有用因子を導入したE株は、有用因子導入前のA株と比較して、約2.9倍高いタンデムscFv226抗体を分泌した。このように有用因子を順に集積することで、低分子抗体の生産性を増加させることに成功した（図５）。 Example 5 Development of a high-producing small molecule antibody strain by accumulating useful factors The effect of promoting the production of small molecule antibodies by the useful factors obtained in the above screening is relatively low, 1.1 to 1.6 times (in tandem scFv226 production). Met. In order to increase the amount of small molecule antibody secreted, the accumulation of useful factors was examined. This time, a total of 8 of the top 4 factors (EF1st-1 to 4, Table 1) obtained from the anti-lysozyme scFv antibody strain and the top 4 factors (EF2nd-1 to 4, Table 2) obtained from the tandem scFv226 antibody strain. Factors were selected. By accumulating these, we aimed to prepare a high-producing strain of tandem scFv226 antibody. Specifically, each of the eight selected factors was introduced into the tandem scFv226-producing parent strain by the method described in (13) above, and the amount of tandem scFv226 antibody secreted was evaluated. The high stock was used as the next-generation parent stock. By repeating the introduction of this useful factor and the evaluation of the amount of tandem scFv226 antibody secreted four times, we aimed to obtain a high-producing strain of tandem scFv226 antibody. In the first generation, one gene-deficient strain (A strain) that produces tandem scFv226 antibody was used as the parent strain, and only the useful factor EF1st-1, which increased the amount of taFv226 antibody secreted most, was introduced into the ARG4 site, and the tandem scFv226 antibody was introduced. The amount of secretion was evaluated. As a result, a strain B in which the amount of tandem scFv226 antibody secreted was increased by about 1.5 times as compared with the strain A was obtained (Table 11). With strain B as the second-generation parent strain, each of the eight factors selected above was introduced into the URA3 site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above. When the amount of tandem scFv226 antibody secreted from the prepared strain was evaluated, the EF2nd-4 introduced strain (C strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain B (Table 12). ). The C strain was used as the third-generation parent strain, and the eight factors selected above were introduced into the GUT1 site, respectively, and a strain in which each factor was overexpressed was prepared by the method described in (13) above. When the amount of tandem scFv226 antibody secreted from the prepared strain was evaluated, the EF1st-4 introduced strain (D strain) secreted the highest amount, and secreted about 1.2 times as much antibody as the parent strain C (Table 13). ). With the D strain as the 4th generation parent strain, each of the 8 factors selected above was introduced into the AOX1 promoter site, and a strain in which each factor was overexpressed was prepared by the method described in (13) above. When the amount of tandem scFv226 antibody secreted from the prepared strain was evaluated, the EF2nd-1-introduced strain had the highest amount of secretion, and secreted about 1.3 times as much antibody as the parent strain D (Table 14). This strain was designated as E strain. In this way, the E strain into which the four useful factors were introduced secreted tandem scFv226 antibody, which was about 2.9 times higher than that of the A strain before the introduction of the useful factors. By accumulating useful factors in order in this way, we succeeded in increasing the productivity of small molecule antibodies (Fig. 5).
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
実施例６　抗体高生産株からの抗体発現カセットの交換
　抗体生産株の抗体発現カセットを抜き差しができれば、実施例５で作製したタンデムscFv226高生産株において異なる抗体の生産が可能となる。そこでCre-loxPシステムを用いて、遺伝子発現カセットの交換を検討した。具体的には、抗体発現カセットのコマガタエラ・ファフィのゲノム導入用配列の両末端にloxP配列（34塩基長）を挿入すると、コマガタエラ・ファフィに導入された発現カセットは両末端にloxP配列を有することとなる（図６）。次にメタノール誘導性のAOX1プロモーターで制御されたCreリコンビナーゼ遺伝子を有するプラスミドpPAP_CP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）を抗体生産株にZeoマーカーにて導入する。この際にAOX1プロモーターのリークレベルでのCreリコンビナーゼ発現でloxP間の塩基配列が削除される。抗体発現カセットの削除は、ゲノム導入部位の両末端に設計したプライマーでのコロニーPCR法また薬剤アッセイにて薬剤マーカーの有無にて評価できる。この抗体カセットを削除した株の同じゲノム挿入部位に、異なる抗体発現ベクターを導入することにより、タンデムscFv226高生産株のタンデムscFv226抗体から異なる抗体への発現が可能となる（図６）。
　モデルケースとして、緑色蛍光タンパク質（GFP）及び赤色蛍光タンパク質遺伝子（RFP）を用いて、タンパク質発現カセットの交換を行った。GFPにはEGFP（配列番号214）、RFPにはE2crimson（配列番号215）をコードする遺伝子を用いた。GFP発現ベクターにはpPGP_GFP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）を用いた。このベクターは、GFPがGAPDHプロモーター制御下で発現するように設計されている。RFP遺伝子は合成DNAをテンプレートにしてプライマー114（配列番号216）及びプライマー115（配列番号217）を用いてPCRで調製した。この核酸断片を、SpeI及びXhoI処理後に核酸断片を調製し、上記PCRにより調製されたRFPをコードする塩基配列の核酸断片と混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、pPGP_RFPを構築した。このベクターは、RFPがGAPDHプロモーター制御下で発現するように設計されている。
　コマガタエラ・ファフィ野生株を用いたGFP発現株からRFP発現株へのレポーター遺伝子の交換過程を図７に示す。まず、コマガタエラ・ファフィ（図７（１））のCCA38473ターミネーター部位にGFP発現カセット（CCA38473ターミネーター配列内のEcoRV部位にて直鎖状に変換されたpPGP_GFP断片）を導入した株を、上記（１３）で記した手法にて作製した（図７（２））。この株はG418耐性であり、フローサイトメトリー（FCM）ではGFP蛍光が確認できた。次に、この株にプラスミドpPAP_CP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）を導入した（図７（３））。Zeocin耐性を有する株が得られたが、この時点でこの株はG418耐性を失っているのが確認できた。Creリコンビナーゼのリーク発現によりG418マーカーを含む発現カセットが脱落したためと考えられた。この株をYPD寒天培地でシングルコロニーアイソレーションを行うことでpPAP_CPを脱落させた（図７（４））。この株は、G418及びZeocin耐性を失っていた。またFCM解析でも、GFP蛍光強度はバックグラウンドレベルに下がっていた。最後にCCA38473ターミネーター部位にRFP発現カセット（CCA38473ターミネーター配列内のEcoRV部位にて直鎖状に変換されたpPGP_RFP断片）を導入した株を上記（１３）で記した手法にて作製した（図７（５））。この株は、G418 耐性を有し、FCM解析でも高いRFP蛍光強度が確認できた。以上の結果より、レポーター発現カセットの交換が上記手法により可能であることが示された。 Example 6 Exchange of antibody expression cassette from antibody-producing strain If the antibody expression cassette of the antibody-producing strain can be inserted and removed, different antibodies can be produced in the tandem scFv226 high-producing strain prepared in Example 5. Therefore, we examined the exchange of gene expression cassettes using the Cre-loxP system. Specifically, when a loxP sequence (34 base length) is inserted at both ends of the genome transfer sequence of Komagataera fafi of the antibody expression cassette, the expression cassette introduced into Komagataera fafi has loxP sequences at both ends. (Fig. 6). Next, a plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) having a Cre recombinase gene regulated by a methanol-induced AOX1 promoter is introduced into an antibody-producing strain using a Zeo marker. .. At this time, Cre recombinase expression at the leak level of the AOX1 promoter deletes the nucleotide sequence between loxPs. The deletion of the antibody expression cassette can be evaluated by the colony PCR method using primers designed at both ends of the genome introduction site or the presence or absence of a drug marker in a drug assay. By introducing a different antibody expression vector into the same genome insertion site of the strain from which this antibody cassette has been deleted, expression from the tandem scFv226 antibody of the tandem scFv226 high-producing strain to a different antibody becomes possible (Fig. 6).
As a model case, the protein expression cassette was exchanged using green fluorescent protein (GFP) and red fluorescent protein gene (RFP). A gene encoding EGFP (SEQ ID NO: 214) was used for GFP, and a gene encoding E2crimson (SEQ ID NO: 215) was used for RFP. As the GFP expression vector, pPGP_GFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was used. This vector is designed for GFP to be expressed under the control of the GAPDH promoter. The RFP gene was prepared by PCR using synthetic DNA as a template and primer 114 (SEQ ID NO: 216) and primer 115 (SEQ ID NO: 217). This nucleic acid fragment is prepared after SpeI and XhoI treatment, mixed with the nucleic acid fragment of the nucleotide sequence encoding RFP prepared by the above PCR, and spliced using the In-fusion HD Cloning Kit to obtain pPGP_RFP. It was constructed. This vector is designed so that RFP is expressed under the control of the GAPDH promoter.
FIG. 7 shows the process of exchanging the reporter gene from the GFP-expressing strain to the RFP-expressing strain using the Komagataera fafi wild strain. First, a strain into which a GFP expression cassette (pPGP_GFP fragment linearly converted at the EcoRV site in the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site of Komagataera fafi (Fig. 7 (1)) was introduced into the above (13). It was produced by the method described in (Fig. 7 (2)). This strain was G418 resistant, and GFP fluorescence was confirmed by flow cytometry (FCM). Next, the plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was introduced into this strain (Fig. 7 (3)). A strain with Zeocin resistance was obtained, but at this point it was confirmed that this strain had lost G418 resistance. It was considered that the expression cassette containing the G418 marker was dropped due to the leak expression of Cre recombinase. This strain was stripped of pPAP_CP by performing single colony isolation on YPD agar medium (Fig. 7 (4)). This strain had lost resistance to G418 and Zeocin. FCM analysis also showed that the GFP fluorescence intensity had dropped to the background level. Finally, a strain into which an RFP expression cassette (pPGP_RFP fragment linearly converted at the EcoRV site in the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site was prepared by the method described in (13) above (Fig. 7 (Fig. 7). 5)). This strain has G418 resistance, and high RFP fluorescence intensity was confirmed by FCM analysis. From the above results, it was shown that the reporter expression cassette can be replaced by the above method.
実施例７　異なる低分子抗体での抗体高生産株の評価
　実施例５で得られたタンデムscFv226抗体高生産株では、抗体発現ベクターのCCA38473ターミネーター部位への導入用配列の両末端にloxP配列を導入していたことから、酵母ゲノムに導入された抗体カセットの両末端にはloxP配列が導入されている（図８）。そのため、酵母細胞内でCreリコンビナーゼが発現すればタンデムscFv226抗体発現カセットを抜くことができる。実際、プラスミドpPAP_CP（Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018）を抗体高生産株に上記（１３）で記した手法にて導入後、シングルコロニーアイソレーションを行った結果、コロニーPCR及びG418を用いた薬剤アッセイにて、抗体発現カセットの削除が確認できた。次に、タンデムscFv226抗体とは異なる低分子抗体として、抗リゾチウムscFv抗体発現ベクターpUC_Paox1_MFα_scFv_Taox1_T38473_G418、ブリナツモマブ抗体発現ベクターpUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418及びミニボディー抗体発現ベクターpUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418の3種類をそれぞれ、制限酵素EcoRVにて直鎖状に変換後、実施例５の高生産株A株、D株及びE株のCCA38473ターミネーター部位に導入した抗体生産株（それぞれA1、A2、A3株、D1、D2、D3株及びE1、E2、E3株）を上記（１３）で記した手法にて作製した。各抗体の分泌量をELISA法及びSDS-PAGEにて評価したところ、宿主株と比較して、抗リゾチウムscFv抗体生産株では10倍以上、ブリナツモマブ抗体生産株では、3倍以上の抗体分泌量が確認された。またミニボディー抗体生産株では、D株由来のD3株では約2倍、E株由来のE3株では約3倍の抗体分泌量が確認された（図９）。以上の結果より、タンデムscFv226抗体の生産性を指標として取得した有用因子の集積株は、タンデムscFv226抗体生産だけでなく、異なる低分子抗体においても高い生産性を有することが示された。 Example 7 Evaluation of antibody high-producing strains with different small molecule antibodies In the tandem scFv226 antibody high-producing strain obtained in Example 5, loxP sequences were introduced at both ends of the sequence for introduction of the antibody expression vector into the CCA38473 terminator site. Therefore, loxP sequences were introduced at both ends of the antibody cassette introduced into the yeast genome (Fig. 8). Therefore, if Cre recombinase is expressed in yeast cells, the tandem scFv226 antibody expression cassette can be removed. In fact, after introducing the plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) into a high antibody-producing strain by the method described in (13) above, single colony isolation was performed. As a result, it was confirmed that the antibody expression cassette was deleted by the drug assay using colony PCR and G418. Next, as the low molecular antibody that is different from the tandem scFv226 antibody, anti-lysozyme scFv antibody expression vector PUC_Paox1_MFarufa_scFv_Taox1_T38473_G418, Burinatsumomabu antibody expression vector pUC_Paox1_MFα (mut) _Blinatumomab_Taox1_loxP_T38473_loxP_G418 and minibody three antibody expression vector pUC_Paox1_MFα (mut) _Minibody_Taox1_loxP_T38473_loxP_G418 respectively, restriction enzyme Antibody-producing strains (A1, A2, A3 strains, D1, D2, D3 strains, respectively) introduced into the CCA38473 terminator site of the high-producing strains A, D, and E of Example 5 after being linearly converted by EcoRV. And E1, E2, E3 strains) were prepared by the method described in (13) above. When the amount of each antibody secreted was evaluated by the ELISA method and SDS-PAGE, the amount of antibody secreted was 10 times or more in the anti-lysothium scFv antibody-producing strain and 3 times or more in the blinatumomab antibody-producing strain as compared with the host strain. confirmed. In addition, among the minibody antibody-producing strains, the amount of antibody secreted was confirmed to be about twice that of the D3 strain derived from the D strain and about three times that of the E3 strain derived from the E strain (Fig. 9). From the above results, it was shown that the stocks of useful factors obtained by using the productivity of the tandem scFv226 antibody as an index have high productivity not only in the production of the tandem scFv226 antibody but also in different small molecule antibodies.
実施例８　ペアスクリーニング用抗体産生促進タンパク質過剰発現細胞ライブラリの作製と高生産株のスクリーニング
　実施例５で示した通り、8種類の有用因子を用いて行った集積実験は、有用因子の集積が低分子抗体の生産および分泌の促進に効果的であることを示した。そこで、得られた有用因子を効率よく集積させる手法として、2種類の有用因子を同時に過剰発現させた酵母ライブラリを作製し、そのライブラリより低分子抗体を最も分泌する株を、実施例３で示したハイスループットスクリーニング法を用いて見出すことを考案した（ペアスクリーニング、図１０）。その実証として、難分泌生産性を示すブリナツモマブ生産株のブリナツモマブ産生向上を目指し、実施例２にて得られた有用因子群を用いて、ペアスクリーニングを２回繰り返した。まず、実施例４で得られたブリナツモマブ分泌を促進した有用因子９種類（EF3rd-1～9、表３）を選び、2種類のプライマーセット（プライマー124（配列番号229）及びプライマー125（配列番号230）、プライマー126（配列番号231）及びプライマー127（配列番号232））を用いたPCR法にて、それぞれのDNA断片（GAPDHプロモーター―有用因子ORFとそのターミネーター配列）を増幅させた。この2種類のPCR産物と、BamHI及びXhoIにて切断したpUC2_NAT_TMRP40が同じDNA分子数になるように混合後、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、2つの有用因子発現カセットがリンカー配列を挟んで逆位に連結された塩基配列を含むプラスミドの集団からなるプラスミドライブラリを作製した（図１０、組み合わせ種類の多様性：81）。作製したプラスミドライブラリを上記（１３）で作製したブリナツモマブ分泌株に導入し、約2,400個の形質転換体を得た。現れた形質転換体より、実施例３に示したハイスループットスクリーニング法にて、96ウェルディープウェルプレート8枚を用いてスクリーニングを行った。培養上清中のブリナツモマブ分泌量評価は、Hisタグを指標としたELISA法にて実施した。スクリーニングにて高生産を示した2株（1st-11株、1st-12株）を解析した結果、宿主株と比較して、約5倍に抗体分泌量が増加していた（表１５）。次に実施例４と同様の手法を用いて、得られた2つの高分泌株に導入された有用因子を同定した結果、共にEF3rd-1が導入されていた。もう一方の有用因子として、EF3rd-2及びEF3rd-5が挿入されていた。また実施例７において有用因子集積実験で得られたブリナツモマブ高分泌株E2株（4有用因子導入株）は、宿主株と比較して抗体分泌量は約3.2倍であったことから、今回ペアスクリーニングで得られた高分泌株（2有用因子導入株）の方が、ブリナツモマブ抗体の分泌量が高いことを示した。
　次に、この2株を親株として2サイクル目のペアスクリーニングを行った。今回、実施例２で得られたブリナツモマブ分泌を促進する有用因子としてEF1st-1～6及びEF2nd-1～4の計10個を選び、組み合わせの最適化を行った。2種類のプライマーセット（プライマー128（配列番号233）及びプライマー129（配列番号234）、プライマー130（配列番号235）及びプライマー131（配列番号236））を用いて有用因子カセット（GAPDHプロモーター―有用因子ORFとそのターミネーター配列）を用いてPCR法にて、DNAをそれぞれ増幅させた。この2種類のPCR産物と、BamHI及びXhoIにて切断したpUC2_Hyg_TARG83が同じDNA分子数になるように混合し、In-fusion HD Cloning Kitを用いて繋ぎ合わせて、2つの有用因子発現カセットがリンカー配列を挟んで逆位に連結された塩基配列を含むプラスミドの集団からなるプラスミドライブラリを作製した（図１０、組み合わせ種類の多様性：100）。作製したプラスミドライブラリを上記で示した2種類のブリナツモマブ高分泌株に導入し、約4,400個の形質転換体を得た。現れた形質転換体より、上記のハイスループットスクリーニング法にて、96ウェルウェルプレート9枚分（864株）をスクリーニングした。スクリーニングにて高分泌を示した4株（2nd_11-7株、2nd_11-8株、2nd_12-7株、2nd_12-2株）を解析した結果、宿主株と比較して、13~15倍高い分泌量を示した（表１５）。次に実施例４と同様の手法を用いて得られた4種類の株に導入された有用因子を同定した結果、すべての株でEF1st-1が導入されていた（表１５）。またもう一方の有用因子は、EF2nd-4（EF3rd-2と同一遺伝子）（2nd_11-7株及び2nd_11-8株）及びEF1st-3（2nd_12-2株及び2nd_12-7株）であった（表１５）。以上の結果では、実施例７で示したタンデムscFv226生産株で最適であった組み合わせとは異なる組み合わせの有用因子がブリナツモマブ抗体高分泌株に含まれていた。低分子抗体のモダリティー及びアミノ酸配列によって最適な有用因子の組み合わせが存在すると考えられる。 Example 8 Preparation of antibody production promoting protein overexpressing cell library for pair screening and screening of high-producing strains As shown in Example 5, the accumulation experiment conducted using eight kinds of useful factors showed low accumulation of useful factors. It was shown to be effective in promoting the production and secretion of molecular antibodies. Therefore, as a method for efficiently accumulating the obtained useful factors, a yeast library in which two kinds of useful factors were overexpressed at the same time was prepared, and a strain that secretes the most small molecule antibody from the library is shown in Example 3. We devised to find out using a high-throughput screening method (pair screening, FIG. 10). As a demonstration, pair screening was repeated twice using the useful factor group obtained in Example 2 with the aim of improving the production of blinatumomab in the blinatumomab-producing strain showing low-secretory productivity. First, 9 types of useful factors (EF3rd-1 to 9, Table 3) that promoted brinattumomab secretion obtained in Example 4 were selected, and 2 types of primer sets (Primer 124 (SEQ ID NO: 229) and Primer 125 (SEQ ID NO: 229) were selected. Each DNA fragment (GAPDH promoter-useful factor ORF and its terminator sequence) was amplified by a PCR method using primer 126 (SEQ ID NO: 231) and primer 127 (SEQ ID NO: 232)). These two types of PCR products and pUC2_NAT_TMRP40 cleaved with BamHI and XhoI are mixed so that they have the same number of DNA molecules, and then spliced together using the In-fusion HD Cloning Kit to obtain two useful factor expression cassettes. A plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 81). The prepared plasmid library was introduced into the blinatumomab secretory strain prepared in (13) above to obtain about 2,400 transformants. The transformants that appeared were screened using eight 96-well deep-well plates by the high-throughput screening method shown in Example 3. The amount of blinatumomab secreted in the culture supernatant was evaluated by the ELISA method using the His tag as an index. As a result of analyzing two strains (1st-11 strain and 1st-12 strain) that showed high production by screening, the amount of antibody secretion increased about 5 times as compared with the host strain (Table 15). Next, as a result of identifying the useful factors introduced into the two highly secreted strains obtained by using the same method as in Example 4, EF3rd-1 was introduced in both of them. As the other useful factor, EF3rd-2 and EF3rd-5 were inserted. In addition, the blinatumomab hypersecretory strain E2 strain (4 useful factor-introduced strains) obtained in the useful factor accumulation experiment in Example 7 had an antibody secretion amount of about 3.2 times that of the host strain. It was shown that the highly secreted strain (2 useful factor-introduced strain) obtained in 1) secreted a higher amount of blinatumomab antibody.
Next, pair screening was performed in the second cycle using these two strains as parent strains. This time, a total of 10 EF1st-1 to 6 and EF2nd-1 to 4 were selected as useful factors for promoting blinatumomab secretion obtained in Example 2, and the combination was optimized. A useful factor cassette (GAPDH promoter-useful factor) using two primer sets (primer 128 (SEQ ID NO: 233) and primer 129 (SEQ ID NO: 234), primer 130 (SEQ ID NO: 235) and primer 131 (SEQ ID NO: 236)). DNA was amplified by PCR using ORF and its terminator sequence). These two types of PCR products and pUC2_Hyg_TARG83 cleaved with BamHI and XhoI are mixed so that they have the same number of DNA molecules, and they are joined using the In-fusion HD Cloning Kit to obtain two useful factor expression cassettes. A plasmid library consisting of a population of plasmids containing nucleotide sequences linked invertedly across the plate was prepared (Fig. 10, variety of combination types: 100). The prepared plasmid library was introduced into the two types of blinatumomab hypersecretory strains shown above to obtain about 4,400 transformants. From the transformants that appeared, 9 96-well well plates (864 strains) were screened by the above high-throughput screening method. As a result of analyzing 4 strains (2nd_11-7 strain, 2nd_11-8 strain, 2nd_12-7 strain, 2nd_12-2 strain) that showed high secretion by screening, the amount of secretion was 13 to 15 times higher than that of the host strain. Was shown (Table 15). Next, as a result of identifying the useful factors introduced into the four types of strains obtained by using the same method as in Example 4, EF1st-1 was introduced into all the strains (Table 15). The other useful factors were EF2nd-4 (same gene as EF3rd-2) (2nd_11-7 strain and 2nd_11-8 strain) and EF1st-3 (2nd_12-2 strain and 2nd_12-7 strain) (Table). 15). In the above results, the blinatumomab antibody hypersecreting strain contained useful factors in a combination different from the optimal combination in the tandem scFv226-producing strain shown in Example 7. It is considered that there is an optimal combination of useful factors depending on the modality and amino acid sequence of the small molecule antibody.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
実施例９　異なる低分子抗体でのペアスクリーニング
　実施例８において、ペアスクリーニング法を2回繰り返し、ブリナツモマブ抗体の高生産株を取得した。またブリナツモマブ高分泌株において最適な組み合わせとして選ばれてきた有用因子は、EF1st-1、EF1st-3、EF2nd-4、EF3rd-1及びEF3rd-5であった。次に、異なる低分子抗体分泌株において、実施例８に示したブリナツモマブ抗体でのペアスクリーニング法を実施し、選ばれる有用因子の特定を試みた。実施例８でのペアスクリーニングの1サイクル目で作製した9種類の有用因子（EF3rd-1～9）から成るプラスミドライブラリを用いて、上記（１３）で作製した低分子抗体（タンデムscFv226）分泌株に導入し、1,200個の形質転換体を得た。現れた形質転換体より、実施例3に示したハイスループットスクリーニング法にて、96ウェルディープウェルプレート4枚分（384株）のスクリーニングを行った。培養上清中のtaFv226分泌量の評価は、上記（１５）に示したELISA法にて実施した。スクリーニングで高い分泌量を示した株を評価した結果、宿主株と比較して、4株（taFv1st-10株、taFv1st-15株、taFv1st-9株、taFv1st-12株）は約2.3倍高い抗体分泌量を示した（表１６）。次に実施例４と同様の手法を用いて、得られた各4種類の株に導入された有用因子を同定した結果、すべての株でEF3rd-2（EF2nd-4）及びEF3rd-3が導入されていた（表１６）。実施例８での1世代目のペアスクリーニングにおいて、最適な有用因子の組み合わせはEF3rd-1及びEF3rd-2（EF2nd-4）、EF3rd-1及びEF3rd-5であり、本実施例のタンデムscFv226分泌株を用いたスクリーニングで得られたEF3rd-2（EF2nd-4）及びEF3rd-3とは異なっていた。すなわち、本結果より、各低分子抗体分泌のために最適な有用因子の組み合わせは異なることが示された。 Example 9 Pair screening with different small molecule antibodies In Example 8, the pair screening method was repeated twice to obtain a high-producing strain of blinatumomab antibody. The useful factors selected as the optimal combination in the blinatumomab hypersecretory strain were EF1st-1, EF1st-3, EF2nd-4, EF3rd-1 and EF3rd-5. Next, in different small molecule antibody-secreting strains, a pair screening method using the blinatumomab antibody shown in Example 8 was carried out to try to identify useful factors to be selected. Small molecule antibody (tandem scFv226) secretory strain prepared in (13) above using a plasmid library consisting of 9 useful factors (EF3rd-1-9) prepared in the first cycle of pair screening in Example 8. Introduced into, 1,200 transformants were obtained. From the transformants that appeared, screening for 4 96-well deep-well plates (384 strains) was performed by the high-throughput screening method shown in Example 3. The amount of taFv226 secreted in the culture supernatant was evaluated by the ELISA method shown in (15) above. As a result of evaluating the strains showing high secretion amount by screening, 4 strains (taFv1st-10 strain, taFv1st-15 strain, taFv1st-9 strain, taFv1st-12 strain) were about 2.3 times higher antibody than the host strain. The amount of secretion was shown (Table 16). Next, as a result of identifying the useful factors introduced into each of the four obtained strains using the same method as in Example 4, EF3rd-2 (EF2nd-4) and EF3rd-3 were introduced in all the strains. It was done (Table 16). In the first generation pair screening in Example 8, the optimal combination of useful factors was EF3rd-1 and EF3rd-2 (EF2nd-4), EF3rd-1 and EF3rd-5, and the tandem scFv226 secretion of this example. It was different from EF3rd-2 (EF2nd-4) and EF3rd-3 obtained by screening using strains. That is, from this result, it was shown that the optimum combination of useful factors for the secretion of each small molecule antibody is different.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　内在性遺伝子の発現が増強された遺伝子組換え細胞を提供することにより、発現が増強された内在性遺伝子の遺伝子ライブラリを構築することが可能となった。また、該ライブラリを用いることによって目的タンパク質の生産を増加させる内在性遺伝子のスクリーニングが可能となった。本出願は、日本で出願された特願2020-050192（出願日：令和2年3月19日）を基礎としており、その内容はすべて本明細書に包含されるものとする。 By providing transgenic cells with enhanced expression of endogenous genes, it has become possible to construct a gene library of endogenous genes with enhanced expression. In addition, the use of the library has made it possible to screen for endogenous genes that increase the production of the target protein. This application is based on Japanese Patent Application No. 2020-050192 (Filing date: March 19, 2nd year of Reiwa), the entire contents of which are incorporated herein by reference.

Claims (18)

  1. 　以下の工程を含む、内在性遺伝子の発現が増強された遺伝子組換え細胞の製造方法。
    （１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
    （２）該直鎖状核酸を宿主細胞に導入する工程、および
    （３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程。     A method for producing a recombinant cell in which the expression of an endogenous gene is enhanced, which comprises the following steps.
    (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
    (2) The step of introducing the linear nucleic acid into a host cell, and (3) the endogenous gene is homologously recombined by the linear nucleic acid, and a highly expressive promoter is operably linked. A step of selecting transgenic cells containing a sex gene.
  2. 　宿主細胞および遺伝子組換え細胞が酵母、細菌、真菌、昆虫細胞、動物細胞または植物細胞である、請求項１に記載の方法。 The method according to claim 1, wherein the host cell and the recombinant cell are yeast, bacteria, fungi, insect cells, animal cells or plant cells.
  3. 　酵母がメタノール資化性酵母、分裂酵母または出芽酵母である、請求項２に記載の方法。 The method according to claim 2, wherein the yeast is methanol-utilizing yeast, fission yeast or budding yeast.
  4. 　メタノール資化性酵母がコマガタエラ属酵母またはオガタエア属酵母である、請求項３に記載の方法。 The method according to claim 3, wherein the methanol-utilizing yeast is a yeast of the genus Komagataera or a yeast of the genus Ogataea.
  5. 　内在性遺伝子が以下の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子である、請求項１～４のいずれか１項に記載の方法。
    （１）配列番号４７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２）配列番号４６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３）配列番号４８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （４）配列番号４９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （５）配列番号５０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （６）配列番号５１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （７）配列番号５２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （８）配列番号５３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （９）配列番号５４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１０）配列番号５５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１１）配列番号５６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１２）配列番号５７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１３）配列番号５８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１４）配列番号５９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１５）配列番号６０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１６）配列番号６１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１７）配列番号６２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１８）配列番号６３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１９）配列番号６４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２０）配列番号６５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２１）配列番号６６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２２）配列番号６７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２３）配列番号６８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２４）配列番号６９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２５）配列番号７０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２６）配列番号７１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２７）配列番号７３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２８）配列番号７５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２９）配列番号７６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３０）配列番号７７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３１）配列番号７８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
    （３２）配列番号７９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。     The method according to any one of claims 1 to 4, wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32).
    (1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47.
    (2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46.
    (3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48.
    (4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
    (5) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50.
    (6) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51.
    (7) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52.
    (8) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53.
    (9) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54.
    (10) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55.
    (11) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56.
    (12) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57.
    (13) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58.
    (14) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59.
    (15) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60.
    (16) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61.
    (17) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 62.
    (18) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63.
    (19) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 64.
    (20) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 65.
    (21) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66.
    (22) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 67.
    (23) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 68.
    (24) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 69.
    (25) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 70.
    (26) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71.
    (27) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73.
    (28) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 75.
    (29) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76.
    (30) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77.
    (31) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78, and (32) the same or substantially the same base as the base sequence represented by SEQ ID NO: 79. An endogenous gene containing a sequence.
  6. 　核酸断片が、以下の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片である、請求項５に記載の方法。
    （i）配列番号２３９で表される塩基配列を含む核酸断片、
    （ii）配列番号２３８で表される塩基配列を含む核酸断片、
    （iii）配列番号２４０で表される塩基配列を含む核酸断片、
    （iv）配列番号２４１で表される塩基配列を含む核酸断片、
    （v）配列番号２４２で表される塩基配列を含む核酸断片、
    （vi）配列番号２４３で表される塩基配列を含む核酸断片、
    （vii）配列番号２４４で表される塩基配列を含む核酸断片、
    （viii）配列番号２４５で表される塩基配列を含む核酸断片、
    （ix）配列番号２４６で表される塩基配列を含む核酸断片、
    （x）配列番号２４７で表される塩基配列を含む核酸断片、
    （xi）配列番号２４８で表される塩基配列を含む核酸断片、
    （xii）配列番号２４９で表される塩基配列を含む核酸断片、
    （xiii）配列番号２５０で表される塩基配列を含む核酸断片、
    （xiv）配列番号２５１で表される塩基配列を含む核酸断片、
    （xv）配列番号２５２で表される塩基配列を含む核酸断片、
    （xvi）配列番号２５３で表される塩基配列を含む核酸断片、
    （xvii）配列番号２５４で表される塩基配列を含む核酸断片、
    （xviii）配列番号２５５で表される塩基配列を含む核酸断片、
    （xix）配列番号２５６で表される塩基配列を含む核酸断片、
    （xx）配列番号２５７で表される塩基配列を含む核酸断片、
    （xxi）配列番号２５８で表される塩基配列を含む核酸断片、
    （xxii）配列番号２５９で表される塩基配列を含む核酸断片、
    （xxiii）配列番号２６０で表される塩基配列を含む核酸断片、
    （xxiv）配列番号２６１で表される塩基配列を含む核酸断片、
    （xxv）配列番号２６２で表される塩基配列を含む核酸断片、
    （xxvi）配列番号２６３で表される塩基配列を含む核酸断片、
    （xxvii）配列番号２６５で表される塩基配列を含む核酸断片、
    （xxviii）配列番号２６７で表される塩基配列を含む核酸断片、
    （xxix）配列番号２６８で表される塩基配列を含む核酸断片、
    （xxx）配列番号２６９で表される塩基配列を含む核酸断片、
    （xxxi）配列番号２７０で表される塩基配列を含む核酸断片、および
    （xxxii）配列番号２７１で表される塩基配列を含む核酸断片。     The method according to claim 5, wherein the nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
    (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239,
    (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238,
    (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240,
    (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
    (V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242,
    (Vi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243,
    (Vii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244,
    (Viii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 245,
    (Ix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 246,
    (X) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247,
    (Xi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248,
    (Xii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249,
    (Xiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250,
    (Xiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251.
    (Xv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252,
    (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253,
    (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254,
    (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255,
    (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256,
    (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
    (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258,
    (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259,
    (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260,
    (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
    (Xxv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262,
    (Xxvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263,
    (Xxvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265,
    (Xxviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267,
    (Xxix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 268,
    (Xxx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269,
    (Xxxi) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 270, and (xxxii) a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 271.
  7. 　内在性遺伝子が核酸断片を含むプラスミドが制限酵素で切断された直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された該内在性遺伝子を含む遺伝子組換え細胞であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、遺伝子組換え細胞。 In a recombinant cell containing the endogenous gene in which a highly expressive promoter is operably linked, in which a plasmid containing a nucleic acid fragment is homologously recombined with a linear nucleic acid in which the endogenous gene is cleaved with a restriction enzyme. The nucleic acid fragment is a base sequence in which a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene, and the partial sequence into which the restriction enzyme recognition site is inserted and the end codon are sequentially linked. Including, recombinant cells.
  8. 　遺伝子組換え細胞が酵母、細菌、真菌、昆虫細胞、動物細胞または植物細胞である、請求項７に記載の遺伝子組換え細胞。 The recombinant cell according to claim 7, wherein the recombinant cell is a yeast, a bacterium, a fungus, an insect cell, an animal cell or a plant cell.
  9. 　酵母がメタノール資化性酵母、分裂酵母または出芽酵母である、請求項８に記載の遺伝子組換え細胞。 The genetically modified cell according to claim 8, wherein the yeast is methanol-utilizing yeast, fission yeast or budding yeast.
  10. 　メタノール資化性酵母がコマガタエラ属酵母またはオガタエア属酵母である、請求項９に記載の遺伝子組換え細胞。 The genetically modified cell according to claim 9, wherein the methanol-utilizing yeast is a yeast of the genus Komagataera or a yeast of the genus Ogataea.
  11. 　内在性遺伝子が以下の（１）～（３２）の内在性遺伝子からなる群から選択される少なくとも１つの内在性遺伝子である、請求項７～１０のいずれか１項に記載の遺伝子組換え細胞。
    （１）配列番号４７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２）配列番号４６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３）配列番号４８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （４）配列番号４９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （５）配列番号５０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （６）配列番号５１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （７）配列番号５２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （８）配列番号５３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （９）配列番号５４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１０）配列番号５５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１１）配列番号５６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１２）配列番号５７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１３）配列番号５８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１４）配列番号５９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１５）配列番号６０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１６）配列番号６１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１７）配列番号６２で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１８）配列番号６３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （１９）配列番号６４で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２０）配列番号６５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２１）配列番号６６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２２）配列番号６７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２３）配列番号６８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２４）配列番号６９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２５）配列番号７０で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２６）配列番号７１で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２７）配列番号７３で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２８）配列番号７５で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （２９）配列番号７６で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３０）配列番号７７で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、
    （３１）配列番号７８で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子、および
    （３２）配列番号７９で表される塩基配列と同一または実質的に同一の塩基配列を含む内在性遺伝子。     The transgenic cell according to any one of claims 7 to 10, wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the following endogenous genes (1) to (32). ..
    (1) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 47.
    (2) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 46.
    (3) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 48.
    (4) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 49.
    (5) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 50.
    (6) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 51.
    (7) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 52.
    (8) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 53.
    (9) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 54.
    (10) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 55.
    (11) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 56.
    (12) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 57.
    (13) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 58.
    (14) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 59.
    (15) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 60.
    (16) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 61.
    (17) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 62.
    (18) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 63.
    (19) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 64.
    (20) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 65.
    (21) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 66.
    (22) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 67.
    (23) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 68.
    (24) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 69.
    (25) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 70.
    (26) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 71.
    (27) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 73.
    (28) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 75.
    (29) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 76.
    (30) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 77.
    (31) An endogenous gene containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 78, and (32) the same or substantially the same base as the base sequence represented by SEQ ID NO: 79. An endogenous gene containing a sequence.
  12. 　核酸断片が、以下の（i）～（xxxii）の核酸断片からなる群から選択される少なくとも１つの核酸断片である、請求項１１に記載の遺伝子組換え細胞。
    （i）配列番号２３９で表される塩基配列を含む核酸断片、
    （ii）配列番号２３８で表される塩基配列を含む核酸断片、
    （iii）配列番号２４０で表される塩基配列を含む核酸断片、
    （iv）配列番号２４１で表される塩基配列を含む核酸断片、
    （v）配列番号２４２で表される塩基配列を含む核酸断片、
    （vi）配列番号２４３で表される塩基配列を含む核酸断片、
    （vii）配列番号２４４で表される塩基配列を含む核酸断片、
    （viii）配列番号２４５で表される塩基配列を含む核酸断片、
    （ix）配列番号２４６で表される塩基配列を含む核酸断片、
    （x）配列番号２４７で表される塩基配列を含む核酸断片、
    （xi）配列番号２４８で表される塩基配列を含む核酸断片、
    （xii）配列番号２４９で表される塩基配列を含む核酸断片、
    （xiii）配列番号２５０で表される塩基配列を含む核酸断片、
    （xiv）配列番号２５１で表される塩基配列を含む核酸断片、
    （xv）配列番号２５２で表される塩基配列を含む核酸断片、
    （xvi）配列番号２５３で表される塩基配列を含む核酸断片、
    （xvii）配列番号２５４で表される塩基配列を含む核酸断片、
    （xviii）配列番号２５５で表される塩基配列を含む核酸断片、
    （xix）配列番号２５６で表される塩基配列を含む核酸断片、
    （xx）配列番号２５７で表される塩基配列を含む核酸断片、
    （xxi）配列番号２５８で表される塩基配列を含む核酸断片、
    （xxii）配列番号２５９で表される塩基配列を含む核酸断片、
    （xxiii）配列番号２６０で表される塩基配列を含む核酸断片、
    （xxiv）配列番号２６１で表される塩基配列を含む核酸断片、
    （xxv）配列番号２６２で表される塩基配列を含む核酸断片、
    （xxvi）配列番号２６３で表される塩基配列を含む核酸断片、
    （xxvii）配列番号２６５で表される塩基配列を含む核酸断片、
    （xxviii）配列番号２６７で表される塩基配列を含む核酸断片、
    （xxix）配列番号２６８で表される塩基配列を含む核酸断片、
    （xxx）配列番号２６９で表される塩基配列を含む核酸断片、
    （xxxi）配列番号２７０で表される塩基配列を含む核酸断片、および
    （xxxii）配列番号２７１で表される塩基配列を含む核酸断片。     The transgenic cell according to claim 11, wherein the nucleic acid fragment is at least one nucleic acid fragment selected from the group consisting of the following nucleic acid fragments (i) to (xxxii).
    (I) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239,
    (Ii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 238,
    (Iii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240,
    (Iv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241.
    (V) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242,
    (Vi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243,
    (Vii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244,
    (Viii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 245,
    (Ix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 246,
    (X) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247,
    (Xi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248,
    (Xii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249,
    (Xiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250,
    (Xiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251.
    (Xv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252,
    (Xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253,
    (Xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254,
    (Xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255,
    (Xix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 256,
    (Xx) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 257.
    (Xxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258,
    (Xxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 259,
    (Xxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 260,
    (Xxiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 261.
    (Xxv) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262,
    (Xxvi) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263,
    (Xxvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265,
    (Xxviii) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267,
    (Xxix) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 268,
    (Xxx) Nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269,
    (Xxxi) A nucleic acid fragment containing the base sequence represented by SEQ ID NO: 270, and (xxxii) a nucleic acid fragment containing the base sequence represented by SEQ ID NO: 271.
  13. 　目的タンパク質をコードする塩基配列をゲノムに含む、請求項７～１２のいずれか１項に記載の遺伝子組換え細胞。 The genetically modified cell according to any one of claims 7 to 12, which contains a base sequence encoding a target protein in its genome.
  14. 　目的タンパク質が異種タンパク質である、請求項１３に記載の遺伝子組換え細胞。 The genetically modified cell according to claim 13, wherein the target protein is a heterologous protein.
  15. 　請求項１３または１４に記載の遺伝子組換え細胞を培養する工程を含む、目的タンパク質の製造方法。 A method for producing a target protein, which comprises the step of culturing the genetically modified cells according to claim 13 or 14.
  16. 　請求項７～１０のいずれか１項に記載の遺伝子組換え細胞を含む、内在性遺伝子過剰発現細胞ライブラリ。 An endogenous gene overexpressing cell library containing the recombinant cell according to any one of claims 7 to 10.
  17. 　以下の工程を含む、目的タンパク質の生産を増強する内在性遺伝子をスクリーニングする方法。
    （１）核酸断片を含むプラスミドを制限酵素で切断して直鎖状の核酸を調製する工程であって、該核酸断片が、高発現性プロモーター、該内在性遺伝子の開始コドンから始まる部分配列であって該制限酵素認識部位が挿入された部分配列および終止コドンが順番に連結された塩基配列を含む、工程、
    （２）該直鎖状核酸を目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞に導入する工程、
    （３）該内在性遺伝子が該直鎖状核酸によって相同的に組み換えられた、高発現性プロモーターが機能可能に連結された内在性遺伝子を含む遺伝子組換え細胞を選抜する工程、
    （４）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞を培養する工程、
    （５）工程（３）で得られた細胞および目的タンパク質をコードする塩基配列をゲノムに含んだ宿主細胞による目的タンパク質の生産量をそれぞれ測定する工程、および
    （６）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。     A method for screening an endogenous gene that enhances the production of a protein of interest, which comprises the following steps.
    (1) A step of preparing a linear nucleic acid by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment is a highly expressive promoter and a partial sequence starting from the stop codon of the endogenous gene. A step comprising a partial sequence into which the restriction enzyme recognition site has been inserted and a base sequence in which stop codons are sequentially linked.
    (2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding a target protein in the genome.
    (3) A step of selecting a recombinant cell containing an endogenous gene in which the endogenous gene is homologously recombined by the linear nucleic acid and to which a highly expressive promoter is operably linked.
    (4) A step of culturing a host cell containing the cell obtained in step (3) and the base sequence encoding the target protein in the genome.
    (5) The step of measuring the production amount of the target protein by the cells obtained in the step (3) and the host cell containing the base sequence encoding the target protein in the genome, respectively, and (6) increasing the production amount of the target protein. The step of identifying the endogenous gene to be caused.
  18. 　以下の工程を含む、目的タンパク質の生産を増強する内在性遺伝子をスクリーニングする方法。
    （１）請求項１６に記載の内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞に目的タンパク質をコードする塩基配列を含む発現ベクターを導入し、培養する工程、
    （２）内在性遺伝子過剰発現細胞ライブラリおよび宿主細胞による目的タンパク質の生産量を測定する工程、および
    （３）目的タンパク質の生産量を増加させる内在性遺伝子を特定する工程。
     
          A method for screening an endogenous gene that enhances the production of a protein of interest, which comprises the following steps.
    (1) A step of introducing and culturing an expression vector containing a base sequence encoding a target protein into the endogenous gene overexpressing cell library according to claim 16 and a host cell.
    (2) A step of measuring the production amount of the target protein by the endogenous gene overexpressing cell library and the host cell, and (3) a step of identifying the endogenous gene that increases the production amount of the target protein.
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