Method for producing a genetically modified cell with enhanced expression of an endogenous gene

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

Technical Field

[0001] The present invention relates to a method for producing a genetically modified cell with enhanced expression of an endogenous gene, a genetically modified cell produced by the method, and a method for screening an endogenous gene that enhances the production of a target protein using an endogenous gene overexpression cell library containing the cell.

Background Art

[0002] For the production of industrially useful biomaterials such as antibodies, enzymes, and cytokines for medical and diagnostic use, genetic recombination methods are widely used. As hosts for producing target proteins by genetic recombination methods, animals such as chickens, animal cells such as CHO, insects such as silkworms, insect cells such as sf9, and microorganisms such as yeast, Escherichia coli, and actinomycetes are used. Among host organisms, yeast can be cultured on an inexpensive medium on a large scale and at a high density, so that the target protein can be produced at low cost. If a signal peptide or the like is used, the target protein can be secreted into the culture medium, which facilitates the purification process of the target protein. In addition, since yeast is a eukaryote, post-translational modifications such as glycosylation are possible. Therefore, yeast is very beneficial and various studies have been conducted. If innovative production technologies that can handle various target proteins can be developed in yeast, in addition to strengthening cost competitiveness through a dramatic improvement in productivity, broad industrial expansion can be expected.

[0003] Komagataella pastoris, a type of yeast, has excellent protein expression ability and can utilize an inexpensive carbon source, methanol, which is advantageous for industrial production. (Mut +)It is yeast. For example, Non-Patent Document 1 reports a method for producing heterologous proteins such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibody using Komagataella pastoris. When producing heterologous proteins in yeast, various attempts have been made to improve productivity, such as adding signal sequences, using strong promoters, codon modification, co-expression of chaperone genes, co-expression of transcription factor genes, inactivation of protease genes derived from the host yeast, and examination of culture conditions.

[0004] 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 amount of heterologous proteins. When attempting to search for endogenous genes of host cells that improve the production amount of heterologous proteins, usually, the target gene has been searched using a cDNA library or a genomic library of the host cell. However, in screening using a cDNA library or a genomic library, bias occurs due to blind library preparation, and the number of samples to be evaluated becomes enormous, requiring a great deal of labor.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a method for producing a genetically modified cell in which the expression of an endogenous gene is enhanced, a genetically modified cell produced by the method, and a method for screening an endogenous gene that enhances the production of a target protein using an endogenous gene overexpression cell library containing the cell.

Means for Solving the Problems

[0007] The inventors have successfully prepared a Komagataella phaffii population in which the expression of each endogenous gene is enhanced, in which a highly expressive promoter is functionally linked to all endogenous genes (5,001 types) of Komagataella phaffii by homologous recombination. Furthermore, the inventors prepared a Komagataella phaffii population in which the expression of each endogenous gene was enhanced from Komagataella phaffii into which a single-chain antibody expression vector was introduced, and identified 35 endogenous genes that increase the production amount of the single-chain antibody. In addition, the inventors succeeded in further increasing the production amount of the single-chain antibody by combining 4 out of these 35 endogenous genes. As a result of further studies based on these findings, the inventors have completed the present invention.

[0008] That is, the present invention provides the following. [1] A method for producing a genetically modified cell in which the expression of an endogenous gene is enhanced, comprising 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 contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a base sequence in which a stop codon is linked in order. (2) A step of introducing the linear nucleic acid into a host cell, and (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene is homologously recombined with the linear nucleic acid and a highly expressive promoter is functionally linked. [2] The method according to [1], wherein the host cell and the genetically modified cell are yeast, bacteria, fungi, insect cells, animal cells or plant cells. [3] The method according to [2], wherein the yeast is a methanol-utilizing yeast, a fission yeast or a budding yeast. [4] The method according to [3], wherein the methanol-utilizing yeast is a yeast belonging to the genus Komagataella or the genus Ogataea. [5] The method according to any one of [1] to [4], wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the endogenous genes (1) to (32) below: (1) an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 46; (2) an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 47; (3) an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 48; (4) an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 49; (5) an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 50; (6) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 51; (7) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 52; (8) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 53; (9) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 54; (10) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 55; (11) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 56; (12) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 57; (13) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 58; (14) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 59; (15) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 60; An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 61 (17) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 62 (18) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 63 (19) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 64 (20) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 65 (21) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (22) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 67 (23) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 68 (24) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 69 (25) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 70 (26) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71 (27) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 73 (28) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 75 (29) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 76 (30) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 77 (31) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 78, and (32) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 79. 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 comprising the nucleotide sequence represented by SEQ ID NO: 238, (ii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 239, (iii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 240, (iv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 241, (v) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 242, (vi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 243, (vii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 244, (viii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 245, (ix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 246, (x) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 247, (xi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 248, (xii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 249, (xiii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 250, (xiv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 251, (xv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 252, (xvi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 253, (xvii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 254, (xviii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 255, (xix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 256, (xx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 257, (xxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 258, (xxii) A nucleic acid fragment comprising 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) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262, (xxvi) A 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) A 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) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 269, (xxxi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 270, and (xxxii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 271. [7] A genetically modified cell containing an endogenous gene to which a highly expressing promoter is operably linked, which is homologously recombined by a linear nucleic acid obtained by cleaving a plasmid in which the endogenous gene contains a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment contains a highly expressing promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a nucleotide sequence in which a stop codon is linked in order. [8] The genetically modified cell according to [7], wherein the genetically modified cell is a yeast, a bacterium, a fungus, an insect cell, an animal cell or a plant cell. [9] The genetically modified cell according to [8], wherein the yeast is a methanol-assimilating yeast, a fission yeast or a budding yeast.

[10] The genetically modified cell according to [9], wherein the methanol-assimilating yeast is a yeast belonging to the genus Komagataella or the genus Ogataea.

[11] The genetically modified cell according to any one of [7] to

[10] , wherein the endogenous gene is at least one endogenous gene selected from the group consisting of the following (1) to (32) endogenous genes. (1) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 46, (2) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 47, (3) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 48, (4) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 49, (5) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 50, (6) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 51, (7) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 52, (8) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 53, (9) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 54, (10) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 55, (11) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 56, (12) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 57, (13) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 58, (14) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 59, (15) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 60, (16) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 61, (17) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 62, (18) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 63, An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 64, (20) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 65, (21) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66, (22) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 67, (23) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 68, (24) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 69, (25) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 70, (26) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71, (27) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 73, (28) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 75, (29) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 76, (30) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 77, (31) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 78, and (32) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 79.

[12] The recombinant 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 comprising the nucleotide sequence represented by SEQ ID NO: 238, (ii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 239, (iii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 240; (iv) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 241; (v) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 242; (vi) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 243; (vii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 244; (viii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 245; (ix) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 246; (x) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 247; (xi) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 248; (xii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 249; (xiii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 250; (xiv) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 251; (xv) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 252; (xvi) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 253; (xvii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 254; (xviii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 255; (xix) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 256; (xx) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 257; (xxi) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 258; (xxii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 259; (xxiii) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 260; (xxiv) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 261; (xxv) a nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 262; (xxvi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 263, (xxvii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 265, (xxviii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 267, (xxix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 268, (xxx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 269, (xxxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 270, and (xxxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 271.

[13] The genetically modified cell according to any one of [7] to

[12] , which contains the nucleotide sequence encoding the target protein in the genome.

[14] The genetically modified cell according to

[13] , wherein the target protein is a heterologous protein.

[15] A method for producing a target protein, which includes the step of culturing the genetically modified cell according to

[13] or

[14] .

[16] An endogenous gene overexpression cell library containing the genetically modified 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 includes the following steps. (1) A step of cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme to prepare a linear nucleic acid, wherein the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and having a restriction enzyme recognition site inserted therein, and a nucleotide sequence in which a stop codon is ligated in order. (2) A step of introducing the linear nucleic acid into a host cell that contains the nucleotide sequence encoding the target protein in the genome. (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene is homologously recombined by the linear nucleic acid and a highly expressive promoter is functionally linked. (4) A step of culturing the cell obtained in step (3) and a host cell that contains the nucleotide sequence encoding the target protein in the genome. (5) Measuring the production amount of the target protein by host cells containing the cells obtained in step (3) and the nucleotide sequence encoding the target protein in the genome, respectively, and (6) Identifying an endogenous gene that increases the production amount of the target protein.

[18] A method for screening an endogenous gene that enhances the production of a target protein, comprising the following steps. (1) Introducing and culturing an expression vector containing the nucleotide sequence encoding the target protein into the endogenous gene overexpression cell library and host cells described in

[16] ; (2) Measuring the production amount of the target protein by the endogenous gene overexpression cell library and host cells; and (3) Identifying an endogenous gene that increases the production amount of the target protein.

Effect of the Invention

[0009] By providing a method for enhancing the expression of each endogenous gene, it has become possible to easily construct a recombinant cell library in which the expression of each endogenous gene is enhanced. In addition, by using this library, it has become possible to screen for endogenous genes that increase the production of the target protein.

Brief Description of the Drawings

[0010]

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[0011] 1. Method for producing genetically modified cells with enhanced expression of endogenous genes The present invention provides a method for producing a genetically modified cell in which expression of an endogenous gene is enhanced (hereinafter, the method for producing a genetically modified cell of the present invention).

[0012] In the method for producing a genetically modified cell of the present invention, a genetically modified cell refers to a cell into which a linear nucleic acid, as described below, has been introduced and into which expression of an endogenous gene has been enhanced by genetic recombination. In this specification, a genetically modified cell before introduction of the linear nucleic acid may also 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.

[0013] The species of the genetically modified cells and host cells (hereinafter referred to as the cells of the present invention) are not particularly limited, and examples include yeast, bacteria, fungi, insect cells, animal cells, and plant cells. Yeast is preferred, and methylotrophic yeast, fission yeast, and budding yeast are more preferred, with methylotrophic yeast being even more preferred. Generally, methylotrophic yeast is defined as yeast that can be cultured using methanol as the sole carbon source. However, yeast that was originally methylotrophic but has lost its methylotrophic performance due to artificial modification or mutation is also included in the methylotrophic yeast in the present invention.

[0014] Examples of methylotrophic yeast cells include yeasts belonging to the genus Pichia, the genus Ogataea, the genus Komagataella, etc. Yeasts belonging to the genus Komagataella or the genus Ogataea are preferred, and yeasts belonging to the genus Komagataella are particularly preferred.

[0015] In the genus Pichia, Pichia methanolica is preferred; in the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea parapolymorpha, Ogataea minuta are preferred; in the genus Komagataella, Komagataella pastoris, Komagataella phaffii are preferred examples. Among them, Komagataella phaffii is the most preferred. It should be noted that both Komagataella pastoris and Komagataella phaffii have the alias of Pichia pastoris.

[0016] Specific cell lines that can be used as the cells of the present invention include Komagataella pastoris NRBC0948 (CBS704, DSMZ 70382), Komagataella pastoris X-33, Komagataella phaffii CBS7435 (Y-11430), and the like. These cell lines can be obtained from the American Type Culture Collection, Thermo Fisher Scientific, and the like.

[0017] In addition, in the present invention, derivative strains from these Komagataella yeast strains can also be used. For example, Komagataella pastoris GS115 strain (available from Thermo Fisher Scientific) with histidine auxotrophy, and the like can be mentioned. Also, as another derivative strain, a non-homologous recombination mechanism disrupted strain (Δku70, Δdnl4) of Komagataella phaffii can also be used. In the present invention, derivative strains and the like from these strains can also be used.

[0018] In the method for producing a genetically modified 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 typically be DNA, and particularly can be genomic DNA. Further, the endogenous gene is not limited by whether it is a different functional region. For example, it may contain only exons, or it may contain exons and introns.

[0019] The endogenous gene is not particularly limited as long as it is a gene possessed by the cell of the present invention. For example, for the purpose of enhancing the production of the target protein described later, at least one endogenous gene selected from the group consisting of the following (1) to (32) endogenous genes is exemplified. (1) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 46 (EF1st-2). (2) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 47 (EF1st-3). (3) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 48 (EF1st-4). (4) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 49 (EF1st-5), (5) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 50 (EF1st-6), (6) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 51 (EF1st-7), (7) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 52 (EF1st-8), (8) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 53 (EF1st-9), (9) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 54 (EF1st-10), (10) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 55 (EF1st-11), (11) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 56 (EF1st-12), (12) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 57 (EF1st-13), (13) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 58 (EF1st-14), (14) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 59 (EF1st-15), (15) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 60 (EF1st-16), (16) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 61 (EF1st-17), (17) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 62 (EF1st-18), (18) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 63 (EF2nd-1); (19) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 64 (EF2nd-2); (20) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 65 (EF2nd-3), (21) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4), (22) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 67 (EF2nd-5), (23) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 68 (EF2nd-6), (24) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 69 (EF2nd-7); (25) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 70 (EF2nd-9), (26) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71 (EF3rd-1); (27) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 73 (EF3rd-3); (28) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 75 (EF3rd-5); (29) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 76 (EF3rd-6); (30) An endogenous gene containing the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 77 (EF3rd-7), (31) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 78 (EF3rd-8), and An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 79 (EF3rd-9).

[0020] At least one endogenous gene selected from the group consisting of the above endogenous genes (1) to (32) may preferably be at least two endogenous genes selected from the group consisting of the following endogenous genes (a) to (g). (a) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 47 (EF1st-3). (b) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 48 (EF1st-4). (c) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 63 (EF2nd-1). (d) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4). (e) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71 (EF3rd-1). (f) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 73 (EF3rd-3), and (g) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 75 (EF3rd-5).

[0021] In addition to at least one endogenous gene selected from the group consisting of the above endogenous genes (1) to (32), the endogenous gene may further contain the following endogenous genes. (33) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 45 (EF1st-1).

[0022] Examples of the endogenous gene include the following combinations. (A) An endogenous gene comprising a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 45 (EF1st-1). An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 48 (EF1st-4), an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 63 (EF2nd-1), and an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4). (B) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 45 (EF1st-1), an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4), an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71 (EF3rd-1), and an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 75 (EF3rd-5). (C) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 45 (EF1st-1), an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 47 (EF1st-3), an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4), and an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 71 (EF3rd-1). (D) An endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 66 (EF2nd-4), and an endogenous gene containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 73 (EF3rd-3).

[0023] Examples of a nucleotide sequence substantially identical to the nucleotide sequences represented by SEQ ID NOs: 45 to 71, 73, and 75 to 79 include nucleotide sequences having an identity of about 85% or more, preferably about 90% or more, and most preferably about 95% or more to the nucleotide sequences represented by SEQ ID NOs: 45 to 71, 73, and 75 to 79. Here, "identity" means the ratio (%) of identical nucleotide sequences to all overlapping nucleotide sequences in an optimal alignment (preferably, the algorithm can consider introduction of gaps into one or both of the sequences for the optimal alignment) when two nucleotide sequences are aligned using a mathematical algorithm known in the art. The identity of nucleotide sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expect value = 10; gaps are allowed; filtering = ON; match score = 1; mismatch score = -3).

[0024] In the method for producing a genetically modified cell of the present invention, the enhanced expression of an endogenous gene refers to a state in which the expression level of mRNA, which is a transcription product of the endogenous gene, or a polypeptide, which is a translation product, is enhanced. The expression level of mRNA can be quantified using methods such as the real-time PCR method, the RNA-Seq method, Northern hybridization, or hybridization methods using DNA arrays. The expression level of a polypeptide can be quantified using an antibody that recognizes the polypeptide, a staining compound that binds to the polypeptide, or the like. In addition to the quantification methods listed above, conventional methods used by those skilled in the art may also be used.

[0025] In the method for producing a genetically modified 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 below is enhanced. However, the expression level of the transcription product or translation product of the endogenous gene is preferably 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.

[0026] The method for producing a genetically modified cell of the present invention includes the following steps. (1) A step of cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme to prepare a linear nucleic acid, wherein the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and having a restriction enzyme recognition site inserted therein, and a base sequence in which a stop codon is sequentially linked. (2) A step of introducing the linear nucleic acid into a host cell, and (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene has been homologously recombined with the linear nucleic acid and a highly expressive promoter is functionally linked.

[0027] The method for producing a genetically modified cell of the present invention includes a step of cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme to prepare a linear nucleic acid (step (1)). Here, the nucleic acid fragment is a nucleic acid fragment containing a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and having a restriction enzyme recognition site inserted therein, and a base sequence in which a stop codon is sequentially linked.

[0028] In 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 appropriate for the cell 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 a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, or the like. When the cell of the present invention is a bacterium belonging to the genus Escherichia, the high-expression promoter of the present invention is preferably a trp promoter, a lac promoter, a recA promoter, a λP L promoter, an lpp promoter, a T7 promoter, etc. When the cell of the present invention is a bacterium belonging to the genus Bacillus, the high-expression promoter of the present invention is preferably an SPO1 promoter, an SPO2 promoter, a penP promoter, etc. When the cell of the present invention is a fungus, the high-expression promoter of the present invention is preferably an ADH promoter, a CMV (cytomegalovirus) promoter, etc. When the cell of the present invention is an insect cell, the high-expression promoter of the present invention is preferably a polyhedrin promoter, a P10 promoter, etc. When the cell of the present invention is an animal cell, the high-expression promoter of the present invention is preferably an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter, an RSV (Rous sarcoma virus) promoter, a MoMuLV (Moloney murine leukemia virus) LTR, an HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. When the cell of the present invention is a plant cell, the high-expression promoter of the present invention is preferably a CaMV (cauliflower mosaic virus) 35S promoter, etc.

[0029] 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, into which a restriction enzyme recognition site has been 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 publicly known databases. For example, when Komagataella phaffii is used as the cell of the present invention, the nucleotide sequence information starting from the start codon of all endogenous genes of Komagataella phaffii can be obtained from the nucleotide sequence information of the four chromosomal DNAs of Komagataella phaffii CBS7435 strain (ACCESSION No. FR839628~FR839631 (J. Biotechnol.154 (4), 312-320 (2011))). Further, when Komagataella pastoris is used as the cell of the present invention, the nucleotide sequence information starting from the start codon of all endogenous genes of Komagataella pastoris can be obtained from the nucleotide sequence information of the four chromosomal DNAs of Komagataella pastoris NBRC 0948 strain (Mattanovich et al., Microbial Cell Factories 8, 29 (2009)), and the nucleotide sequence information of the four chromosomal DNAs of Komagataella pastoris GS115 strain (ACCESSION No. FN392319~FN392322 (Nat. Biotechnol. 27 (6), 561-566 (2009))). Based on the nucleotide sequence information thus obtained, a partial sequence starting from the start codon of the endogenous gene can be designed.

[0030] The length of the partial sequence of the present invention is not particularly limited as long as homologous recombination occurs between the endogenous gene and the linear nucleic acid. For example, it is 20 bases or longer, 50 bases or longer, 100 bases or longer, 150 bases or longer. Further, the length of the partial sequence of the present invention is, for example, 1000 bases or shorter, 750 bases or shorter, 500 bases or shorter, or 250 bases or shorter.

[0031] In addition, the partial sequence of the present invention is a partial sequence into 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 that exists only in the partial sequence among the entire base sequences of the plasmid containing the above nucleic acid fragment. Examples of the restriction enzyme recognition site inserted into the partial sequence of the present invention include sites recognized by type IIS restriction enzymes such as BspQI, BbsI, BsaI, and BsmBI, where the recognition site and the cleavage site are different.

[0032] The above restriction enzyme recognition site may be inserted at any site within the partial sequence of the present invention, but is preferably inserted at a position where the partial sequence of the present invention is divided to such an extent that homologous recombination occurs between the endogenous gene. The insertion position of the restriction enzyme recognition site within 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 the endogenous gene and having a length of 183 bases, the restriction enzyme recognition site is inserted by substituting the 92nd base.

[0033] The number of the above restriction enzyme recognition sites inserted into the partial sequence of the present invention is not particularly limited as long as it exists only in the partial sequence among the entire base sequences of the plasmid containing the above nucleic acid fragment, but is usually 1 to several, preferably 1 or 2.

[0034] In step (1), the stop codon (hereinafter, the stop codon of the present invention) is any one of TAA, TAG, or TGA.

[0035] In step (1), the nucleic acid fragment (hereinafter, the nucleic acid fragment of the present invention) contains a nucleotide sequence in which the highly expressing promoter of the present invention, the partial sequence of the present invention, and the stop codon of the present invention are linked in this order. That is, in the nucleic acid fragment of the present invention, the carbon at the 3'-end of the highly expressing 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 carbon at the 3'-end of the partial sequence of the present invention and the carbon at the 5'-end of the stop codon of the present invention form a phosphodiester bond. Note that a spacer sequence may be inserted between the highly expressing 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 long.

[0036] In step (1), the nucleic acid fragment of the present invention can be directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method") using the genomic DNA fraction prepared from the cells of the present invention as a template and designing primers from the nucleotide sequence information of the highly expressing promoter and the endogenous gene described in a known database. 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-directed mutagenesis method. Alternatively, as in the examples described later, the nucleic acid fragment of the present invention can also be obtained by commissioning the production to Agilent Technologies, Inc.

[0037] The length of the nucleic acid fragment of the present invention is not particularly limited, and is, for example, 20 bases or longer, 50 bases or longer, 100 bases or longer, 150 bases or longer, or 200 bases or longer. In addition, the length of the partial sequence of the present invention is, for example, 1000 bases or shorter, 500 bases or shorter, or 400 bases or shorter.

[0038] When the endogenous gene is at least one endogenous gene selected from the group consisting of the above (1) to (32) endogenous genes, the nucleic acid fragment of the present invention is at least one nucleic acid fragment selected from the group consisting of the following (i) to (xxxii) nucleic acid fragments. (i) A 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) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 241 (EF1st-5 OLS), (v) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 242 (EF1st-6 OLS), (vi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 243 (EF1st-7 OLS), (vii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 244 (EF1st-8 OLS), (viii) A 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) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 247 (EF1st-11 OLS), (xi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 248 (EF1st-12 OLS), (xii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 249 (EF1st-13 OLS), (xiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 250 (EF1st-14 OLS), (xiv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 251 (EF1st-15 OLS), (xv) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 252 (EF1st-16 OLS), (xvi) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 253 (EF1st-17 OLS), (xvii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 254 (EF1st-18 OLS), (xviii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS), (xix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 256 (EF2nd-2 OLS), (xx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 257 (EF2nd-3 OLS), (xxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS), (xxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 259 (EF2nd-5 OLS), (xxiii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 260 (EF2nd-6 OLS), (xxiv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 261 (EF2nd-7 OLS), (xxv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 262 (EF2nd-9 OLS), (xxvi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS), (xxvii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS), (xxviii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 267 (EF3rd-5 OLS), (xxix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 268 (EF3rd-6 OLS), (xxx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 269 (EF3rd-7 OLS), (xxxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 270 (EF3rd-8 OLS), and (xxxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 271 (EF3rd-9 OLS).

[0039] At least one nucleic acid fragment selected from the group consisting of the nucleic acid fragments of (i) to (xxxii) above may preferably be at least two nucleic acid fragments selected from the group consisting of the following nucleic acid fragments (a’) to (g’). (a’) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS), (b’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), (c’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS), (d’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS), (e’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS), (f’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS), and (g’) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267 (EF3rd-5 OLS).

[0040] In addition to at least one nucleic acid fragment selected from the group consisting of the above nucleic acid fragments (i) to (xxxii), the nucleic acid fragment may further contain the following nucleic acid fragments. (xxxiii) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS).

[0041] Examples of the nucleic acid fragment include the following combinations. (A) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 240 (EF1st-4 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 255 (EF2nd-1 OLS), and A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS). (B) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS), and A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 267 (EF3rd-5 OLS). A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 237 (EF1st-1 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 239 (EF1st-3 OLS), A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS), and A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 263 (EF3rd-1 OLS). (D) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 258 (EF2nd-4 OLS), and A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 265 (EF3rd-3 OLS).

[0042] The nucleic acid fragments obtained as described above are contained in plasmids. In the present invention, a plasmid is an artificially constructed nucleic acid molecule. The nucleic acid molecule constituting the plasmid is usually DNA, preferably double-stranded DNA. Examples of plasmids include YEp vectors, YRp vectors, YCp vectors, pPICHOLI, pHIP (Journal of General Microbiology (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha), pHRP (see the above-mentioned literature cited for pHIP), pHARS (Molecular and General Genetics MGG February 1986, Volume 202, Issue 2, pp 302-308, Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors), Escherichia coli-derived plasmid vectors (pUC18, pUC19, pBR322, pBluescript, pQE), Bacillus subtilis-derived plasmid vectors (pHY300PLK, pMTLBS72), etc. can be used.

[0043] In addition to the nucleic acid fragment of the present invention, the above plasmid can further contain a cloning site containing one or more restriction enzyme recognition sites, an overlap region for using cloning systems such as Clontech's In-Fusion cloning system and New England Biolabs' Gibson Assembly system, the nucleotide sequence of a selectable marker gene (such as an auxotrophic complementation gene, a drug resistance gene), and the like. Examples of auxotrophic complementation genes include the URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, and the like. Examples of drug resistance genes include the G418 resistance gene, Zeocin (trademark) resistance gene, hygromycin resistance gene, Clone NAT resistance gene, blasticidin S resistance gene, nourseothricin resistance gene, and the like.

[0044] 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 prepare a linear nucleic acid (hereinafter, the linear nucleic acid of the present invention). By cleaving the plasmid containing the nucleic acid fragment of the present invention with a restriction enzyme, a linear nucleic acid can be prepared in which the latter half (homologous sequence 1) of the partial sequence of the present invention linked to a stop codon is arranged at the 5'-end and the former half (homologous sequence 2) of the partial sequence of the present invention linked to a highly expressive promoter is arranged at the 3'-end.

[0045] 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. The method for introducing the linear nucleic acid of the present invention into a host cell, i.e., the transformation method, can be any known method. For example, when yeast cells are used as the host cell, methods include, but are not limited to, electroporation, the lithium acetate method, and the spheroplast method. For example, a common method for transforming Komagataella phaphii is the electroporation method described in "High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol" (Biotechniques. 2004 Jan;36(1):152-4).

[0046] The method for producing a genetically modified cell of the present invention includes a step (step (3)) of selecting genetically modified cells containing an endogenous gene operably linked to a highly expressing promoter, the endogenous gene being homologously recombined with the linear nucleic acid of the present invention.

[0047] In step (3), the linear nucleic acid of the present invention introduced into the host cell undergoes homologous recombination (single crossover recombination) between the second half of the partial sequence of the present invention (homologous sequence 1) linked to the stop codon located at the 5' end and the first half of the partial sequence of the present invention (homologous sequence 2) linked to the high-expression promoter located at the 3' end, which serve as homologous regions to the endogenous gene. As a result of homologous recombination, the linear nucleic acid of the present invention is inserted between homologous sequences 1 and 2 contained in the endogenous gene, and the endogenous gene originally present in the host cell is rendered nonfunctional by the stop codon contained in the linear nucleic acid of the present invention. Instead, a new endogenous gene operably linked to the high-expression promoter contained in the linear nucleic acid of the present invention is generated downstream of the nonfunctional gene. As a result, in the genetically modified cell of the present invention, expression of the endogenous gene is enhanced by the high-expression promoter.

[0048] In step (3), when selecting genetically engineered cells containing an endogenous gene operably linked to a highly expressible promoter (hereinafter referred to as the genetically engineered cells of the present invention), it is preferable to use a selection marker gene such as an auxotrophy-complementing gene or a drug resistance gene. The selection marker is not particularly limited. However, if the host cell is a Komagataella yeast, auxotrophy-complementing genes such as the URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, or ARG4 gene can be used to select genetically engineered cells of the present invention by restoring the prototrophic phenotype in a strain auxotrophic for uracil, leucine, adenine, histidine, or arginine, respectively. Furthermore, drug resistance genes such as the G418 resistance gene, Zeocin™ resistance gene, hygromycin resistance gene, Clone NAT resistance gene, and Blasticidin S resistance gene can be used to select genetically engineered cells of the present invention by their resistance on media containing G418, Zeocin™, hygromycin, Clone NAT, or Blasticidin S, respectively. The auxotrophic selection marker used to prepare a genetically modified yeast cannot be used unless the selection marker is disrupted in the host yeast. In this case, the selection marker in the host yeast can be disrupted by any method known to those skilled in the art.

[0049] 2. Genetically engineered cells with enhanced expression of endogenous genes The present invention provides genetically modified cells in which expression of an endogenous gene is enhanced (hereinafter referred to as the genetically modified cells of the present invention).

[0050] The recombinant cell of the present invention can be prepared by the method for producing a recombinant cell of the present invention. Specifically, the recombinant cell of the present invention is a recombinant cell containing an endogenous gene homologously recombined with a linear nucleic acid obtained by cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme, and a highly expressive promoter is functionally linked to the endogenous gene. The nucleic acid fragment is a recombinant cell containing a nucleotide sequence in which a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and having a restriction enzyme recognition site inserted therein, and a stop codon are linked in sequence.

[0051] In the recombinant cell of the present invention, the recombinant cell, endogenous gene, nucleic acid fragment, plasmid, restriction enzyme, highly expressive promoter, partial sequence, etc. may be the same as those described in the method for producing a recombinant cell of the present invention.

[0052] The genetically engineered cell of the present invention may contain a nucleotide sequence encoding a target protein in its genome. In the present invention, the target protein is a protein produced by a cell containing a nucleotide sequence encoding the target protein in its genome, which may be an endogenous protein or a heterologous protein of the cell. Examples of the target protein include enzymes derived from microorganisms, proteins produced by animals and plants which are multicellular organisms, etc. For example, phytase, protein A, protein G, protein L, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, nuclease, oxidase, lactase, xylanase, trypsin, pectinase, isomerase, fibroin, and fluorescent protein, etc. may be mentioned, but are not limited thereto. Particularly, proteins for human and / or animal therapy are preferred. Specific examples of proteins for human and / or animal therapy include hepatitis B virus surface antigen, hirudin, antibody, human antibody, partial antibody, human partial antibody, serum albumin, human serum albumin, epidermal growth factor, human epidermal growth factor, insulin, growth hormone, erythropoietin, interferon, blood coagulation factor VIII, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin, IL-1, IL-6, tissue plasminogen activator (TPA), urokinase, leptin, and stem cell growth factor (SCF), etc.

[0053] Here, the antibody refers to a heterotetramer protein composed of two polypeptide chains each of L chain and H chain linked by disulfide bonds, and is not particularly limited as long as it has the ability to bind to a specific antigen.

[0054] Here, the partial antibody refers to Fab antibody, (Fab)2 antibody, scFv antibody, diabody antibody, camel VHH antibody, and derivatives thereof, etc. It is not particularly limited as long as it has the ability to bind to a specific antigen. The Fab antibody refers to a heteromeric protein in which the L chain and Fd chain of the antibody are bound by an S-S bond, or a heteromeric protein in which the L chain and Fd chain of the antibody associate without containing an S-S bond, and it is not particularly limited as long as it has the ability to bind to a specific antigen.

[0055] The amino acids constituting the above-mentioned target protein may be natural, non-natural, or modified. Also, the amino acid sequence of the protein may be artificially modified or de-novo designed.

[0056] The base sequence encoding the above-mentioned target protein is contained in the expression vector and integrated into an arbitrary site of the genome of the genetically modified cell of the present invention by homologous recombination. The expression vector can be produced, for example, by excising a DNA fragment containing the base sequence encoding the above-mentioned target protein and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. As the expression vector, plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194); plasmids derived from yeast (e.g., pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animal cell expression plasmids (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; insect virus vectors such as baculovirus (e.g., BmNPV, AcNPV); animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. are used. As the promoter, any promoter appropriate for the host used for gene expression may be used, and for example, the high-expression promoter of the present invention is preferably mentioned.

[0057] As expression vectors, in addition to the above, those containing enhancers, splicing signals, polyA addition signals, selection markers, SV40 replication origins, etc. as desired can also be used. Examples of selection markers include the dihydrofolate reductase (dhfr) gene, ampicillin resistance gene, neomycin resistance gene, etc. In particular, when using dhfr gene-deficient Chinese hamster cells and using the dhfr gene as a selection marker, the target gene can also be selected using a medium that does not contain thymidine. Also, if necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host cell may be added to the 5'-terminal side of the base sequence encoding the target protein (or substituted with the native signal codon). For example, when the host cell is a bacterium of the genus Escherichia, the PhoA signal sequence, OmpA signal sequence, etc. are used; when the host cell is a bacterium of the genus Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. are used; when the host cell is yeast, the MFα signal sequence, SUC2 signal sequence, etc. are used; when the host cell is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. are used respectively. Also, the expression vector contains a partial sequence of the genome of the recombinant cell of the present invention, which includes a partial sequence into which a restriction enzyme recognition site is inserted. The linear expression vector cleaved by a restriction enzyme is introduced into the recombinant cell of the present invention, and the latter half (homologous sequence 1) of the partial sequence arranged at the 5'-end and the first half (homologous sequence 2) of the partial sequence arranged at the 3'-end are used as homologous regions to the partial sequence of the genome of the recombinant cell of the present invention, and homologous recombination (single crossover recombination) occurs. 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.

[0058] There is provided a method for producing a target protein (hereinafter, the method for producing the target protein of the present invention) including the step of culturing the recombinant cell of the present invention containing the base sequence encoding the target protein in the genome.

[0059] The cell culture conditions are not particularly limited and may be appropriately selected according to the cells. In this culture, any medium containing a nutrient source that the cells can assimilate can be used. As the nutrient source, saccharides such as glucose, sucrose, 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, oils such as soybean oil and rapeseed oil, or a carbon source such as a mixture thereof, ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, etc. A nitrogen source, and furthermore, a normal medium appropriately mixed with other inorganic salts, nutrient sources such as vitamins, etc. can be used. Also, the culture can be either batch culture or continuous culture.

[0060] As a preferred embodiment of the present invention, when Komagataella yeast or Ogataea yeast is used for yeast, the carbon source may be one kind of glucose, glycerol, or methanol, or two or more kinds thereof. Also, these carbon sources may be present from the initial stage of the culture or may be added during the culture.

[0061] By culturing the gene 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 cells or in the culture solution. Regarding the method for recovering the target protein, known purification methods can be appropriately combined and used. For example, first, the gene recombinant cell of the present invention containing the base sequence encoding the target protein in the genome is cultured in an appropriate medium, and the cells are removed from the culture supernatant by centrifugation of the culture solution or by filtration treatment. The obtained culture supernatant is subjected to salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation method using acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, ultrafiltration, etc. The target protein is recovered from the culture supernatant by using these methods alone or in combination.

[0062] Cell culture can usually be carried out under general conditions. For example, in the case of yeast, it can be carried out by aerobic culture for 10 hours to 10 days within the range of pH 2.5 to 10.0 and temperature range of 10°C to 48°C.

[0063] The recovered target protein can be used as it is, or it can also be used after being modified to bring about pharmacological changes such as PEGylation, or modified to add functions such as enzymes or isotopes. In addition, various formulation treatments may be used.

[0064] 4. Provided are a cell library in which the expression of each endogenous gene is enhanced (hereinafter, the endogenous gene overexpression cell library of the present invention) containing the gene recombinant cell of the present invention, and a method for screening an endogenous gene that enhances the production of a target protein using the endogenous gene overexpression cell library of the present invention (hereinafter, screening methods 1 and 2 of the present invention).

[0065] In the gene recombinant cell of the present invention, the endogenous gene originally possessed by 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 gene that has lost its function, a new gene (the same gene as the endogenous gene) that is functionally linked to the high-expression promoter contained in the linear nucleic acid of the present invention is generated. As a result, in the gene recombinant cell of the present invention, the expression of the endogenous gene is enhanced by the high-expression promoter. Therefore, the population of gene recombinant cells of the present invention in which the expression of each gene of all the 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.

[0066] 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 contains a high-expression promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a base sequence in which a stop codon is linked in sequence. (2) A step of introducing the linear nucleic acid into a host cell containing a base sequence encoding the target protein in its genome. (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene has been homologously recombined by the linear nucleic acid and a highly expressive promoter is operably linked. (4) A step of culturing the cells obtained in step (3) and a host cell containing a base sequence encoding the target protein in its genome. (5) A step of measuring the production amounts of the target protein by the cells obtained in step (3) and a host cell containing a base sequence encoding the target protein in its genome, respectively, and (6) A step of identifying an endogenous gene that increases the production amount of the target protein.

[0067] The screening method 1 of the present invention includes a step of cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme to prepare a linear nucleic acid, wherein the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a base sequence in which a stop codon is linked in order (step (1)). Step (1) in the screening method 1 of the present invention may be the same as step (1) in the method for producing a genetically modified cell of the present invention.

[0068] The screening method 1 of the present invention includes a step of introducing the linear nucleic acid of the present invention into a host cell (hereinafter, the target protein-expressing cell of the present invention) containing a base sequence encoding the target protein in its genome (step (2)). The method of 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 genetically modified cell of the present invention. The target protein may be the same as the target protein contained in the genome of the genetically modified cell of the present invention. Also, the host cell may be the same as the host cell used in the method for producing a genetically modified cell of the present invention.

[0069] The screening method 1 of the present invention includes a step (step (3)) of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene has been homologously recombined with the linear nucleic acid of the present invention and a highly expressive promoter is operably linked. Step (3) in the screening method 1 of the present invention may be the same as step (3) in the method for producing a genetically modified cell of the present invention.

[0070] The screening method 1 of the present invention includes a step (step (4)) of culturing the cells obtained in 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 a genetically modified cell of the present invention.

[0071] The screening method 1 of the present invention includes a step (step (5)) of measuring the production amounts of the target protein by the cells obtained in step (3) and the target protein-expressing cells, respectively.

[0072] The measurement of the production amount of the target protein by the cells obtained in step (3) and the target protein-expressing cells of the present invention can be performed by measuring the expression level of mRNA, which is a transcription product of the target protein, or a polypeptide, which is a translation product. The expression level of mRNA can be quantified using methods such as the real-time PCR method, the RNA-Seq method, Northern hybridization, or hybridization methods using DNA arrays, and the expression level of the polypeptide can be quantified using antibodies that recognize the polypeptide, staining compounds that bind to the polypeptide, etc. In addition to the quantification methods listed above, conventional methods used by those skilled in the art may also be used.

[0073] 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.

[0074] 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, it can be determined that the cells contain an endogenous gene that increases the production amount of the target protein. The increase in the production amount of the target protein in the cells obtained in step (3) may be, for example, 1.01-fold, 1.02-fold, 1.03-fold, 1.04-fold, 1.05-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more, or 100-fold, 90-fold, 80-fold, 70-fold, 60-fold, 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, or 5-fold or less, compared to the production amount of the target protein in the target protein-expressing cells of the present invention. When the target protein is secreted and produced, the total protein secretion production amount from the cells can be easily determined by methods known to those skilled in the art, such as the Bladford method, Lowry method, and BCA method, using the cell culture supernatant or the like. The secretion production amount of a specific target protein from the cells can be easily determined by methods such as the ELISA method, using the cell culture supernatant or the like.

[0075] After identifying the gene recombinant cells that increase the expression of the target protein from the cells obtained in step (3) as described above, the endogenous gene whose expression is enhanced can be identified by known means. For example, the genome is extracted from the cells, fragmented with restriction enzymes, and then the fragmented genome is self-ligated. The genomic fragment containing the target endogenous gene circularized by self-ligation can be screened with a drug resistance gene. The endogenous gene can be specifically identified by determining the nucleotide sequence of the screened genomic fragment containing the target endogenous gene by the Sanger method.

[0076] In another embodiment, Screening Method 2 of the present invention includes the following steps. (1) A step of introducing an expression vector containing a nucleotide sequence encoding a target protein into the endogenous gene overexpression cell library and host cells of the present invention and culturing them. (2) Measuring the production amount of the target protein by the endogenous gene overexpression cell library and the host cell, and (3) Identifying the endogenous gene that increases the production amount of the target protein.

[0077] The screening method 2 of the present invention includes the step of introducing an expression vector containing a nucleotide sequence encoding the target protein into the endogenous gene overexpression cell library and the host cell of the present invention and culturing (step (1)). The method of introducing an expression vector containing a nucleotide sequence encoding the target protein into the endogenous gene overexpression cell library and the host cell of the present invention and culturing may be the same as those described in the method of introducing the linear nucleic acid of the present invention into the host cell and the method of culturing the genetically modified cell of the present invention.

[0078] The screening method 2 of the present invention includes the step of measuring the production amount of the target protein by the endogenous gene overexpression cell library and the host cell (step (2)). The method for measuring the production amount of the target protein by the endogenous gene overexpression 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.

[0079] The screening method 2 of the present invention includes the step of identifying the endogenous gene that increases the production amount of the target protein (step (3)). The method for identifying the endogenous gene that increases the production amount 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.

Examples

[0080] The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

[0081] Materials and Methods (1) Preparation of various genes used for vector preparation Detailed operation methods and the like regarding the recombinant DNA technology used in the following examples are described in the following books: Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience). Also, in the following examples, the plasmid used for yeast transformation was prepared by introducing the constructed vector into Escherichia coli DH5α competent cells (manufactured by Takara Bio Inc.) and culturing and amplifying the obtained transformant. The plasmid was prepared from the plasmid-bearing strain using Wizard (registered trademark) Plus SV Minipreps DNA Purification Systems (manufactured by Promega). The 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 its downstream sequence (SEQ ID NO: 6), GUT1 gene (SEQ ID NO: 7), the gene encoding KAR2 derived from Komagataella phaffii with a terminator linked thereto (SEQ ID NO: 8), and the gene encoding PDI1 (SEQ ID NO: 9) used in the construction of the vector were prepared by PCR using a mixture of chromosomal DNA of Komagataella phaffii CBS7435 strain (the nucleotide sequence is described in EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) as a template. The genes (SEQ ID NOs: 45 to 79) encoding the antibody expression promoting protein group derived from Komagataella phaffii with a terminator linked thereto (amino acid sequences represented by SEQ ID NOs: 10 to 44) were prepared by PCR using the genome of Komagataella phaffii obtained by screening as a template.The GAP promoter was prepared by PCR using primer 1 (SEQ ID NO: 80) and primer 2 (SEQ ID NO: 81), the AOX1 promoter was prepared by PCR using primer 3 (SEQ ID NO: 82) and primer 4 (SEQ ID NO: 83), the AOX1 terminator was prepared by PCR using primer 5 (SEQ ID NO: 84) and primer 6 (SEQ ID NO: 85), the CCA38473 terminator was prepared by PCR using primer 7 (SEQ ID NO: 86) and primer 8 (SEQ ID NO: 87), the ARG4 gene controlled by the promoter was prepared by PCR using primer 9 (SEQ ID NO: 88) and primer 10 (SEQ ID NO: 89), the URA3 gene lacking the start codon and having an AscI-PmeI recognition site added therein was prepared by PCR using primer 11 (SEQ ID NO: 90) and mutagenesis primer 12 (SEQ ID NO: 91), mutagenesis primer 13 (SEQ ID NO: 92) and primer 14 (SEQ ID NO: 93), the GUT1 gene lacking the start codon and having an AscI-PmeI recognition site added therein was prepared by PCR using primer 15 (SEQ ID NO: 94) and mutagenesis primer 16 (SEQ ID NO: 95), mutagenesis primer 17 (SEQ ID NO: 96) and primer 18 (SEQ ID NO: 97), and the gene encoding the antibody expression promoting protein (amino acid sequence shown by SEQ ID NOs: 10 to 44) linked with the terminator was prepared by PCR using forward primer 19 (SEQ ID NO: 98) and respective reverse primers 20 to 55 (SEQ ID NOs: 99 to 134). In the construction of the vector, the secreted signal MFα gene (SEQ ID NO: 135) used was prepared by PCR using primer 56 (SEQ ID NO: 136) and primer 57 (SEQ ID NO: 137) with a chromosomal DNA mixture of Saccharomyces cerevisiae BY4741 strain (the nucleotide sequence is described in ACCESSION No. BK006934 to BK006949) as a template. Also, the two amino acid substitution mutant (L42S / V50A) (SEQ ID NO: 138) of the secreted signal MFα gene was prepared by PCR using synthetic DNA as a template and primer 56 (SEQ ID NO: 136) and primer 57 (SEQ ID NO: 137). The Zeocin (trademark) resistance gene (SEQ ID NO: 139) controlled by a promoter, which was used in the construction of the vector, was prepared by PCR using synthetic DNA as a template. The G418 resistance gene (SEQ ID NO: 140) controlled by a promoter, which was used in the construction of the vector, was prepared by PCR using synthetic DNA as a template. The hygromycin resistance gene (SEQ ID NO: 141) controlled by a promoter, which was used in the construction of the vector, was prepared by PCR using synthetic DNA as a template. The nourseothricin resistance gene (SEQ ID NO: 142) controlled by a promoter, which was used in the construction of the vector, was prepared by PCR using synthetic DNA as a template. The blasticidin resistance gene (SEQ ID NO: 143) controlled by a promoter, which was used in the construction of the vector, was prepared by PCR using synthetic DNA as a template. The anti-lysozyme 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), which were used in the construction of the vector, were prepared by PCR using synthetic DNA as a template. Also, the KAR2 gene (SEQ ID NO: 8) and PDI1 gene (SEQ ID NO: 9), which were used in the construction of the vector, were prepared by PCR using the genome of Komagataella phaffii CBS7435 strain as a template. For PCR, PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) etc. were used, and the reaction conditions were carried out according to the method described in the attached manual. The preparation of chromosomal DNA was carried out using the Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka) etc. from Komagataella pastoris ATCC76273 strain or Saccharomyces cerevisiae BY4741 strain under the conditions described therein.

[0082] (2) Construction of the 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 fully synthesized and inserted between the HindIII-EcoRI sites of pUC19 (manufactured by Takara Bio Inc., Code No. 3219) to prepare pUC-1. A nucleic acid fragment with EcoRI recognition sequences added to both ends of a G418 resistance gene (SEQ ID NO: 140) controlled by a promoter was prepared by PCR using primer 58 (SEQ ID NO: 149) and primer 59 (SEQ ID NO: 150), and inserted into the EcoRI site of pUC-1 after EcoRI treatment to construct pUC_G418. Next, a nucleic acid fragment of the CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using primer 7 (SEQ ID NO: 86) and primer 8 (SEQ ID NO: 87), mixed with the nucleic acid fragment obtained by treating the above pUC_G418 with XbaI, and ligated using the In-fusion HD Cloning Kit (manufactured by Clontech) to construct pUC_T38473_G418. Next, a nucleic acid fragment with 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), treated with BamHI and SpeI, and inserted between the BamHI-BglII sites of pUC_T38473_G418 to construct pUC_Paox1_T38473_G418. Next, a nucleic acid fragment with an MluI recognition sequence and a 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), and inserted between the MluI-BglII sites of pUC_Paox1_T38473_G418 after MluI and BglII treatment to construct pUC_Paox1_Taox1_T38473_G418. Next, nucleic acid fragments with SpeI recognition sequences and BglII recognition sequences added to the ends of the secretion signal MFα gene (SEQ ID NO: 135) and its two-amino acid substitution mutant (L42S / V50A) (SEQ ID NO: 138) were prepared by PCR using 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)_Taox1_T38473_G418.

[0083] (3) Construction of anti-lysozyme single-chain antibody expression vector The anti-lysozyme single-chain antibody gene (SEQ ID NO: 144) was prepared by PCR using primer 60 (SEQ ID NO: 151) and primer 61 (SEQ ID NO: 152) with synthetic DNA as a template. This nucleic acid fragment has the downstream terminal region of the secretion signal MFα gene sequence added as an overlap region upstream of the base sequence encoding the anti-lysozyme single-chain antibody, and the upstream terminal region of the AOX1 terminator sequence added as an overlap region downstream. Also, a base sequence encoding a His tag (SEQ ID NO: 153) is added between the base sequence encoding the anti-lysozyme antibody and the upstream terminal region of the AOX1 terminator sequence. After treating pUC_Pgap_MFα_Taox1_T38473_G418 constructed in (2) above with XhoI and MluI, a nucleic acid fragment was prepared, mixed with the nucleic acid fragment of the base sequence encoding the anti-lysozyme single-chain antibody prepared by the above PCR, and ligated using the In-fusion HD Cloning Kit to construct pUC_Paox1_MFα_scFv_Taox1_T38473_G418. This vector is designed such that the anti-lysozyme single-chain antibody is expressed under the control of the AOX1 promoter.

[0084] (4) Construction of bispecific antibody (tandem scFv226) expression vector The gene of the bispecific antibody (tandem scFv226; taFv226) (SEQ ID NO: 145) in which an anti-CD3 single-chain antibody and an anti-EGFR single-chain antibody are fused was prepared by PCR using primer 62 (SEQ ID NO: 154) and primer 63 (SEQ ID NO: 155) with synthetic DNA as a template. This nucleic acid fragment has, as an overlap region upstream of the nucleotide sequence encoding the taFv226 antibody, the downstream terminal region of the secretion signal MFα gene sequence (a 2-amino acid substitution mutant (L42S / V50A)), and, as an overlap region downstream, the upstream terminal region of the AOX1 terminator sequence added thereto. Further, between the nucleotide sequence encoding the taFv226 antibody and the upstream terminal region of the AOX1 terminator sequence, a nucleotide sequence encoding a c-Myc tag (SEQ ID NO: 156) and a His tag (SEQ ID NO: 153) is added. After treating pUC_Paox1_MFα(mut)_Taox1_T38473_G418 constructed in (2) above with XhoI and MluI, a nucleic acid fragment was prepared, mixed with the nucleic acid fragment of the nucleotide sequence encoding the bispecific antibody (taFv226) prepared by the above PCR, and ligated using an In-fusion HD Cloning Kit to construct pUC_Paox1_MFα(mut)_taFv226_Taox1_T38473_G418. Next, a nucleic acid fragment of the CCA38473 terminator (SEQ ID NO: 4) was prepared by PCR using primer 64 (SEQ ID NO: 157) and primer 65 (SEQ ID NO: 158), mixed with the nucleic acid fragment obtained by treating the above pUC_Paox1_MFα(mut)_taFv226_Taox1_T38473_G418 with XbaI, and ligated 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 such that the bispecific antibody (taFv226) is expressed under the control of the AOX1 promoter.

[0085] (5) Construction of the blinatumomab expression vector The Blinatumomab gene (SEQ ID NO: 146), which is the only bispecific small molecule antibody currently on the market, was prepared by PCR using primers 66 (SEQ ID NO: 159) and 67 (SEQ ID NO: 160) with synthetic DNA as a template. This nucleic acid fragment has, as an overlapping region upstream of the nucleotide sequence encoding the Blinatumomab antibody, the downstream terminal region of the secretion signal MFα gene sequence (a 2 - amino acid substitution variant (L42S / V50A)), and, as an overlapping region downstream, the upstream terminal region of the AOX1 terminator sequence. Also, 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. After treating pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above with XhoI and MluI, a nucleic acid fragment was prepared, mixed with the nucleic acid fragment of the nucleotide sequence encoding the Blinatumomab antibody prepared by the above PCR, and ligated using the In - fusion HD Cloning Kit to construct pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418. This vector is designed such that the Blinatumomab antibody is expressed under the control of the AOX1 promoter.

[0086] (6) Construction of the minibody antibody expression vector The minibody gene (SEQ ID NO: 147), which is a small molecule antibody formed by fusing the anti - EGFR single - chain antibody gene and a part of the Fc region, was prepared by PCR using primers 68 (SEQ ID NO: 161) and 69 (SEQ ID NO: 162) with synthetic DNA as a template. This nucleic acid fragment has, as an overlapping region upstream of the nucleotide sequence encoding the minibody antibody, the downstream terminal region of the secretion signal MFα gene sequence (a 2-amino acid substitution variant (L42S / V50A)), and, as an overlapping region downstream, the upstream terminal region of the AOX1 terminator sequence added thereto. Also, 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. The nucleic acid fragment was prepared by treating pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above with XhoI and MluI, mixed with the nucleic acid fragment of the nucleotide sequence encoding the minibody antibody prepared by the above PCR, and ligated using the In-fusion HD Cloning Kit to construct pUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418. This vector is designed such that the minibody antibody is expressed under the control of the AOX1 promoter.

[0087] (7) Construction of a vector for producing a gene overexpression cell library To delete the region between the XbaI and BspQI sites of plasmid pUC19, after treatment with XbaI and BspQI, the ends were blunted by treatment with the Klenow fragment (manufactured by Takara Bio Inc.), and then self-ligation was performed using a ligation kit (DNA Ligation kit <Mighty Mix>) to prepare pUC_del. Next, a nucleic acid fragment with EcoRI recognition sequences added to both ends of the Zeocin (trademark) resistance gene (SEQ ID NO: 139) controlled by a promoter was prepared by PCR using primer 70 (SEQ ID NO: 163) and primer 71 (SEQ ID NO: 164), and this was inserted between the EcoRI sites of pUC_del using the In-fusion HD Cloning Kit to construct pUC_del_Zeo. 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), two nucleic acid fragments corresponding to the first half and the second half of the GAPDH promoter were prepared by PCR using primer 72 (SEQ ID NO: 165) and mutagenesis primer 73 (SEQ ID NO: 166), mutagenesis primer 74 (SEQ ID NO: 167) and primer 75 (SEQ ID NO: 168), respectively. After treating pPGP_EGFP with BamHI and SpeI, a nucleic acid fragment was prepared, mixed with the two nucleic acid fragments prepared by the above PCR, and ligated using the In-fusion HD Cloning Kit to prepare pPGPdel_EGFP. Next, to prepare a GAPDH promoter and an EGFP gene fragment with the BspQI site disrupted, a nucleic acid fragment was prepared by PCR using pPGPdel_EGFP as a template and primers 76 (SEQ ID NO: 169) and 77 (SEQ ID NO: 170). Also, to prepare a nucleic acid fragment of the CYC1 terminator (SEQ ID NO: 171) derived from Saccharomyces cerevisiae, it was prepared by PCR using the Saccharomyces cerevisiae BY4741 strain as a template and primers 78 (SEQ ID NO: 172) and 79 (SEQ ID NO: 173). After treating pUC_del_Zeo with SacI and BamHI, a nucleic acid fragment was prepared, mixed with the two nucleic acid fragments prepared by the above PCR, and ligated using the In-fusion HD Cloning Kit to prepare pUC_del_Zeo_Pgap-EGFP-CYC1t. This vector is designed to be a vector for constructing a gene overexpression cell library using Komagataella phaffii by replacing the EGFP portion sandwiched between SpeI and XhoI with the following OLS sequence designed from the genomic sequence information of Komagataella phaffii.

[0088] (8) Construction of KAR2 and PDI1 gene overexpression vectors A nucleic acid fragment with EcoRI recognition sequences added to both ends of the Zeocin (trademark) resistance gene (SEQ ID NO: 139) controlled by a promoter was prepared by PCR using primer 70 (SEQ ID NO: 163) and primer 71 (SEQ ID NO: 164), and this was inserted between the EcoRI sites of pUC19 to construct pUC_Zeo. Next, nucleic acid fragments of the first half and the second half of the ARG4 gene (SEQ ID NO: 5) controlled by a promoter were prepared by PCR using primer 9 (SEQ ID NO: 88) and primer 80 for mutation insertion (SEQ ID NO: 174), primer 81 (SEQ ID NO: 175) and primer 10 (SEQ ID NO: 89), mixed with pUC_Zeo treated with HindIII and PstI, and ligated using the In-fusion HD Cloning Kit to construct pUC_Arg4_Zeo having a sequence in which an AscI-FseI-PmeI restriction enzyme site (SEQ ID NO: 176) was introduced into the center of the Arg4 gene. Next, a nucleic acid fragment with a HindIII recognition sequence added to one end of the EGFP expression cassette (GAPDH promoter - EGFP gene - AOX1t) was prepared by PCR using primer 82 (SEQ ID NO: 177) and 83 (SEQ ID NO: 178) with pPGP_EGFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) as a template, mixed with pUC_Arg4_Zeo treated with BamHI, and ligated using the In-fusion HD Cloning Kit to construct pUC_Arg4_Pgap_EGFP_Taox1_Zeo. Next, a nucleic acid fragment with SpeI recognition sequences and HindIII recognition sequences added to both ends of the genes encoding the KAR2 and PDI1 proteins (SEQ ID NOs: 179 and 180, respectively) linked to a terminator was prepared by PCR using primer 84 (SEQ ID NO: 181) and primer 85 (SEQ ID NO: 182), primer 86 (SEQ ID NO: 183) and primer 87 (SEQ ID NO: 184). It was mixed with pUC_Arg4_Pgap_EGFP_Taox1_Zeo treated with SpeI and HindIII, and ligated using the In-fusion HD Cloning Kit to construct pUC_Arg4_Pgap_KAR2_T37552_Zeo and pUC_Arg4_Pgap_PDI1_T37552_Zeo, respectively. This vector is designed such that KAR2 or PDI1 as a model protein is overexpressed under the control of the GAPDH promoter.

[0089] (9) Construction of the antibody production promoting protein overexpression vector 1 Nucleic acid fragments with SpeI recognition sequences and HindIII recognition sequences added to both ends of the genes encoding the antibody production promoting protein group (amino acid sequences represented by SEQ ID NOs: 10 to 44) linked to a terminator (SEQ ID NOs: 45 to 79) were prepared by PCR using primer 19 (SEQ ID NO: 98) and primers 20 to 55 (SEQ ID NOs: 99 to 134), respectively. They were mixed with pUC_Arg4_Pgap_EGFP_Taox1_Zeo treated with SpeI and HindIII, and ligated using the In-fusion HD Cloning Kit to construct 36 types from pUC_Arg4_Pgap_EF1st-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo. This vector is designed such that the antibody production promoting proteins (amino acid sequences represented by SEQ ID NOs: 10 to 44) are each expressed under the control of the GAPDH promoter.

[0090] (10) Construction of the antibody production promoting protein overexpression vector 2 A nucleic acid fragment with EcoRI recognition sequences added to both ends of the hygromycin resistance gene (SEQ ID NO: 141) controlled by a promoter was prepared by PCR using primer 88 (SEQ ID NO: 185) and primer 89 (SEQ ID NO: 186), and this was inserted between the EcoRI sites of pUC_Arg4_Pgap_EGFP_Taox1_Zeo to construct pUC_Arg4_Pgap_EGFP_Taox1_Hyg. Next, nucleic acid fragments of the first half and the second half of the URA3 gene (SEQ ID NO: 6) with the start codon deleted were prepared by PCR using primer 90 (SEQ ID NO: 187) and mutagenesis primer 91 (SEQ ID NO: 188), mutagenesis primer 92 (SEQ ID NO: 189) and primer 93 (SEQ ID NO: 190) respectively, mixed with pUC_Arg4_Pgap_EGFP_Taox1_Hyg treated with NheI and PstI, and ligated using the In-fusion HD Cloning Kit to construct pUC_URA3_Pgap_EGFP_Taox1_Hyg having a sequence with an AscI-PmeI restriction enzyme site (SEQ ID NO: 191) introduced into the center of the URA3 gene. Next, a nucleic acid fragment with SpeI recognition sequences and HindIII recognition sequences added to both ends of the gene (SEQ ID NOs: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) ligated with a terminator was prepared by PCR using forward primer 19 (SEQ ID NO: 98) and respective reverse primers 20 to 27 (SEQ ID NOs: 99 to 106), mixed with pUC_URA3_Pgap_EGFP_Taox1_Hyg treated with SpeI and HindIII, and ligated using the In-fusion HD Cloning Kit to construct 8 kinds from pUC_URA3_Pgap_EF1st-1_Hyg to pUC_URA3_Pgap_EF2nd-4_Hyg. This vector is designed such that the antibody production promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) is expressed under the control of the GAPDH promoter.

[0091] (11) Construction of the antibody production promoting protein overexpression vector 3 A nucleic acid fragment with EcoRI recognition sequences added to both ends of the nourseothricin resistance gene (SEQ ID NO: 142) controlled by a promoter was prepared by PCR using primer 88 (SEQ ID NO: 185) and primer 94 (SEQ ID NO: 192), and this was inserted between the EcoRI sites of pUC_Arg4_Pgap_EGFP_Taox1_Zeo to construct pUC_Arg4_Pgap_EGFP_Taox1_NAT. Next, nucleic acid fragments of the first half and the second half of the GUT1 gene (SEQ ID NO: 7) with the start codon deleted were prepared by PCR using primer 95 (SEQ ID NO: 193) and mutagenesis primer 96 (SEQ ID NO: 194), and mutagenesis primer 97 (SEQ ID NO: 195) and primer 98 (SEQ ID NO: 196), respectively. They were mixed with pUC_Arg4_Pgap_EGFP_Taox1_NAT treated with NheI and PstI, and ligated using the In-fusion HD Cloning Kit to construct pUC_Gut1_Pgap_EGFP_Taox1_NAT having a sequence with an AscI-PmeI restriction enzyme site (SEQ ID NO: 191) introduced into the center of the Gut1 gene and its downstream sequence (0.7 Kb). Next, a nucleic acid fragment with SpeI recognition sequences and HindIII recognition sequences added to both ends of the gene (SEQ ID NOs: 45 to 52) encoding the antibody production promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) ligated with a terminator was prepared by PCR using forward primer 19 (SEQ ID NO: 98) and respective reverse primers 20 to 27 (SEQ ID NOs: 99 to 106). It was mixed with pUC_Gut1_Pgap_EGFP_Taox1_NAT treated with SpeI and HindIII, and ligated using the In-fusion HD Cloning Kit to construct 8 kinds from pUC_GUT1_Pgap_EF1st-1_NAT to pUC_GUT1_Pgap_EF2nd-4_NAT. This vector is designed such that the antibody production promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) is expressed under the control of the GAPDH promoter.

[0092] (12) Construction of an overexpression vector for antibody production-promoting protein 4 A nucleic acid fragment with EcoRI recognition sequences added to both ends of a blastocidin resistance gene (SEQ ID NO: 143) controlled by a promoter was prepared by PCR using primer 88 (SEQ ID NO: 185) and primer 99 (SEQ ID NO: 197), and this was 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 pUC_Arg4_Pgap_EGFP_Taox1_bsd treated with NheI and PstI, and ligated using the In-fusion HD Cloning Kit to construct pUC_Paox1_Pgap_EGFP_Taox1_bsd having an AOX1 promoter sequence as a genomic integration site. Next, a nucleic acid fragment with SpeI recognition sequence and HindIII recognition sequence added to both ends of a gene (SEQ ID NOs: 45 to 52) encoding an antibody production-promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) linked to a terminator was prepared by PCR using forward primer 19 (SEQ ID NO: 98) and respective reverse primers 20 to 27 (SEQ ID NOs: 99 to 106), mixed with pUC_Paox1_Pgap_EGFP_Taox1_bsd treated with SpeI and HindIII, and ligated using the In-fusion HD Cloning Kit to construct 8 kinds from pUC_Paox1_Pgap_EF1st-1_bsd to pUC_Paox1_Pgap_EF2nd-4_bsd. This vector is designed such that an antibody production-promoting protein (8 kinds of amino acid sequences represented by SEQ ID NOs: 10 to 17) is expressed under the control of the GAPDH promoter.

[0093] (13) Obtaining of transformed yeast Using the anti-lysozyme single-chain antibody expression vector pUC_Paox1_MFα_scFv_Taox1_T38473_G418 constructed in (3) above, the tandem scFv226 antibody expression vector pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above, the blinatumomab antibody expression vector pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418 constructed in (5) above, and the minibody antibody vector pUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above, Komagataella phaffii was transformed as follows. Komagataella phaffii Dnl4-deficient and histidine-requiring strain (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was cultured with shaking at 30 °C for 16 hours in 2 ml of YPD medium (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose), then subcultured by 10-fold dilution in fresh YPD medium and further cultured with shaking at 30 °C for 4 hours. After culturing, the yeast cells were collected by centrifugation, washed (suspended by adding 6 ml of sterile water and the yeast cells were collected by centrifugation), and then the yeast cells were resuspended in the sterile water remaining on the test tube wall to obtain a competent cell solution. Using the anti-lysozyme single-chain antibody expression vector pUC_Paox1_MFα_scFv_Taox1_T38473_G418 constructed in (3) above, the tandem scFv226 antibody expression vector pUC_Paox1_MFα(mut)_taFv226_Taox1_loxP_T38473_loxP_G418 constructed in (4) above, the blinatumomab antibody expression vector pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418 constructed in (5) above, and the minibody antibody vector pUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418 constructed in (6) above, Escherichia coli was transformed. The obtained transformant was cultured in 5 ml of LB medium containing ampicillin (1% tryptone (manufactured by Nacalai Tesque), 0.5% dried yeast extract (manufactured by Nacalai Tesque), 1% sodium chloride, 0.01% sodium ampicillin (manufactured by Nacalai Tesque)). A plasmid was obtained from the resulting bacterial cells using the Plasmid Plus Midi kit (manufactured by Qiagen). This plasmid was linearized by EcoRV treatment using the EcoRV recognition sequence within the CCA38473 terminator. 42 μl of the competent cell solution was mixed with 20 μg each of 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)_Minibody_Taox1_loxP_T38473_loxP_G418, 8 μl of a 10 mg / ml carrier DNA (manufactured by Takara Bio Inc.) solution, 8 μl of a 1 M DTT solution, 4 μl of a 4 M lithium acetate solution, and 100 μl of a 60% polyethylene glycol solution, and the mixture was allowed to stand at 42°C for 20 minutes. After standing for 20 minutes, the yeast cells were 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 allowed to stand at 30°C for 2 hours. After standing for 2 hours, the yeast cells were spread on a YPDG418 selection agar plate (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate (manufactured by Nacalai Tesque)), and strains that grew in static culture at 30°C for 3 days were selected to obtain yeast expressing anti-lysozyme single-stranded antibody, yeast expressing tandem scFv226 antibody, yeast expressing blinatumomab antibody, and yeast expressing minibody antibody. Confirmation of gene activation was performed by amplification nucleic acid fragment fragment length and internal sequence sequence analysis, gene expression analysis, etc. of PCR using the chromosomal DNA of the transformed yeast as a template. As a result, it was confirmed that the desired gene was activated in the transformed yeast obtained in Example 7.

[0094] (14) Culture of Transformed Yeast Each low-molecular-weight antibody-expressing yeast obtained in the above (13) and the low-molecular-weight antibody-expressing yeasts prepared in Examples 4, 5, and 7 were inoculated into 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 potassium phosphate (pH 6.0), 2% Glycerol), and this was cultured with shaking at 30°C, 170 rpm for 24 hours to obtain a preculture solution. 200 μl of the preculture solution was subcultured into 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, 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 for 48 hours, and then the culture supernatant was collected by centrifugation (12,000 rpm, 5 minutes, 4°C).

[0095] (15) Measurement of the secretion amount of each low-molecular-weight antibody by ELISA The measurement of the secretion expression amount of the anti-lysozyme single-chain antibody, tandem scFv226 antibody, blinatumomab antibody, or minibody antibody in the culture supernatant obtained in the above (14) was performed by the following method using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method. ELISA plate (Coaster Assay Plate, 96well Clear, Easywash TM(manufactured by Corning)) was dissolved in immobilization buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.15 g / L monosodium phosphate (anhydrous), 0.2 g / L potassium dihydrogen phosphate (anhydrous), 1 mM magnesium chloride) to a concentration of 1 μM / mL of lysozyme (manufactured by Wako Pure Chemical Industries, Ltd., against anti-lysozyme antibody) or 2 μg / mL of Protein L (Ray Biotech, against tandem scFv226 antibody, brinatumomab antibody, minibody antibody), and 50 μL of each was added to 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 Dainippon Sumitomo Pharma Co., Ltd.), and allowed to stand at room temperature for 1 hour. After washing 3 times with PBST buffer (8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.15 g / L monosodium phosphate (anhydrous), 0.2 g / L potassium dihydrogen phosphate (anhydrous), 0.1% Tween 20), 50 μL of each serially diluted standard antibody and diluted culture supernatant were added to each well and reacted at room temperature for 1 hour. After washing 3 times with PBST buffer, 50 μL of secondary antibody solution (secondary antibody: Anti-6X His tag antibody (HRP) (manufactured by Abcam)) diluted 120,000-fold with PBST buffer was added to each well and reacted at room temperature 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 allowed to stand at room temperature for 3 minutes. After adding 50 μL of 1 M hydrochloric acid solution (manufactured by Nacalai Tesque) to stop the reaction, the absorbance at 450 nm was measured using a microplate reader (Envison; manufactured by PerkinElmer). Quantification of each low molecular weight antibody in the culture supernatant was performed using the calibration curve of each standard antibody.

[0096] (16) Evaluation of the secretion amount of each low molecular weight antibody by SDS-PAGE Evaluation of the secretion amount of anti-lysozyme single-chain antibody, tandem scFv226 antibody, brinatumomab antibody or minibody antibody into the culture supernatant obtained in (14) above was performed by SDS-PAGE according to the method shown below. Each culture supernatant solution was mixed with 2x sample buffer, treated at 100 °C for 10 minutes, and then gel electrophoresis was performed using a gel for 15% SDS-PAGE (e·PAGE, manufactured by ATTO Corporation). The gel after electrophoresis was stained with CBB Stain One (manufactured by Nacalai Tesque Inc.), and the band at the target position was confirmed.

[0097] (17) Construction of an expression vector for promoting antibody production of the protein for pair screening A gene fragment (SEQ ID NO: 218) having a multiple cloning site of HindIII-NotI-BamHI-SpeI-XhoI-BglII-XbaI-EcoRI was completely synthesized and inserted between the HindIII-EcoRI sites of pUC19 (manufactured by Takara Bio Inc., Code No. 3219) to prepare pUC-2. Nucleic acid fragments with EcoRI recognition sequences added to both ends of the promoter-controlled nourseothricin resistance gene (SEQ ID NO: 142) and hygromycin resistance gene (SEQ ID NO: 141) were prepared by PCR using primers 88 (SEQ ID NO: 185) and 94 (SEQ ID NO: 192) and primers 88 (SEQ ID NO: 185) and 89 (SEQ ID NO: 186), respectively, 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 prepared by PCR using primer 116 (SEQ ID NO: 220) and primer 117 for mutagenesis (SEQ ID NO: 221), primer 118 for mutagenesis (SEQ ID NO: 222) and primer 119 (SEQ ID NO: 223) respectively, mixed with pUC2_NAT treated with BglII and XbaI, ligated using the In-fusion HD Cloning Kit, and pUC2_NAT_TMRP40 having a sequence with an AscI restriction enzyme site (GGCGCGCC) introduced into the center of the MRP40 terminator was constructed. Next, the nucleic acid fragments of the first half and the second half of the ARG83 terminator (SEQ ID NO: 224) were prepared by PCR using primer 120 (SEQ ID NO: 225) and primer 121 for mutagenesis (SEQ ID NO: 226), primer 122 for mutagenesis (SEQ ID NO: 227) and primer 123 (SEQ ID NO: 228) respectively, mixed with pUC2_Hyg treated with BglII and XbaI, ligated using the In-fusion HD Cloning Kit, and pUC2_Hyg_TARG83 having a sequence with an AscI restriction enzyme site (GGCGCGCC) introduced into the center of the MRP40 terminator was constructed.

[0098] Example 1 Preliminary study for the preparation of a Komagataella phaffii gene overexpression cell library As a gene overexpression cell library replacing the conventional cDNA library and genomic library, a method for preparing a yeast gene overexpression cell library was developed, which is characterized by replacing the endogenous promoter of all yeast genes with a high-expression type (GAPDH promoter) (Figure 1). The following shows the specific method for preparing the library ((i)-(iv) correspond to (i)-(iv) in Figure 1). (i) Using the sequence information of all genes (GeneX, 5,001 types) of Komagataella phaffii, 5,001 types of single-stranded DNA sequences (229 base pairs in length) (Oligonucleotide Library Synthesis (OLS) sequences) are designed and prepared. In each OLS sequence, two BspQI recognition sites are arranged so as to be sandwiched by BspQI cleavage sites (BspQI is a type IIS restriction enzyme with different recognition and cleavage sites). Also, when a BspQI recognition site exists within base numbers 1 to 91 in each OLS sequence, the bases are substituted so that no amino acid substitution occurs, and the BspQI recognition site has been previously disrupted. (ii) Next, the above OLS sequences are made into double-stranded DNA by the PCR method. The pUC_del_Zeo_Pgap-EGFP-CYC1t prepared in (7) above is treated with SpeI and XhoI, mixed with the above double-stranded OLS sequences, and ligated using an In-fusion HD Cloning Kit to prepare a plasmid library. (iii) The plasmid of the above library is linearized with the restriction enzyme BspQI, and the linear plasmid is inserted into the target position (each GeneX) on the genome of Komagataella phaffii 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 a high-expression type (Pgap), and a gene overexpression cell library (5,001 types) of Komagataella phaffii can be obtained. Whether the above method can actually produce a gene-overexpressing Komagataella phaffii strain was examined using the genes KAR2 and PDI1 derived from Komagataella phaffii as model proteins. The nucleotide sequences from the 1st to the 91st bases and from the 93rd to the 183rd bases from the start codon of the Komagataella phaffii gene KAR2 or PDI1 were extracted, and the 3'-terminal sequence of the GAPDH promoter, the restriction enzyme SpeI recognition site, the nucleotide sequence from the 1st to the 91st bases from the start codon, two restriction enzyme BspQI recognition sites (the two juxtaposed BspQI recognition sites are ligated so as to be sandwiched between two BspQI cleavage sites), the nucleotide sequence from the 93rd to the 183rd bases, the stop codon (TGA), the restriction enzyme recognition cleavage site XhoI, and the 5'-terminal sequence of the CYC1 terminator derived from Saccharomyces cerevisiae were ligated in this order to prepare an OLS sequence for KAR2 overexpression and an OLS sequence for PDI1 overexpression (Figure 2, SEQ ID NO: 200 (KAR2), SEQ ID NO: 201 (PDI1)). After treating the vector pUC_del_Zeo_Pgap-EGFP-CYC1t for preparing the overexpression cell library of (7) above with SpeI and XhoI, a nucleic acid fragment was prepared, mixed with the OLS sequence for KAR2 overexpression and the OLS sequence for PDI1 overexpression prepared as described above, respectively, and ligated 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 KAR2 or PDI1 overexpression is arranged downstream of the GAPDH promoter derived from Komagataella phaffii, and the nucleotide sequence thereof was confirmed by the Sanger method to be appropriate. After converting these plasmids into linear forms with the restriction enzyme BspQI, they were each introduced into Komagataella phaffii DNL4-deficient and histidine-requiring strains (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) and inserted into the target site (KAR2 gene or PDI1 gene) by homologous recombination. Primer sequences 102 (SEQ ID NO: 202) and 103 (SEQ ID NO: 203) that flank the KAR2 gene on the genome, and primer sequences 104 (SEQ ID NO: 204) and 105 (SEQ ID NO: 205) that flank the PDI1 gene were designed, and colony PCR was used to verify the insertion of the above plasmids into the target site of the Komagataella phaffii genome. From the positions of the bands of the PCR products in agarose gel electrophoresis, it was shown that each plasmid had been inserted into the target site on the genome. Furthermore, whether the KAR2 or PDI1 gene of the inserted strain was overexpressed was evaluated by measuring the transcription levels of KAR2 and PDI1 using the RT-qPCR method. Total RNA of Komagataella phaffii wild type strain, KAR2 overexpression strain, and PDI1 overexpression strain was obtained using the RNeasy kit (Qiagen). After that, reverse transcription reaction was carried out using a reverse transcription kit (ReverTraAce qPCR RT Master Mix, TOYOBO), and the transcription levels of KAR2 and PDI1 in each strain were quantified using a quantitative PCR kit (KOD SYBR® qPCR Mix, TOYOBO). Primer sequences 106 (SEQ ID NO: 206) and 107 (SEQ ID NO: 207) were used for quantification of the KAR2 transcription level, and primer sequences 108 (SEQ ID NO: 208) and 109 (SEQ ID NO: 209) were used for quantification of the PDI1 transcription level. The ACT1 gene of Komagataella phaffii was used as a reference for qPCR, and primer sequences 110 (SEQ ID NO: 210) and 111 (SEQ ID NO: 211) were used. KAR2 overexpression strain and PDI1 overexpression strain as controls (each having 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 used as comparison targets. As a result of RT-qPCR, it was shown that the KAR2 and PDI1 overexpression strains prepared by the above method overexpressed their respective genes, similar to their respective control strains (Figure 3). The above results indicated that it was possible to prepare a Komagataella phaffii gene overexpression cell library by this method.

[0099] Example 2 Preparation of Komagataella phaffii gene overexpression cell library In response to the results of Example 1, the above OLS sequences were designed for all Komagataella phaffii genes (5,001 genes) (Figure 2) and prepared (by Agilent Technologies). To convert all the obtained OLS sequences into double-stranded DNA, DNA was amplified by PCR using primers 112 (SEQ ID NO: 212) and 113 (SEQ ID NO: 213) complementary to both ends of the OLS sequence. The double-stranded OLS sequences were ligated into the vector pUC_del_Zeo_Pgap-EGFP-CYC1t for preparing the overexpression cell library described in (7) above using the In fusion method, and Escherichia coli DH5α strain was transformed with the obtained plasmid. The obtained transformant colonies (4x10 5 individuals) were collected from multiple plates, and plasmids were extracted using the Plasmid Plus Midi kit (Qiagen) to obtain a plasmid library. In addition, 12 transformant colonies obtained here were cultured in LB medium, plasmids were extracted, and the nucleotide sequences of the OLS sequences contained in the plasmids were confirmed by the Sanger method. As a result, all had different nucleotide sequences. When analyzed based on the designed OLS sequences, 6 out of 12 strains had the sequences as designed. However, among the remaining 6 strains, 2 strains had a single nucleotide substitution (G→T), 1 strain had a single nucleotide insertion, 1 strain had two nucleotide substitutions (both G→T) and a single nucleotide deletion, 1 strain had a single nucleotide substitution (G→T) and two nucleotide deletions, and 1 strain could not be analyzed. The obtained plasmid library was digested with the restriction enzyme BspQI, and after DNA purification, it was introduced into the Komagataella phaffii strain secreting low molecular weight antibodies (anti-lysozyme scFv antibody, tandem scFv226, blinatumomab antibody) using the electroporation method.

[0100] Example 3 High-throughput screening using the secretion amount of low molecular weight antibodies as an index The transformant group (gene overexpression cell library) obtained by the electroporation method in Example 2 was aligned in a 96-well format on a square plate of YPD agar medium supplemented with Zeocin using a colony picker (PM-2, manufactured by Microtech Nichion) and cultured at 30°C to form colonies, which was used as a master plate. Using a 96-pin, 96 strains were simultaneously inoculated from the master plate into a deep well plate containing 0.5 mL of BMGY medium. After stirring culture 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 BMMY medium, and further stirring culture was performed at 30°C for 48 hours. After the culture, the deep well plate was centrifuged, and the supernatant was diluted with PBS, and the amount of low molecular weight antibody was evaluated by ELISA using the His tag or c-myc tag as an index. Using the above method, gene overexpression cell libraries were prepared for each strain producing anti-lysozyme scFv antibody, tandem scFv226 antibody, and blinatumomab antibody, and strains with increased low molecular weight antibody amount (screening positive strains) were screened from gene overexpression strains (about 19,200 strains) corresponding to 200 deep well plates for each strain.

[0101] Example 4 Identification of Useful Factors The gene overexpressed in the obtained screening positive strains was identified by the following method (Figure 4) ((a) to (f) below correspond to (a) to (f) of Figure 4). (a) Genomic DNA was extracted from the screening positive strains using Gen Takara (manufactured by Takara Bio Inc.). (b) The genomic DNA was fragmented by simultaneous treatment with multiple restriction enzymes (any of the following (i) to (iii) was used). (i) After treating the genomic DNA with 5'-protruding end restriction enzymes EcoRI, SalI, NheI, BamHI, and ClaI, the 5'-protruding ends of the fragmented DNA were made blunt ends using Klenow fragment. (ii) The genomic DNA was treated with 5'-protruding end restriction enzymes BamHI, BclI, and BglII (the 5'-protruding ends have the same base sequence (GATC)). (iii) The genomic DNA was treated with blunt end restriction enzymes BsaAI, BsaBI, BstZ17I, HpaI, PmlI, SnaBI, and StuI. (c) The fragmented DNA was circularized by self-ligation. (d) The circularized DNA was used to transform the Escherichia coli DH5α strain on an LB agar medium supplemented with ampicillin and zeocin. (e) Plasmids were extracted from the transformants. (f) The nucleotide sequence of the OLS sequence of the plasmid inserted into the genome of Komagataella phaffii was determined from the plasmid extracted by the Sanger method. Using the above method, 36 genes overexpressed in the screening-positive strains were determined. However, overexpression of these genes does not necessarily result in an increase in the amount of the low-molecular-weight antibody. Therefore, overexpression vectors of the 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 introduced into the Arg4 site of each low-molecular-weight antibody-producing strain used in Example 2 by the method described in (13) above to prepare strains, and it was examined whether the secretion amount of the low-molecular-weight antibody increased. As a result, in the anti-lysozyme scFv antibody-producing strains into which each overexpressed gene was introduced, 18 strains were obtained in which the antibody secretion amount increased 1.1-fold to 1.7-fold compared to the host strain (Table 1). In the tandem scFv226-producing strains into which each overexpressed gene was introduced, 9 strains were obtained in which the antibody secretion amount increased 1.1-fold to 1.6-fold (Table 2). Furthermore, in the blinatumomab-producing strains into which each overexpressed factor was introduced, 9 strains were obtained in which the antibody secretion amount increased 1.2-fold to 2.3-fold (Table 3). From these results, it was confirmed that overexpression of each of the above 36 genes is effective in promoting the production of low-molecular-weight antibodies, and the 36 genes are useful factors for promoting antibody production (Table 4). Among the useful factors obtained in this screening, only two (EF2nd-8 (KAR2) and EF3rd-4 (KIN2)) were factors for which prior studies had been conducted in Komagataella phaffii (Damasceno et al., Appl. Microbiol. Biotechnol. Vol. 74, 2007 (KAR2), Gasser et al. Appl. Environ. Microbiol. Vol. 73, No. 20 2007 (KIN2)). Also, EF2nd-4 and EF3rd-2 were the same gene (YCK). EF1st-1 has also been separately reported by the present inventors. From the above, among the genes described in Table 4, 32 factors excluding EF1st-1, EF2nd-8, EF3rd-2, and EF3rd-4 were newly found in this study. Next, it was examined whether the useful factors obtained from 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 an anti-lysozyme scFv antibody-producing strain were introduced into the ARG4 sites of tandem scFv226 antibody and blinatumomab antibody-producing strains, respectively, to prepare strains. As a result of evaluating the antibody secretion amount of each strain by ELISA, compared with the host strain, the antibody secretion amount in the tandem scFv226 antibody-producing strain was 0.9 to 1.6 times (Table 5), and in the blinatumomab antibody-producing strain was 1.0 to 2.3 times (Table 6). Similarly, nine useful factor expression vectors (9 types from pUC_Arg4_Pgap_EF2nd-1_T37552_Zeo1 to pUC_Arg4_Pgap_EF2nd-9_T37552_Zeo) obtained from a tandem scFv antibody-producing strain were introduced into the ARG4 sites of anti-lysozyme scFv antibody and blinatumomab antibody-producing strains, respectively, to prepare strains. As a result of evaluating the antibody secretion amount of each strain by ELISA, compared with the host strain, the antibody secretion amount in the anti-lysozyme scFv antibody-producing strain was 1.0 to 1.2 times (Table 7), and in the blinatumomab antibody-producing strain was 1.0 to 1.8 times (Table 8). Also similarly, nine useful factor expression vectors (from pUC_Arg4_Pgap_EF3rd-1_T37552_Zeo to pUC_Arg4_Pgap_EF3rd-9_T37552_Zeo) obtained from a blinatumomab antibody-producing strain were introduced into the ARG4 sites of anti-lysozyme scFv antibody and tandem scFv226 antibody-producing strains, respectively, to prepare strains. As a result of evaluating the antibody secretion amount of each strain by ELISA, compared with the host strain, the antibody secretion amount in the anti-lysozyme scFv antibody-producing strain was 1.0 to 1.7 times (Table 9), and in the tandem scFv226 antibody-producing strain was 0.8 to 1.5 times (Table 10). The group of useful factors obtained from the tandem scFv antibody-producing strain was not effective in increasing the production of anti-lysozyme scFv antibody. On the other hand, many of the useful factors obtained from the anti-lysozyme scFv antibody and blinatumomab antibody-producing strains were also confirmed to be effective in promoting the production of different low-molecular antibodies.

[0102]

Table 1

[0103]

Table 2

[0104]

Table 3

[0105]

Table 4

[0106]

Table 5

[0107]

Table 6

[0108]

Table 7

[0109]

Table 8

[0110]

Table 9

[0111]

Table 10

[0112] Example 5 Development of a high-producing strain of low-molecular antibodies by accumulation of useful factors The promoting effect of the useful factors obtained from the above screening on the production of low-molecular antibodies was relatively low, being 1.1 to 1.6 times (in the case of tandem scFv226 production). In order to increase the secretion amount of low-molecular antibodies, the accumulation of useful factors was investigated. This time, a total of 8 factors were selected, namely 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. By accumulating these factors, we aimed to create a high-producing strain of tandem scFv226 antibody. Specifically, each of the selected 8 factors was introduced one by one into the tandem scFv226-producing parental strain by the method described in (13) above, and the secretion amount of the tandem scFv226 antibody was evaluated. Among them, the strain with the highest secretion amount was used as the parental strain for the next generation. By repeating the introduction of these useful factors and the evaluation of the secretion amount of the tandem scFv226 antibody 4 times, we aimed to obtain a high-producing strain of tandem scFv226 antibody. In the first generation, using the single-gene deletion strain (strain A) that produces the tandem scFv226 antibody as the parental strain, only the useful factor EF1st-1 that most increased the secretion amount of the taFv226 antibody was introduced into the ARG4 site, and the secretion amount of the tandem scFv226 antibody was evaluated. As a result, strain B, in which the secretion amount of the tandem scFv226 antibody increased by about 1.5 times compared to strain A, was obtained (Table 11). Using strain B as the parental strain for the second generation, each of the 8 factors selected above was introduced into the URA3 site, and strains in which each factor was overexpressed were prepared by the method described in (13) above. When the secretion amount of the tandem scFv226 antibody of the prepared strains was evaluated, the EF2nd-4-introduced strain (strain C) had the highest secretion amount and secreted about 1.2 times more antibody compared to the parental strain B (Table 12). Using strain C as the parental strain for the third generation, each of the 8 factors selected above was introduced into the GUT1 site, and strains in which each factor was overexpressed were prepared by the method described in (13) above. When the secretion amount of the tandem scFv226 antibody of the prepared strains was evaluated, the EF1st-4-introduced strain (strain D) had the highest secretion amount and secreted about 1.2 times more antibody compared to the parental strain C (Table 13). Using strain D as the parental strain for the fourth generation, each of the 8 factors selected above was introduced into the AOX1 promoter site, and strains in which each factor was overexpressed were prepared by the method described in (13) above.When the secretion amount of the tandem scFv226 antibody of the prepared strain was evaluated, the EF2nd-1-introduced strain had the highest secretion amount and secreted about 1.3 times more antibodies compared to the parental D strain (Table 14). This strain was designated as strain E. Thus, strain E into which four useful factors were introduced secreted about 2.9 times more tandem scFv226 antibodies compared to strain A before the introduction of the useful factors. By sequentially accumulating the useful factors in this way, we succeeded in increasing the productivity of the low-molecular-weight antibody (Figure 5).

[0113]

Table 11

[0114]

Table 12

[0115]

Table 13

[0116]

Table 14

[0117] Example 6 Replacement of the Antibody Expression Cassette from the High-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, the Cre-loxP system was used to examine the replacement of gene expression cassettes. Specifically, when loxP sequences (34 bases in length) are inserted at both ends of the genomic introduction sequence of Komagataella phaffii for the antibody expression cassette, the expression cassette introduced into Komagataella phaffii will have loxP sequences at both ends (Figure 6). Next, the plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) having the Cre recombinase gene controlled by the methanol-inducible AOX1 promoter is introduced into the antibody-producing strain with the Zeo marker. At this time, the nucleotide sequence between loxP is deleted by the expression of Cre recombinase at the leak level of the AOX1 promoter. The deletion of the antibody expression cassette can be evaluated by the presence or absence of the drug marker by colony PCR using primers designed at both ends of the genomic insertion site or by drug assay. By introducing different antibody expression vectors into the same genomic insertion site of the strain with the antibody cassette deleted, it becomes possible to express different antibodies from the tandem scFv226 antibody of the tandem scFv226 high-producing strain (Figure 6). As a model case, the protein expression cassette was exchanged using green fluorescent protein (GFP) and red fluorescent protein gene (RFP). For GFP, a gene encoding EGFP (SEQ ID NO: 214) was used, and for RFP, a gene encoding E2crimson (SEQ ID NO: 215) was used. As the GFP expression vector, pPGP_GFP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) was used. This vector is designed such that GFP is expressed under the control of the GAPDH promoter. The RFP gene was prepared by PCR using synthetic DNA as a template and primers 114 (SEQ ID NO: 216) and 115 (SEQ ID NO: 217). After treating this nucleic acid fragment with SpeI and XhoI, a nucleic acid fragment was prepared, mixed with the nucleic acid fragment encoding RFP prepared by the above PCR, and ligated using the In-fusion HD Cloning Kit to construct pPGP_RFP. This vector is designed such that RFP is expressed under the control of the GAPDH promoter. The process of replacing the reporter gene from a GFP-expressing strain to an RFP-expressing strain using the Komagataella phaffii wild strain is shown in Fig. 7. First, a strain in which a GFP expression cassette (a linearized pPGP_GFP fragment at the EcoRV site within the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site of Komagataella phaffii (Fig. 7(1)) was prepared by the method described in (13) above (Fig. 7(2)). This strain was resistant to G418, and GFP fluorescence was confirmed by flow cytometry (FCM). Next, 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, and it was confirmed that this strain had lost G418 resistance at this point. This was thought to be due to the leaky expression of Cre recombinase causing the expression cassette containing the G418 marker to drop out. This strain was subjected to single colony isolation on YPD agar medium to remove pPAP_CP (Fig. 7(4)). This strain had lost resistance to G418 and Zeocin. Also, in the FCM analysis, the GFP fluorescence intensity had dropped to the background level. Finally, a strain in which an RFP expression cassette (a linearized pPGP_RFP fragment at the EcoRV site within the CCA38473 terminator sequence) was introduced into the CCA38473 terminator site was prepared by the method described in (13) above (Fig. 7(5)). This strain had G418 resistance, and high RFP fluorescence intensity was confirmed by FCM analysis. From the above results, it was shown that the replacement of the reporter expression cassette was possible by the above method.

[0118] Example 7 Evaluation of antibody high-producing strains with different single-chain 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 into the CCA38473 terminator site of the antibody expression vector. Therefore, loxP sequences were introduced at both ends of the antibody cassette introduced into the yeast genome (Figure 8). Therefore, if Cre recombinase is expressed in yeast cells, the tandem scFv226 antibody expression cassette can be excised. In fact, after introducing plasmid pPAP_CP (Ito et al., FEMS Yeast Research, Vol. 18, No. 7, 2018) into the antibody high-producing strain by the method described in (13) above and performing single colony isolation, deletion of the antibody expression cassette was confirmed by colony PCR and drug assay using G418. Next, as low-molecular antibodies different from the tandem scFv226 antibody, three types of anti-lysozyme scFv antibody expression vector pUC_Paox1_MFα_scFv_Taox1_T38473_G418, blinatumomab antibody expression vector pUC_Paox1_MFα(mut)_Blinatumomab_Taox1_loxP_T38473_loxP_G418, and minibody antibody expression vector pUC_Paox1_MFα(mut)_Minibody_Taox1_loxP_T38473_loxP_G418 were each linearized with the restriction enzyme EcoRV and then introduced into the CCA38473 terminator site of the high-producing strains A, D, and E of Example 5 to prepare antibody-producing strains (A1, A2, A3 strains, D1, D2, D3 strains, and E1, E2, E3 strains, respectively) by the method described in (13) above. When the secretion amount of each antibody was evaluated by ELISA and SDS-PAGE, it was confirmed that the anti-lysozyme scFv antibody-producing strain had an antibody secretion amount more than 10 times, and the blinatumomab antibody-producing strain had an antibody secretion amount more than 3 times that of the host strain. In addition, in the minibody antibody-producing strain, the antibody secretion amount was about 2 times in D3 strain derived from D strain and about 3 times in E3 strain derived from E strain (Figure 9). From the above results, it was shown that the integrated strain of useful factors obtained using the productivity of the tandem scFv226 antibody as an index has high productivity not only in the production of the tandem scFv226 antibody but also in different low-molecular antibodies.

[0119] Example 8 Preparation of a Cell Library Overexpressing an Antibody Production-Promoting Protein for Pair Screening and Screening of High-Producing Strains As shown in Example 5, the integration experiment conducted using eight types of useful factors indicated that the integration of useful factors is effective in promoting the production and secretion of low-molecular antibodies. Therefore, as a method for efficiently integrating the obtained useful factors, a yeast library in which two types of useful factors were simultaneously overexpressed was prepared, and a strain that secretes the most low-molecular antibodies from that library was devised to be found using the high-throughput screening method shown in Example 3 (pair screening, Figure 10). As proof thereof, aiming to improve the production of blinatumomab in the blinatumomab-producing strain showing poor secretion productivity, pair screening was repeated twice using the group of useful factors obtained in Example 2. First, nine types of useful factors (EF3rd-1 to 9, Table 3) that promoted blinatumomab secretion obtained in Example 4 were selected, and each DNA fragment (GAPDH promoter - useful factor ORF and its terminator sequence) was amplified by PCR using two types of primer sets (primer 124 (SEQ ID NO: 229) and primer 125 (SEQ ID NO: 230), primer 126 (SEQ ID NO: 231) and primer 127 (SEQ ID NO: 232)). After mixing these two types of PCR products and pUC2_NAT_TMRP40 cut with BamHI and XhoI so that they had the same number of DNA molecules, they were ligated using the In-fusion HD Cloning Kit to prepare a plasmid library consisting of a population of plasmids containing a nucleotide sequence in which two useful factor expression cassettes were ligated in reverse with a linker sequence in between (Figure 10, diversity of combination types: 81). The prepared plasmid library was introduced into the blinatumomab-secreting strain prepared in (13) above, and approximately 2,400 transformants were obtained. Screening was performed on the emerged transformants using eight 96-well deep-well plates by the high-throughput screening method shown in Example 3. Evaluation of the blinatumomab secretion amount in the culture supernatant was performed by ELISA using the His tag as an index. As a result of analyzing two strains (1st-11 strain, 1st-12 strain) that showed high production in the screening, the antibody secretion amount had increased by approximately five times compared to the host strain (Table 15). Next, using the same method as in Example 4, the useful factors introduced into the two high-secreting strains obtained were identified, and as a result, EF3rd-1 was introduced in both cases.As another useful factor, EF3rd-2 and EF3rd-5 were inserted. In addition, in Example 7, the high-secreting strain E2 strain (4 useful factor-introduced strain) obtained in the useful factor accumulation experiment had an antibody secretion amount about 3.2 times that of the host strain. Therefore, it was shown that the high-secreting strain (2 useful factor-introduced strain) obtained by the pair screening this time had a higher secretion amount of the blinatumomab antibody. Next, using these two strains as parental strains, the second cycle of pair screening was performed. This time, a total of 10 factors, EF1st-1 to 6 and EF2nd-1 to 4, were selected as the useful factors that promote blinatumomab secretion obtained in Example 2, and the combination was optimized. Two types of 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)) were used to amplify DNA by PCR using a useful factor cassette (GAPDH promoter - useful factor ORF and its terminator sequence). These two types of PCR products were mixed with pUC2_Hyg_TARG83 digested with BamHI and XhoI so that they had the same number of DNA molecules, and were ligated using the In-fusion HD Cloning Kit to prepare a plasmid library consisting of a population of plasmids containing a nucleotide sequence in which two useful factor expression cassettes were inversely linked with a linker sequence in between (Figure 10, diversity of combination types: 100). The prepared plasmid library was introduced into the two types of blinatumomab high-secreting strains shown above, and approximately 4,400 transformants were obtained. From the resulting transformants, 9 plates (864 strains) of 96-well plates were screened by the above high-throughput screening method. As a result of analyzing the 4 strains (2nd_11-7 strain, 2nd_11-8 strain, 2nd_12-7 strain, 2nd_12-2 strain) that showed high secretion in the screening, compared with the host strain, they showed a secretion level 13 to 15 times higher (Table 15). Next, as a result of identifying the useful factors introduced into the 4 types of strains obtained using the same method as in Example 4, EF1st-1 was introduced in all the strains (Table 15). The other useful factor was EF2nd-4 (the 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). From the above results, the useful factors in a combination different from the combination that was optimal in the tandem scFv226-producing strain shown in Example 7 were included in the blinatumomab antibody high-secreting strain. 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.

[0120]

Table 15

[0121] 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. Also, the useful factors that had been selected as the optimal combination in the blinatumomab high-secreting strain were EF1st-1, EF1st-3, EF2nd-4, EF3rd-1, and EF3rd-5. Next, in different small molecule antibody-secreting strains, the pair screening method with the blinatumomab antibody shown in Example 8 was carried out to attempt to identify the selected useful factors. Using the plasmid library consisting of 9 types of useful factors (EF3rd-1 to 9) prepared in the first cycle of pair screening in Example 8, it was introduced into the small molecule antibody (tandem scFv226)-secreting strain prepared in (13) above, and 1,200 transformants were obtained. From the emerging transformants, screening of 4 plates (384 strains) of 96-well deep-well plates was carried out by the high-throughput screening method shown in Example 3. Evaluation of the secretion amount of taFv226 in the culture supernatant was carried out by the ELISA method shown in (15) above. As a result of evaluating the strains showing a high secretion amount in the screening, 4 strains (taFv1st-10 strain, taFv1st-15 strain, taFv1st-9 strain, taFv1st-12 strain) showed an antibody secretion amount approximately 2.3 times higher than that of the host strain (Table 16). Next, using the same method as in Example 4, as a result of identifying the useful factors introduced into each of the 4 types of obtained strains, EF3rd-2 (EF2nd-4) and EF3rd-3 were introduced in all the strains (Table 16). In the first-generation pair screening in Example 8, the optimal combinations of useful factors were EF3rd-1 and EF3rd-2 (EF2nd-4), EF3rd-1 and EF3rd-5, which were different from EF3rd-2 (EF2nd-4) and EF3rd-3 obtained by the screening using the tandem scFv226-secreting strain of this example. That is, from this result, it was shown that the optimal combinations of useful factors for each small molecule antibody secretion were different.

[0122]

Table 16

Industrial Applicability

[0123] By providing a genetically modified cell in which the expression of an endogenous gene is enhanced, it has become possible to construct a gene library of the endogenous gene with enhanced expression. In addition, by using the library, it has become possible to screen for an endogenous gene that increases the production of a target protein. This application is based on Japanese Patent Application No. 2020-050192 (filing date: March 19, 2020), the content of which is hereby incorporated by reference in its entirety.

Claims

**Claim 1** A method for producing a genetically modified cell with enhanced expression of an endogenous gene, comprising 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 contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and having a restriction enzyme recognition site inserted therein, and a base sequence in which a stop codon is sequentially linked, (2) A step of introducing the linear nucleic acid into a host cell, and (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene has been homologously recombined by the linear nucleic acid and a highly expressive promoter is functionally linked. **Claim 2** The method according to claim 1, wherein the host cell and the genetically modified cell are yeast, bacteria, fungi, insect cells, animal cells or plant cells. **Claim 3** The method according to claim 2, wherein the yeast is a methanol-assimilating yeast, fission yeast or budding yeast. **Claim 4** The method according to claim 3, wherein the methanol-assimilating yeast is a yeast belonging to the genus Komagataella or Ogataea. **Claim 5** 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 a base sequence identical to the base sequence represented by SEQ ID NO: 47, or an endogenous gene containing a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 47, and encoding a protein that enhances the production of the target protein, (2) An endogenous gene containing a base sequence identical to the base sequence represented by SEQ ID NO: 46, or an endogenous gene containing a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 46, and encoding a protein that enhances the production of the target protein, (3) An endogenous gene containing a base sequence identical to the base sequence represented by SEQ ID NO: 48, or an endogenous gene containing a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 48, and encoding a protein that enhances the production of the target protein, An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 49, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 49, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 50, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 50, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 51, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 51, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 52, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 52, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 53, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 53, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 54, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 54, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 55, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 55, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 56, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 56, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 57, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 57, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 58, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 58, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 59, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 59, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 60, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 60, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 61, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 61, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 62, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 62, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 63, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 63, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 64, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 64, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 65, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 65, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 66, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 66, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 67, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 67, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 68, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 68, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 69, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 69, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 70, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 70, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 71, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 71, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 73, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 73, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 75, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 75, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 76, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 76, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 77, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 77, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 78, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 78, and encoding a protein that enhances the production of the target protein, and An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 79, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 79, and encoding a protein that enhances the production of the target protein. **Claim 6** 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) A 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) A 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 comprising the nucleotide sequence represented by SEQ ID NO: 249, (xiii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 250, (xiv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 251, (xv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 252, (xvi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 253, (xvii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 254, (xviii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 255, (xix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 256, (xx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 257, (xxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 258, (xxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 259, (xxiii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 260, (xxiv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 261, (xxv) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 262, (xxvi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 263, (xxvii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 265, (xxviii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 267, (xxix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 268, (xxx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 269, (xxxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 270, and (xxxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO:

271. [

7. ] A genetically modified cell comprising an endogenous gene functionally linked to a highly expressive promoter, which is homologously recombined by a linear nucleic acid obtained by cleaving a plasmid in which the endogenous gene contains a nucleic acid fragment with a restriction enzyme, wherein the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a nucleotide sequence in which a stop codon is sequentially linked. [

8. ] The genetically modified cell according to claim 7, wherein the genetically modified cell is a yeast, a bacterium, a fungus, an insect cell, an animal cell, or a plant cell. [

9. ] The genetically modified cell according to claim 8, wherein the yeast is a methanol-assimilating yeast, a fission yeast, or a budding yeast. [

10. ] The genetically modified cell according to claim 9, wherein the methanol-assimilating yeast is a yeast belonging to the genus Komagataella or the genus Ogataea.

11. The genetically modified 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 a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 47, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 47, and encoding a protein that enhances the production of the target protein. (2) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 46, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 46, and encoding a protein that enhances the production of the target protein. (3) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 48, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 48, and encoding a protein that enhances the production of the target protein. (4) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 49, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 49, and encoding a protein that enhances the production of the target protein. (5) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 50, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 50, and encoding a protein that enhances the production of the target protein. (6) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 51, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 51, and encoding a protein that enhances the production of the target protein. (7) An endogenous gene containing a nucleotide sequence identical to the nucleotide sequence represented by SEQ ID NO: 52, or an endogenous gene containing a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 52, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 53, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 53, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 54, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 54, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 55, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 55, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 56, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 56, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 57, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 57, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 58, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 58, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 59, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 59, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 60, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 60, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 61, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 61, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 62, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 62, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 63, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 63, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 64, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 64, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 65, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 65, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 66, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 66, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 67, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 67, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 68, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 68, and encoding a protein that enhances the production of the target protein. An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 69, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 69, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 70, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 70, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 71, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 71, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 73, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 73, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 75, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 75, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 76, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 76, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 77, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 77, and encoding a protein that enhances the production of the target protein An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 78, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 78, and encoding a protein that enhances the production of the target protein, and An endogenous gene containing the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 79, or an endogenous gene containing a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 79, and encoding a protein that enhances the production of the target protein.

12. The genetically modified 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) A 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) A 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) A 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) A nucleic acid fragment containing the nucleotide sequence represented by SEQ ID NO: 262, (xxvi) A 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) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 267, (xxix) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 268, (xxx) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 269, (xxxi) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO: 270, and (xxxii) A nucleic acid fragment comprising the nucleotide sequence represented by SEQ ID NO:

271. (Claim 13) The genetically modified cell according to any one of Claims 7 to 12, which contains in its genome a nucleotide sequence encoding a target protein. (Claim 14) The genetically modified cell according to Claim 13, wherein the target protein is a heterologous protein. (Claim 15) A method for producing a target protein, which includes the step of culturing the genetically modified cell according to Claim 13 or 14. (Claim 16) An endogenous gene overexpression cell library containing the genetically modified cell according to any one of Claims 7 to 10. (Claim 17) A method for screening an endogenous gene that enhances the production of a target protein, which includes the following steps. (1) A step of cleaving a plasmid containing a nucleic acid fragment with a restriction enzyme to prepare a linear nucleic acid, wherein the nucleic acid fragment contains a highly expressive promoter, a partial sequence starting from the start codon of the endogenous gene and into which a restriction enzyme recognition site is inserted, and a nucleotide sequence in which a stop codon is ligated in order. (2) A step of introducing the linear nucleic acid into a host cell containing in its genome a nucleotide sequence encoding a target protein. (3) A step of selecting a genetically modified cell containing an endogenous gene in which the endogenous gene has been homologously recombined by the linear nucleic acid and a highly expressive promoter is operably linked. (4) A step of culturing the cell obtained in step (3) and a host cell containing in its genome a nucleotide sequence encoding a target protein. (5) A step of measuring the production amounts of the target protein by the cell obtained in step (3) and a host cell containing in its genome a nucleotide sequence encoding a target protein, respectively, and (6) A step of identifying an endogenous gene that increases the production amount of the target protein. (Claim 18) A method for screening an endogenous gene that enhances the production of a target protein, which includes the following steps. (1) A step of introducing an expression vector containing a nucleotide sequence encoding a target protein into the endogenous gene overexpression cell library according to Claim 16 and a host cell, and culturing them. (2) a step of measuring the production amount of a target protein by an endogenous gene overexpression cell library and a host cell, and (3) a step of identifying an endogenous gene that increases the production amount of the target protein.

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