JP7239205B2 - Novel host cell and method for producing target protein using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel host cell in which target protein productivity is improved by inactivating a specific gene, a vector for inactivating the gene, and a method for producing a target protein using the novel host cell.

Recombinant methods are widely used to produce industrially useful biomaterials such as antibodies, enzymes, and cytokines for medical and diagnostic applications. Hosts for producing target proteins by genetic recombination include 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. used. Among host organisms, yeast can be cultured on a large scale and at high density using an inexpensive medium, so it is possible to produce the target protein at low cost. Since it can be expressed, it facilitates the purification process of the target protein, and since it is a eukaryotic organism, post-translational modifications such as glycosylation are possible. there is If we can develop an innovative production technology that can handle various target proteins in yeast, we can expect a wide range of industrial development, in addition to strengthening cost competitiveness by dramatically improving productivity.

Komagataella pastoris, a kind of yeast, is a methanol-assimilating yeast that has an excellent ability to express proteins and can utilize an inexpensive carbon source that is advantageous for industrial production. For example, Non-Patent Document 1 reports a method for producing foreign proteins such as green fluorescent protein, human serum albumin, hepatitis B virus surface antigen, human insulin, and single-chain antibodies using Komagataella pastoris. ing. Addition of signal sequence, use of strong promoter, modification of codons, co-expression of chaperone genes, co-expression of transcription factor genes, protease derived from host yeast to improve productivity when producing foreign proteins in yeast Various attempts such as gene inactivation and examination of culture conditions have been made.

FEMS Microbiology Reviews 24 (2000) 45-66.

An object of the present invention is to provide a new means for improving the productivity of a target protein.

The present inventors identified a novel protein by comprehensive analysis of the nucleotide sequences of chromosomal DNA of yeast of the genus Komagataella. Then, the present inventors have found that the amount of target protein secreted in host cells is improved by inactivating the gene encoding the novel protein, and have completed the present invention. That is, the present invention includes the following aspects.

A host cell in which any of the following genes (a) to (d) is inactivated:
(a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1, a gene comprising the nucleotide sequence shown in SEQ ID NO: 3, a gene comprising the nucleotide sequence shown in SEQ ID NO: 5, a gene comprising the nucleotide sequence shown in SEQ ID NO: 7, and SEQ ID NO: 9 and/or a gene comprising the nucleotide sequence shown in SEQ ID NO: 11,
(b) a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7;
A gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9 and/or a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 11 a gene comprising a nucleic acid sequence that hybridizes under stringent conditions to
(c) a gene containing a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1;
a gene containing a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3;
a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 5;
a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 7;
A gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 9, and/or a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 11,
(d) a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8;
A gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and/or a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12 .

The host cell comprises a nucleotide sequence having a region homologous to any of the genes (a) to (d), a nucleotide sequence having a region homologous to the promoter of any of the genes (a) to (d), and / Or it is preferably obtained by transformation with a vector containing a nucleotide sequence having a region homologous to the terminator of any of the genes (a) to (d).

In the host cell, the vector preferably contains a nucleotide sequence encoding the target protein upstream or downstream of the nucleotide sequence.

The host cell preferably contains a vector containing a nucleotide sequence encoding the protein of interest.

Preferably said host cells are yeast, bacteria, fungi, insect cells, animal cells or plant cells.

The yeast is preferably methanol-assimilating yeast, fission yeast, or budding yeast.

It is preferable that the methanol-assimilating yeast is a yeast belonging to the genus Komagataella or a yeast belonging to the genus Ogataea.

The present invention also relates to a method for producing a target protein, comprising the steps of culturing the host cell and recovering the target protein from the culture medium.

Preferably, said protein of interest is a heterologous protein.

In the culture step, it is preferable to use a culture medium containing one or more carbon sources selected from the group consisting of glucose, glycerol and methanol.

By inactivating the gene according to the present invention, there is provided an efficient method for producing a target protein using yeast of the genus Komagataella as a host.

The present invention will now be described in detail using preferred embodiments.

In one aspect, the present invention provides a host cell in which any one of the following genes (a) to (d) has been inactivated:
(a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1, a gene comprising the nucleotide sequence shown in SEQ ID NO: 3, a gene comprising the nucleotide sequence shown in SEQ ID NO: 5, a gene comprising the nucleotide sequence shown in SEQ ID NO: 7, and SEQ ID NO: 9 and/or a gene comprising the nucleotide sequence shown in SEQ ID NO: 11,
(b) a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5;
a gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7;
A gene comprising a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9 and/or a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 11 a gene comprising a nucleic acid sequence that hybridizes under stringent conditions to
(c) a gene containing a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1;
a gene containing a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3;
a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 5;
a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 7;
A gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 9, and/or a gene comprising a nucleotide sequence having 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 11,
(d) a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6;
a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8;
A gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and/or a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12 , concerning.

In the present invention, two nucleic acids hybridize under stringent conditions means, for example, the following. For example, using a filter immobilized with nucleic acid X, after hybridization with nucleic acid Y at 65°C in the presence of 0.7 to 1.0 M NaCl, a double concentration SSC solution (composition of a single concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate), and the filter is washed at 65°C. and a nucleic acid that hybridizes under stringent conditions". Alternatively, it can be said that nucleic acid X and nucleic acid Y "hybridize with each other under stringent conditions". For nucleic acid Y, the filter is preferably washed at 65°C with a 0.5-fold concentration SSC solution, more preferably at 65°C with a 0.2-fold concentration SSC solution, and still more preferably at 65°C with a 0.1-fold concentration SSC solution. It is a nucleic acid that can be obtained as a nucleic acid bound on a filter by washing. The reference nucleic acid X may be colony- or plaque-derived nucleic acid X.

In the present invention, the sequence identity of base sequences and amino acid sequences can be determined using methods, sequence analysis software and the like well known to those skilled in the art. Examples thereof include the blastn program and blastp program of the BLAST algorithm, and the fasta program of the FASTA algorithm. In the present invention, the "sequence identity" of a base sequence to be evaluated with a base sequence X means that the base sequence X and the base sequence to be evaluated are aligned (aligned), gaps are introduced as necessary, It is a value expressed in % of the frequency at which the same base appears at the same site in the base sequence including the gap portion when the degree of base matching between the two is maximized. In the present invention, the "sequence identity" of a given amino acid sequence to be evaluated with an amino acid sequence X means that the amino acid sequence X and the amino acid sequence to be evaluated are aligned (aligned), gaps are introduced as necessary, It is the value expressed in % of the frequency of appearance of the same amino acid at the same site in the amino acid sequence including the gap portion when the degree of amino acid identity between the two is maximized. The sequence identity between the gene and the nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, and/or 11 is 85% or more, preferably 90% or more, more preferably 95% or more, and 96 % or more is even more preferred, 97% or more is particularly preferred, and 98% or more, or 99% or more is most preferred.

In the present invention, "nucleic acid" can also be referred to as "polynucleotide" and refers to DNA or RNA, typically DNA.

In the present invention, the term "nucleotide sequence encoding an amino acid sequence" refers to a nucleotide sequence designed based on the codon table for a polypeptide consisting of an amino acid sequence, and the nucleotide sequence is used to generate a polypeptide by transcription and translation. bring.

In the present invention, the term "polypeptide" refers to a peptide bond of two or more amino acids, and includes proteins as well as short-chain peptides and oligopeptides.

In the present invention, "inactivation" refers to a state in which the function of a gene is lost or reduced, and the expression level of mRNA, which is the transcription product of the gene, or polypeptide, which is the translation product It also includes a state in which the protein is reduced and a state in which it does not function normally as mRNA or protein. The expression level of mRNA can be quantified using real-time PCR, RNA-Seq, Northern hybridization, or hybridization using a DNA array. It can be quantified using an antibody, 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.

As means for inactivating genes, DNA mutation treatment using drugs or ultraviolet rays, partial specific mutagenesis using PCR, RNAi, protease, homologous recombination, and the like can be used. When inactivating a gene, modification (deletion, substitution, addition, insertion) of the nucleotide sequence within the ORF of the gene, and/or transcription initiation or termination of the promoter region, enhancer region, terminator region, etc. are controlled. Modification (deletion, substitution, addition, insertion) of the nucleotide sequence within the region is performed. The sites for deletion, substitution, addition, and insertion, and the base sequences to be deleted, substituted, added, and inserted, are not particularly limited as long as the normal function of the gene can be lost. The gene to be inactivated may be a gene on the chromosome of the host cell or a gene extrachromosomal in the host cell. Among these, it is preferable to inactivate the gene on the chromosome.

"Modification of base sequence" in the present invention can be carried out using a technique of gene insertion or site-directed mutagenesis using homologous recombination. For example, replacement of the gene upstream promoter with a promoter with lower activity, modification to codons not suitable for host cells, etc. Examples include introduction of a vector linked with a downstream sequence.

In the present invention, the degree of decrease in expression level is not particularly limited as long as the productivity of the target protein is improved, but is 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60%. It is preferable that the reduction is 70% or more, 80% or more, 90% or more, 95% or more. It is well known to those skilled in the art that even if the expression level is not reduced, as long as the normal function of the gene product can be lost, it can be inactivated as described above. The degree of is only one of the criteria for inactivation.

As used herein, the term "host cell" refers to a cell into which a vector is introduced and transformed, and is also called a "host" or a "transformant". Host cells, before and after transformation, are sometimes referred to herein simply as "cells." Cells used as hosts are not particularly limited as long as they are cells into which the vector can be introduced.

"Gene containing the base sequence shown in SEQ ID NO: 1, gene containing the base sequence shown in SEQ ID NO: 3, gene containing the base sequence shown in SEQ ID NO: 5, gene containing the base sequence shown in SEQ ID NO: 7, and SEQ ID NO: 9 A gene containing a nucleotide sequence and/or a gene containing the nucleotide sequence shown in SEQ ID NO: 11" is the nucleotide sequence of four chromosomal DNAs of Komagataella pastoris (ATCC76273 strain: ACCESSION No. FR839628 to FR839631 (J. Biotechnol. 154 (4), 312-320 (2011)), and GS115 strain: ACCESSION No. FN392319 to FN392322 (Nat. Biotechnol. 27 (6), 561-566 (2009))). Specifically, the present inventors searched for a polypeptide whose inactivation improves the productivity of the target protein, and a polynucleotide containing a nucleotide sequence encoding the amino acid sequence. As a result, the following polynucleotides were found.

A polynucleotide comprising a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2 (ACCESSION No. CCA39734) in the ATCC76273 strain, and a nucleotide sequence represented by SEQ ID NO: 1 encoding the polypeptide;
A polynucleotide comprising a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 4 (ACCESSION No. CCA38267) in the ATCC76273 strain, and a nucleotide sequence represented by SEQ ID NO: 3 encoding the polypeptide;
A polynucleotide comprising a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 6 (ACCESSION No. CCA37956) in the ATCC76273 strain, and a nucleotide sequence represented by SEQ ID NO: 5 encoding the polypeptide;
A polynucleotide comprising a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 8 (ACCESSION No. CCA37582) in the ATCC76273 strain, and a nucleotide sequence represented by SEQ ID NO: 7 encoding the polypeptide;
A polynucleotide comprising a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 10 (ACCESSION No. CCA39503) in the ATCC76273 strain, and a nucleotide sequence represented by SEQ ID NO: 9 encoding the polypeptide;
A polynucleotide comprising the polypeptide represented by the amino acid sequence represented by SEQ ID NO: 12 (ACCESSION No. CCA38927) in strain ATCC76273 and the base sequence represented by SEQ ID NO: 11 encoding the polypeptide.

In the examples described later, the present inventors introduced a vector that inactivates the gene consisting of the above nucleotide sequence into a host, and confirmed that the productivity of the target protein was improved.

Gene containing the base sequence shown in SEQ ID NO: 1, gene containing the base sequence shown in SEQ ID NO: 3, gene containing the base sequence shown in SEQ ID NO: 5, gene containing the base sequence shown in SEQ ID NO: 7, base shown in SEQ ID NO: 9 A gene containing the sequence and/or a gene containing the nucleotide sequence shown in SEQ ID NO: 11, or a modified gene thereof may be inactivated singly, and multiple genes may be inactivated in one host cell. may have been When more than one of the genes is inactivated, the number of genes to be inactivated may be 2, 3, 4, 5, or 6. Specific examples of inactivation of multiple genes include inactivation of a gene containing the nucleotide sequence shown in SEQ ID NO: 1 and a gene containing the nucleotide sequence shown in SEQ ID NO: 3, or modified genes thereof, SEQ ID NO: Inactivation of the gene comprising the nucleotide sequence shown in SEQ ID NO: 1, the gene comprising the nucleotide sequence shown in SEQ ID NO: 3, and the gene comprising the nucleotide sequence shown in SEQ ID NO: 5, or their modified genes, and the nucleotide sequence shown in SEQ ID NO: 1 , the gene containing the nucleotide sequence shown in SEQ ID NO: 3, and the gene containing the nucleotide sequence shown in SEQ ID NO: 7, or their modified genes, the gene containing the nucleotide sequence shown in SEQ ID NO: 1, the sequence Examples include inactivation of the gene containing the nucleotide sequence shown in No. 3, the gene containing the nucleotide sequence shown in SEQ ID NO: 5, the gene containing the nucleotide sequence shown in SEQ ID NO: 7, or modified genes thereof.

As used herein, the increase in protein production from the host cell is, for example, 1.01-fold, 1.02-fold, 1.03-fold, 1.04-fold, 1.05-fold, 1.1-fold relative to the protein production in the parent cell or wild-type strain. , 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, or 5x or more, or 100x It may be 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. When a protein is produced by secretion, the amount of total protein secreted from cells can be easily determined by methods known to those skilled in the art, such as the Bradford method, Lowry method, and BCA method, using cell culture supernatants and the like. be able to. The amount of a specific protein secreted from a cell can be easily determined by ELISA or the like using cell culture supernatant or the like.

As used herein, the term "parent cell", "wild-type strain", or "host cell before introduction" refers to the expression of a gene containing the nucleotide sequence shown in any of (a) to (d) above. means a host cell or strain that has not been subjected to such treatment. Therefore, the "parent cell", "wild-type strain", or "host cell before introduction" described herein includes genes other than the nucleotide sequence shown in any of (a) to (d) above. Also included are genetically modified host cells or strains.

Gene containing the base sequence shown in SEQ ID NO: 1, gene containing the base sequence shown in SEQ ID NO: 3, gene containing the base sequence shown in SEQ ID NO: 5, gene containing the base sequence shown in SEQ ID NO: 7, base shown in SEQ ID NO: 9 As an example of inactivating the gene containing the sequence and/or the gene containing the nucleotide sequence shown in SEQ ID NO: 11, as in the examples described later, the complementary sequence and 100% sequence of the promoter located upstream of the gene A nucleotide sequence having identity and a nucleotide sequence having 100% sequence identity with the complementary sequence of the terminator located downstream of the gene are prepared as PCR products using primers 1 to 46, and the PCR products are used as vectors. Examples include a method of transforming into Komagataella yeast.

"Gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, sequence A gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in No. 6, a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10 and/or a gene encoding an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12" A gene consisting of a nucleotide sequence designed with reference to the codon table based on a polypeptide consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, and 12. , and combinations thereof. A gene consisting of a base sequence designed with reference to the codon table based on a polypeptide consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, and 12 include, for example, the genes represented by SEQ ID NOs: 1, 3, 5, 7, 9 and 11, respectively. By inactivating the gene, the yield of the target protein can be improved. The sequence identity is 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more, or 99% or more. preferable.

The host cell of the present invention comprises a nucleotide sequence having a region homologous to the gene according to any one of (a) to (d) above, and a promoter and homologous region of the gene according to any one of (a) to (d) above. and / or a nucleotide sequence having a region homologous to the gene terminator according to any one of (a) to (d) above. is preferred.

The present invention provides a nucleotide sequence having a region homologous to the gene according to any one of (a) to (d) above, and a nucleotide having a region homologous to the promoter of the gene according to any one of (a) to (d) above. by transforming a vector containing at least one of the sequence and a nucleotide sequence having a region homologous to the terminator of the gene according to any one of (a) to (d) above, on the chromosome of the host cell The gene according to any one of (a) to (d) can be inactivated. A vector may be used in which all nucleotide sequences having respective homologous regions are incorporated, or vectors may be used in which each nucleotide sequence having homologous regions is incorporated one by one.

The vectors of the present invention can be circular vectors, linear vectors, plasmids, artificial chromosomes, and the like.

A vector in the present invention is an artificially constructed nucleic acid molecule. A nucleic acid molecule constituting a vector of the present invention is usually DNA, preferably double-stranded DNA, and may be circular or linear. The vector of the present invention usually has a predetermined base sequence (the base sequence of any of the above (a) to (d), homologous to the promoter of the gene of any of the above (a) to (d)). A base sequence having a region, a base sequence having a region homologous to the gene terminator described in any one of (a) to (d) above, or a plurality of these base sequences linked together (other Cloning site containing one or more restriction enzyme recognition sites, In-Fusion cloning system from Clontech, Gibson Assembly system from New England Biolabs, etc. Overlapping region for utilizing , base sequence of endogenous gene, base sequence encoding polypeptide consisting of target protein amino acid sequence, base sequence of selectable marker gene (auxotrophic complementary gene, drug resistance gene, etc.), etc. can include Examples of linear vectors include auxotrophic complementary genes such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, G418 resistance gene, Zeocin (trademark) resistance gene, hygromycin resistance gene, Clone NAT resistance. DNA, a PCR product having a base sequence of a drug-resistant gene such as a blasticidin S-resistant gene, or a circular vector or plasmid cleaved with an appropriate restriction enzyme to form a linear chain. Examples of plasmids include YEp vector, YRp vector, YCp vector, pPICHOLI (http://www.mobitec.com/cms/products/bio/04_vector_sys/p_piccholi_shuttle_vector.html), pHIP (Journal of General Microbioogy (1992), 138, 2405-2416. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha), pHRP (see above references 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), and the like can be used. Examples of artificial chromosomes generally refer to artificial chromosome vectors containing centromere DNA sequences, telomere DNA sequences, autonomously replicating sequences (ARS), and yeast artificial chromosomes (YAC vectors) for yeast, such as Saccharomyces cerevisiae and Developed in Schizosaccharomyces pombe and others. In Komagataella pastoris, an artificial chromosome can be constructed by using the centromere DNA sequence described in WO 2016/088824.

In the present invention, "transformation" refers to introduction of the vector into cells. A method for introducing a vector into a host cell, that is, a transformation method, can appropriately use known methods. For example, when yeast cells are used as a host, electroporation method, lithium acetate method, spheroplast method, etc. are not particularly limited to these. For example, as a transformation method of Komagataera pastoris, the electroporation method described in High efficiency transformation by electroporation of Pichia pastoris pretreated with lithiumacetate and dithiothreitol (Biotechniques. 2004 Jan;36(1):152-4.) is common.

In the present invention, when a vector is transformed into a yeast of the genus Komagataella, it is preferable to use a selectable marker gene such as an auxotrophic complementary gene or a drug resistance gene. Selection markers are not particularly limited, but in the case of yeast of the genus Komagata, uracil, leucine, adenine, histidine, and arginine in the case of auxotrophic complementary genes such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, and ARG4 gene, respectively. Transformants can be selected by restoration of the prototrophic strain phenotype in an auxotrophic strain of . For drug resistance genes such as G418 resistance gene, Zeocin (trademark) resistance gene, hygromycin resistance gene, Clone NAT resistance gene, blasticidin S resistance gene, G418, Zeocin (trademark), hygromycin, Clone Transformants can be selected by resistance on a medium containing NAT, blasticidin S. The auxotrophic selectable marker used when producing the yeast host cannot be used unless the selectable marker is destroyed in the host. In this case, the selection marker may be destroyed in the host, and methods known to those skilled in the art can be used.

In the present invention, when a vector is to be integrated into a chromosome, it is preferably prepared as a DNA having a chromosomal homologous region at the same time as in this example. The method of vector integration onto the chromosome may be non-specific integration without using a homologous region, integration using a single homologous region, or double crossover type using two homologous regions. may be incorporated.

In the present invention, in the case of integration using homologous regions, transformation can be carried out with homologous regions at both ends of the selectable marker gene sequence, as in this example. For example, when a gene is desired to be deleted, it can be incorporated using a vector in which the promoter of the gene to be deleted is linked upstream of the selectable marker gene and the terminator of the gene to be deleted is linked downstream.

In the present invention, the number of copies of the vector per chromosome is not particularly limited. In addition, the position on the chromosome where the vector is integrated is not particularly limited as long as the gene of the present invention is inactivated. Regarding the integration sites of transformants in which two or more copies of the vector are integrated, a plurality of vectors may be integrated at the same site, or one or more copies may be integrated at different sites.

The "nucleotide sequence having a homologous region" of the present invention may be a nucleotide sequence having at least a part of the nucleotide sequence of the target nucleotide sequence and having a sequence identity of 50% or more, more preferably 70 % or more, more preferably 80% or more, particularly preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, most preferably 100%. In addition, the length of the base sequence having the homologous region is not particularly limited as long as it is a base sequence of 20 bp or more having the homologous region with the target base sequence because homologous recombination can be performed. 0.1% or more of the length of the target base sequence is preferable, 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more % or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, 7.5% or more, 8% or more, 8.5 % or more, 9% or more, 9.5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55 % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100%.

The "promoter of any of the genes (a) to (d)" of the present invention is present upstream of any of the genes (a) to (d) on the chromosome and increases the expression level of the gene product. A nucleotide sequence to be adjusted, specifically, a nucleotide sequence up to 5000 bp upstream from the initiation codon of any of genes (a) to (d).

The "terminator of any of the genes (a) to (d)" of the present invention is present downstream of any of the genes (a) to (d) on the chromosome and regulates the expression level of the gene product. A nucleotide sequence to be adjusted, specifically, a nucleotide sequence up to 5000 bp downstream from the termination codon of any of genes (a) to (d).

The host cell of the present invention comprises a nucleotide sequence having a region homologous to the gene according to any one of (a) to (d) above, and a promoter and homologous region of the gene according to any one of (a) to (d) above. and / or a nucleotide sequence having a region homologous to the gene terminator according to any one of (a) to (d) above, upstream or downstream of the nucleotide sequence It is preferably obtained by transforming a vector containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of the target protein.

A nucleotide sequence having a region homologous to the gene according to any one of (a) to (d), a nucleotide sequence having a region homologous to the promoter of the gene according to any one of (a) to (d), and/ Alternatively, a vector in which a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of the target protein is arranged upstream or downstream of the nucleotide sequence having a region homologous to the terminator of the gene according to any one of (a) to (d). A host cell with improved productivity of the target protein can be obtained by transformation. Furthermore, a transformation operation is performed by placing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of the target protein upstream and/or downstream of the nucleotide sequence in a vector containing the nucleotide sequence having each homologous region. can improve sexuality.

In the present invention, the "target protein" is a protein produced by a cell into which the vector of the present invention has been introduced, and may be an endogenous protein of the host or a heterologous protein. Examples of target proteins include enzymes derived from microorganisms, proteins produced by multicellular organisms such as animals and plants, and the like. Examples include 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. However, it is not limited to these. A specific example of a glucosidase is BGL1. A specific example of a fluorescent protein is mUkG1. In particular, human and/or animal therapeutic proteins are preferred.

Specific examples of human and/or animal therapeutic proteins 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);

Here, "antibody" refers to a heterotetrameric protein composed of two polypeptide chains, L chain and H chain, formed by disulfide bonds, and if it has the ability to bind to a specific antigen , is not particularly limited.

Here, "partial antibody" refers to Fab-type antibody, (Fab) 2-type antibody, scFv-type antibody, diabody-type antibody, and derivatives thereof, etc., as long as they have the ability to bind to a specific antigen. is not particularly limited. Fab type antibody refers to a heteromeric protein in which the L chain and Fd chain of an antibody are bound by an S-S bond, or a heteromeric protein in which an L chain and an Fd chain of an antibody are associated without an S-S bond, and a specific antigen is not particularly limited as long as it has the ability to bind to Specific examples of partial antibodies include anti-lysozyme single-chain antibodies.

Amino acids constituting the target protein may be natural or non-natural, or may be modified. In addition, the amino acid sequence of the protein may be artificially modified or designed de-novo.

The host cell of the present invention comprises a nucleotide sequence having a region homologous to the gene according to any one of (a) to (d) above, and a promoter and homologous region of the gene according to any one of (a) to (d) above. and/or a vector containing at least one nucleotide sequence having a region homologous to the gene terminator according to any one of (a) to (d) above, and the amino acid sequence of the target protein It is preferably obtained by transforming a vector containing a nucleotide sequence encoding a polypeptide.

A nucleotide sequence having a region homologous to the gene according to any one of (a) to (d), a nucleotide sequence having a region homologous to the promoter of the gene according to any one of (a) to (d), and/ or a vector containing at least one nucleotide sequence having a region homologous to the terminator of the gene according to any one of (a) to (d) above, and a base encoding a polypeptide consisting of the amino acid sequence of the target protein. By transforming with a vector containing the sequence, a host cell with improved productivity of the desired protein can be obtained. Furthermore, by separating the vector to be inactivated from the vector containing the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of the target protein, the vector containing the nucleotide sequence encoding the target protein can be obtained from (a) to Increasing the expression level by introducing one or more copies at a position other than the position of the gene described in any of (d), Plasmids and artificials in which one or more copies of a base sequence encoding a polypeptide consisting of the amino acid sequence of the target protein are present Introducing a chromosome to increase the expression level, and disrupting the auxotrophic complementary gene or drug resistance gene used for inactivating the gene according to any one of (a) to (d) in breeding improvement Since the base sequence encoding the polypeptide consisting of the amino acid sequence of the target protein is also prevented from being destroyed at the same time, the productivity of the target protein can be efficiently improved.

As used herein, "expression" refers to the transcription and translation of a nucleotide sequence that results in the production of a polypeptide. Moreover, the expression may be almost constant without depending on external stimuli, growth conditions, etc., or may depend on it. A promoter that drives expression is not particularly limited as long as it drives expression of a nucleotide sequence encoding a polypeptide.

The biological species of the host cell is not particularly limited, but includes yeast, bacteria, fungi, insect cells, animal cells, and plant cells. Yeast is preferred, and methanol-assimilating yeast, fission yeast, and budding yeast are more preferred. Methanol-assimilating yeast is more preferred. In general, methanol-utilizing yeast is defined as yeast that can be cultured using methanol as the sole carbon source. Yeast that has lost its performance is also included in the methanol-assimilating yeast of the present invention.

Methanol-utilizing yeast includes yeast belonging to the genus Pichia, Ogataea, Candida, Torulopsis, Komagataella, and the like. Pichia methanolica in the genus Pichia, Ogataea angusta in the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea parapolymorpha, Ogataea minuta ), Candida boidinii in the genus Candida, and Komagataella pastoris and Komagataella phaffii in the genus Komagataella.

Among the above methanol-assimilating yeasts, yeast of the genus Komagataella or yeast of the genus Ogataea are particularly preferred.

As the yeast of the genus Komagataella, Komagataella pastoris and Komagataella phaffii are preferred. Both Komagataera pastoris and Komagataera fafi are also known as Pichia pastoris.

Specific examples of strains that can be used as hosts include strains such as Komagataella pastoris ATCC76273 (Y-11430, CBS7435) and Komagataella pastoris X-33. These strains are available from the American Type Culture Collection, Thermo Fisher Scientific, and the like.

Preferred yeasts of the genus Ogataea are Ogataea angusta, Ogataea polymorpha, and Ogataea parapolymorpha. These three are closely related species, all also known as Hansenula polymorpha, or Pichia angusta.

Specific usable strains include strains such as Ogataea angusta NCYC495 (ATCC14754), Ogataea polymorpha 8V (ATCC34438), and Ogataea parapolymorpha DL-1 (ATCC26012). These strains are available from the American Type Culture Collection and the like.

In addition, in the present invention, strains derived from these strains of the genus Komagataella yeast and the genus Ogataea yeast can also be used. As auxotrophic strains, BY4329 derived from NCYC495, BY5242 derived from 8V, BY5243 derived from DL-1 (these are available from the National BioResource Project), and the like can be mentioned. In the present invention, derivative strains and the like from these strains can also be used.

In one aspect, the present invention relates to a method for producing a target protein, comprising the steps of culturing the cells described above and recovering the target protein from the culture medium.

As used herein, the term "culture solution" means culture supernatant, cultured cells, cultured bacterial bodies, or disrupted cells or bacterial bodies. Therefore, as a method of producing a target protein using the transformed yeast of the present invention, a method of culturing the above-described cells and accumulating them in the cells, or a method of secreting and accumulating them in the culture supernatant. and preferably a method of secreting and accumulating in the culture supernatant.

Cell culture conditions are not particularly limited, and may be appropriately selected according to the cells. In the culture, any medium can be used as long as it contains nutrients that can be assimilated by the cells. The nutrient sources include sugars 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, soybean oil, Oils such as rapeseed oil, or carbon sources such as mixtures thereof, nitrogen sources such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, and other inorganic salts, vitamins, etc. A normal medium in which nutrients are appropriately mixed and blended can be used. In addition, the culture can be either batch culture or continuous culture.

In a preferred embodiment of the present invention, when yeast of the genus Komagataella or yeast of the genus Ogataea is used, the carbon source may be one of glucose, glycerol, and methanol, or two or more of them. Moreover, these carbon sources may be present from the beginning of the culture, or may be added during the culture.

By culturing a transformant obtained by introducing the vector of the present invention into a cell, the target protein can be accumulated in the host or in the culture medium and recovered. As for the recovery method of the target protein, known purification methods can be appropriately combined and used. For example, first, the transformed yeast is cultured in an appropriate medium, and the culture supernatant is centrifuged or filtered to remove cells from the culture supernatant. The obtained culture supernatant is subjected to salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography. The target protein is recovered from the culture supernatant by using techniques such as lithography, affinity chromatography, reversed-phase chromatography, ultrafiltration, etc. alone or in combination.

Cells can be cultured under general conditions. For example, yeast is cultured aerobically at a pH of 2.5 to 10.0 and a temperature range of 10°C to 48°C for 10 hours to 10 days. It can be done by

The recovered target protein can be used as it is, or can be used after being subjected to modifications such as PEGylation that bring about pharmacological changes, and modifications that add functions such as enzymes and isotopes. Various formulation processes may also be used.

When a non-secretable target protein is to be extracellularly secreted, a nucleotide sequence encoding a signal sequence may be introduced into the 5' end of the target protein gene. The nucleotide sequence encoding the signal sequence is not particularly limited as long as it encodes a signal sequence that can be secreted and expressed by yeast. Nucleotide sequences encoding Komagataella pastoris acid phosphatase (PHO1), Saccharomyces cerevisiae invertase (SUC2), Saccharomyces cerevisiae PLB1, bovine serum albumin (BSA), human serum albumin (HSA), and immunoglobulin signal sequences etc.

As used herein, the "target protein" that increases production may be an endogenous protein of the host, a heterologous protein, or a foreign protein. As used herein, the term "endogenous protein" refers to a protein produced by a non-genetically modified host cell during culture. On the other hand, the term "heterologous protein" or "foreign protein" as used herein refers to a protein that is not normally expressed in host cells that have not undergone genetic modification, or that, even if expressed, the amount of expression or production is insufficient. say.

The promoter that expresses the target protein is not particularly limited, as long as a promoter that allows expression with the selected carbon source is appropriately used.

When the carbon source is methanol, the promoter for expressing the polypeptide includes, but is not limited to, AOX1 promoter, AOX2 promoter, CAT promoter, DHAS promoter, FDH promoter, FMD promoter, GAP promoter, MOX promoter and the like.

When the carbon source is glucose or glycerol, promoters for polypeptide expression include, but are not limited to, GAP promoter, TEF promoter, LEU2 promoter, URA3 promoter, ADE promoter, ADH1 promoter, PGK1 promoter, and the like.

As used herein, the term "promoter" refers to a nucleotide sequence region located upstream of the above-described predetermined nucleotide sequence, and RNA polymerase and various transcriptional regulatory factors involved in the promotion and suppression of transcription bind to or act on the region. By doing so, the above-mentioned predetermined nucleotide sequence, which is a template, is read and complementary RNA is synthesized (transcribed).

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these.

<Example 1: Preparation of various genes used for vector preparation>
Detailed operating procedures for the recombinant DNA techniques used in the following examples are described in the following publications: Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene et al., 1989). Publishing Associates and Wiley-Interscience).

In addition, in the following examples, the plasmid used for yeast transformation was obtained by introducing the constructed vector into Escherichia coli E. coli DH5α competent cells (manufactured by Takara Bio Inc.) and culturing the resulting transformant for amplification. It was prepared by A plasmid was prepared from the plasmid-carrying strain using FastGene Plasmid Mini Kit (manufactured by Nippon Genetics).

The AOX1 promoter (SEQ ID NO: 71), AOX1 terminator (SEQ ID NO: 72), GAP promoter (SEQ ID NO: 73), and CCA38473 terminator (SEQ ID NO: 74) used in the construction of the vector are the chromosomal DNA of Komagataella pastoris ATCC76273 strain (nucleotide sequence is EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) mixture was used as a template and prepared by PCR. AOX1 promoter is primer 59 (SEQ ID NO: 77) and primer 60 (SEQ ID NO: 78), AOX1 terminator is primer 61 (SEQ ID NO: 79) and primer 62 (SEQ ID NO: 80), GAP promoter is primer 63 (SEQ ID NO: 81) and primer 64 (SEQ ID NO: 82), the CCA38473 terminator was prepared by PCR using primer 65 (SEQ ID NO: 83) and primer 66 (SEQ ID NO: 84).

A promoter-controlled Zeocin™ resistance gene (SEQ ID NO: 75) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. A promoter-controlled G418 resistance gene (SEQ ID NO: 76) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. A promoter-controlled hygromycin resistance gene (SEQ ID NO: 92) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. A promoter-controlled Clone NAT resistance gene (SEQ ID NO: 93) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. A promoter-controlled blasticidin S resistance gene (SEQ ID NO: 94) used in the construction of the vector was prepared by PCR using synthetic DNA as a template. The anti-lysodium single-chain antibody gene (SEQ ID NO: 89) having a mating factor α pre-pro signal sequence, which was used in constructing the vector, was prepared by PCR using a synthetic DNA as a template. The BGL1 gene (SEQ ID NO: 90) having a mating factor α pre-pro signal sequence, which was used in constructing the vector, was prepared by PCR using synthetic DNA as a template. The mUkG1 gene (SEQ ID NO: 91) having a mating factor α pre-pro signal sequence, which was used in constructing the vector, was prepared by PCR using synthetic DNA as a template.

Prime STAR HS DNA Polymerase (manufactured by Takara Bio Inc.) or the like was used for PCR, and the reaction conditions were performed according to the method described in the attached manual. Chromosomal DNA was prepared from Komagataella pastoris ATCC76273 strain using Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka) or the like under the conditions described therein.

<Example 2: Construction of anti-lysozyme single-chain antibody expression vector, BGL1 expression vector, and mUkG1 expression vector>
A gene fragment (SEQ ID NO: 95) having a HindIII-BamHI-BglII-XbaI-EcoRI multicloning site was totally synthesized and inserted between the HindIII-EcoRI sites of pUC19 (Takara Bio Inc., Code No. 3219). and constructed pUC-1.

In addition, a nucleic acid fragment with BamHI recognition sequences added to both sides of the AOX1 promoter (SEQ ID NO: 71) was prepared by PCR using primers 59 (SEQ ID NO: 77) and 60 (SEQ ID NO: 78). was inserted into the BamHI site to construct pUC-Paox.

In addition, a nucleic acid fragment with BamHI recognition sequences added to both sides of the GAP promoter (SEQ ID NO: 73) was prepared by PCR using primers 63 (SEQ ID NO: 81) and 64 (SEQ ID NO: 82), and treated with BamHI. was inserted into the BamHI site to construct pUC-Pgap.

Next, a nucleic acid fragment with EcoRI recognition sequences added to both sides of the promoter-controlled G418 resistance gene (SEQ ID NO: 76) was prepared by PCR using primers 69 (SEQ ID NO: 87) and 70 (SEQ ID NO: 88). , was inserted into the EcoRI site of pUC-Paox or pUC-Pgap after EcoRI treatment to construct pUC-PaoxG418 or pUC-PgapG418.

Next, a nucleic acid fragment having XbaI and SpeI recognition sequences added to the CCA38473 terminator (SEQ ID NO: 74) was prepared by PCR using primers 65 (SEQ ID NO: 83) and 66 (SEQ ID NO: 84), and treated with XbaI and SpeI. It was inserted into the XbaI site of pUC-PaoxG418 or pUC-PgapG418 to construct pUC-PaoxT38473G418 or pUC-PgapT38473G418.

Next, a nucleic acid fragment with XbaI recognition sequences added to both sides of the AOX1 terminator (SEQ ID NO: 72) was prepared by PCR using primers 61 (SEQ ID NO: 79) and 62 (SEQ ID NO: 80). It was inserted into the XbaI site of PaoxT38473 or pUC-PgapT38473G418 to construct pUC-PaoxTaoxT38473G418 or pUC-PgapTaoxT38473G418.

Next, a nucleic acid fragment with BglII recognition sequences added to both sides of the anti-lysozyme single-chain antibody gene (SEQ ID NO: 89) to which the mating factor α pre-pro signal sequence was added was used with primers 67 (SEQ ID NO: 85) and 68 (SEQ ID NO: 86). ), and inserted into the BglII site of pUC-PaoxTaoxT38473G418 or pUC-PgapTaoxT38473G418 after BglII treatment to construct pUC-PaoxscFvTaoxT38473G418 or pUC-PgapscFvTaoxT38473G418. This pUC-PaoxscFvTaoxT38473G418 is designed to express an anti-lysozyme single chain antibody under control of the AOX1 promoter. This pUC-PgapscFvTaoxT38473G418 is designed to express an anti-lysodium single chain antibody under the control of the GAP promoter.

Next, a nucleic acid fragment having BglII recognition sequences added to both sides of the BGL1 gene (SEQ ID NO: 90) to which the mating factor α prepro signal sequence was added was subjected to PCR using primers 67 (SEQ ID NO: 85) and 71 (SEQ ID NO: 96). and inserted into the BglII site of pUC-PaoxTaoxT38473G418 after BglII treatment to construct pUC-PaoxBGL1TaoxT38473G418. This pUC-PaoxBGL1TaoxT38473G418 is designed to express BGL1 under the control of the AOX1 promoter.

Next, a nucleic acid fragment with BglII recognition sequences added to both sides of the mUkG1 gene (SEQ ID NO: 91) to which the mating factor α prepro signal sequence was added was subjected to PCR using primers 67 (SEQ ID NO: 85) and 72 (SEQ ID NO: 97). and inserted into the BglII site of pUC-PaoxTaoxT38473G418 after BglII treatment to construct pUC-PaoxmUkG1TaoxT38473G418. This pUC-PaoxmUkG1TaoxT38473G418 is designed to express mUkG1 under the control of the AOX1 promoter.

<Example 3: Construction of gene inactivation vector consisting of nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11>
The upstream sequence (promoter) of each gene was prepared by PCR using a chromosomal DNA mixture of Komagataella pastoris ATCC76273 strain as a template. Table 1 shows the vector ID, the ACCESSION No. of the polypeptide to be inactivated, the amino acid sequence of the polypeptide, the nucleotide sequence encoding the polypeptide, and the numbers and sequences of the primers (Fw and Re) used to amplify the nucleic acid fragment. .

Next, downstream sequences (terminators) of each gene were prepared by PCR using a chromosomal DNA mixture of Komagataella pastoris ATCC76273 strain as a template. Table 2 shows the vector ID, the ACCESSION No. of the polypeptide to be inactivated, and the numbers and sequences of the primers (Fw and Re) used to amplify the nucleic acid fragment.

Next, as drug resistance genes, a promoter-controlled hygromycin resistance gene (SEQ ID NO: 92), a promoter-controlled Clone NAT resistance gene (SEQ ID NO: 93), a promoter-controlled blasticidin S resistance gene ( SEQ ID NO: 94), a promoter-controlled Zeocin (trademark) resistance gene (SEQ ID NO: 75) as a template, and homologous regions of the gene upstream sequence and downstream sequence for overlap PCR are added to both sides of the gene fragment, Prepared by PCR. Table 3 shows the vector ID, the ACCESSION No. of the polypeptide to be inactivated, the numbers and sequences of the primers (Fw and Re) used to amplify the nucleic acid fragment, and the template.

Next, the upstream nucleic acid fragment, downstream nucleic acid fragment, and gene fragment obtained above are mixed for each vector ID, and the mixture is used as a template, and homologous regions of the gene upstream sequence and downstream sequence for transformation are further added on both sides. The added gene fragment was prepared by overlap PCR. Table 4 shows the vector ID, the ACCESSION No. of the polypeptide to be inactivated, and the numbers and sequences of the primers (Fw and Re) used to amplify the nucleic acid fragment.

This vector is the upstream nucleotide sequence (promoter) and downstream nucleotide sequence of the gene consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11 of Komagataella pastoris ATCC76273 strain. (terminator) as a homologous region, a vector added to both sides of a promoter-controlled drug resistance gene. It is designed to inactivate the genes consisting of the nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11.

<Example 4: Acquisition of transformed yeast>
Using the anti-lysozyme single-chain antibody expression vector pUC-PaoxscFvTaoxT38473G418 or pUC-PgapscFvTaoxT38473G418, the BGL1 expression vector pUC-PaoxBGL1TaoxT38473G418, and the mUkG1 expression vector pUC-PaoxmUkG1TaoxT38473G418 constructed in Example 2, Komagataella pastis was transformed as follows. .

Komagataella pastoris ATCC76273 strain was inoculated into 3 ml of YPD medium (1% yeast extract bacto (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical Co., Ltd.), 2% glucose) and cultured overnight at 30°C with shaking. , to obtain a pre-culture. 500 μl of the resulting preculture was inoculated into 50 ml of YPD medium, cultured with shaking until OD600 reached 1 to 1.5, harvested (3000×g, 10 minutes, 20°C), and added to 250 μl of 1M DTT (final concentration 25 mM) in 10 ml of 50 mM potassium phosphate buffer, pH 7.5.

After incubating this suspension at 30°C for 15 minutes, the cells were harvested (3000 x g, 10 minutes, 20°C) and added to 50 ml of ice-cooled STM buffer (270 mM sucrose, 10 mM Tris-HCl, 1 mM magnesium chloride, pH 7). .5). The washed solution was collected (3000×g, 10 minutes, 4° C.), washed again with 25 ml of STM buffer, and collected (3000×g, 10 minutes, 4° C.). Finally, they were suspended in 250 μl of ice-cold STM buffer to obtain a competent cell solution.

E. coli was transformed with the anti-lysodium single-chain antibody expression vector pUC-PaoxscFvTaoxT38473G418 or pUC-PgapscFvTaoxT38473G418 constructed in Example 2, and the resulting transformant was added to 5 ml of ampicillin-containing 2YT medium (1.6% tryptone bacto (Becton Dickinson), 1% yeast extract bacto (Becton Dickinson), 0.5% sodium chloride, 0.01% ampicillin sodium (Wako Pure Chemical Industries, Ltd.)), and the resulting cells were extracted with the FastGene Plasmid Mini Kit ( Nippon Genetics) was used to obtain pUC-PaoxscFvTaoxT38473G418 or pUC-PgapscFvTaoxT38473G418. This plasmid was linearized by restriction enzyme treatment.

60 μl of this competent cell solution and 1 μl of linear pUC-PaoxscFvTaoxT38473G418 solution or 1 μl of pUC-PgapscFvTaoxT38473G418 solution were mixed and transferred into an electroporation cuvette (disposable cuvette electrode, electrode spacing 2 mm (manufactured by BM Instruments)), After being subjected to 7.5 kV/cm, 25 μF and 200 Ω, the cells were suspended in 1 ml of YPD medium and allowed to stand at 30° C. for 1 hour. After standing for 1 hour, cells were collected (3000×g, 5 minutes, 20° C.), and 961 μl of the supernatant was discarded. After resuspension with the remaining solution, YPDG418 selective agar plate (1% yeast extract (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical), 2% glucose, 1.5% agarose, 0.05% G418 disulfate ( manufactured by Nacalai Tesque)), and strains that grow in stationary culture at 30°C for 3 days were selected to obtain anti-lysozyme single-chain antibody-expressing yeast.

Similarly, BGL1-expressing yeast was obtained using BGL1-expressing vector pUC-PaoxBGL1TaoxT38473G418, and mUkG1-expressing yeast was obtained using mUkG1-expressing vector pUC-PaoxmUkG1TaoxT38473G418.

Subsequently, gene inactivation vectors (CCA39734_Hyg, CCA38267_Hyg, CCA38267_Hyg, CCA37956_Hyg, CCA37582_Hyg, CCA39503_Hyg, CCA38927_Hyg) were used to transform anti-lysozyme single-chain antibody-expressing yeast, BGL1-expressing yeast, and mUkG1-expressing yeast as follows.

Inoculate 3 ml of YPD medium (1% yeast extract bacto (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical Co., Ltd.), 2% glucose) with anti-lysozyme single-chain antibody-expressing yeast, BGL1-expressing yeast, and mUkG1-expressing yeast. and cultured with shaking overnight at 30°C to obtain a preculture. 500 μl of the resulting preculture was inoculated into 50 ml of YPD medium, cultured with shaking until OD600 reached 1 to 1.5, harvested (3000×g, 10 minutes, 20°C), and added to 250 μl of 1M DTT (final concentration 25 mM) in 10 ml of 50 mM potassium phosphate buffer, pH 7.5.

After incubating this suspension at 30°C for 15 minutes, the cells were harvested (3000 x g, 10 minutes, 20°C) and added to 50 ml of ice-cooled STM buffer (270 mM sucrose, 10 mM Tris-HCl, 1 mM magnesium chloride, pH 7). .5). The washed solution was collected (3000×g, 10 minutes, 4° C.), washed again with 25 ml of STM buffer, and collected (3000×g, 10 minutes, 4° C.). Finally, they were suspended in 250 μl of ice-cold STM buffer to obtain a competent cell solution.

60 μl of this competent cell solution and a gene inactivation vector consisting of the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11 constructed in Example 3 ( 3 μl of CCA39734_Hyg, CCA38267_Hyg, CCA37956_Hyg, CCA37582_Hyg, CCA39503_Hyg, CCA38927_Hyg) was mixed, transferred to an electroporation cuvette (disposable cuvette electrode, electrode spacing 2 mm (manufactured by BM Co., Ltd.), and subjected to 7.5 kV/cm, 25 μF, 200 Ω. After that, the cells were suspended in 1 ml of YPD medium and allowed to stand for 1 hour at 30° C. After standing for 1 hour, the cells were harvested (3000×g, 5 minutes, 20° C.), and 963 μl of the supernatant was discarded. After resuspension with the remaining solution, YPDHyg selective agar plate (1% yeast extract (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical Co., Ltd.), 2% glucose, 1.5% agarose, 100 mg/L hygromycin B (manufactured by Wako Pure Chemical Industries, Ltd.)) and select strains that grow in static culture at 30 ° C. for 3 days. Anti-lysozyme single-chain antibody-expressing yeast, BGL1-expressing yeast, and mUkG1-expressing yeast in which the gene consisting of the nucleotide sequence shown in No. 11 was inactivated were obtained.

Subsequently, using the gene inactivation vectors (CCA39734_NAT, CCA37956_bsd, CCA37582_Zeo, CCA39503_bsd) consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 constructed in Example 3, Anti-lysozyme single-chain antibody-expressing yeast, BGL1-expressing yeast, and mUkG1-expressing yeast were further transformed as follows.

3 ml of YPD medium (1% yeast extract bacto (Becton Dickinson company), 2% polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.), 2% glucose) and cultured with shaking overnight at 30°C to obtain a preculture solution. 500 μl of the resulting preculture was inoculated into 50 ml of YPD medium, cultured with shaking until OD600 reached 1 to 1.5, harvested (3000×g, 10 minutes, 20°C), and added to 250 μl of 1M DTT (final concentration 25 mM) in 10 ml of 50 mM potassium phosphate buffer, pH 7.5.

After incubating this suspension at 30°C for 15 minutes, the cells were harvested (3000 x g, 10 minutes, 20°C) and added to 50 ml of ice-cooled STM buffer (270 mM sucrose, 10 mM Tris-HCl, 1 mM magnesium chloride, pH 7). .5). The washed solution was collected (3000×g, 10 minutes, 4° C.), washed again with 25 ml of STM buffer, and collected (3000×g, 10 minutes, 4° C.). Finally, they were suspended in 250 μl of ice-cold STM buffer to obtain a competent cell solution.

60 μl of this competent cell solution and a gene inactivation vector (CCA39734_NAT, CCA37956_bsd, CCA37582_Zeo, CCA39503_bsd) consisting of the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 constructed in Example 3 3 μl was mixed, transferred to an electroporation cuvette (disposable cuvette electrode, electrode spacing 2 mm (manufactured by BM Equipment Co., Ltd.)), subjected to 7.5 kV/cm, 25 μF, 200 Ω, and then the cells were added to 1 ml of YPD medium. It was suspended and allowed to stand at 30°C for 1 hour. After standing for 1 hour, cells were collected (3000×g, 5 minutes, 20° C.), and 963 μl of the supernatant was discarded. After resuspending with the remaining solution, YPDNAT selective agar plate (1% yeast extract (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical), 2% glucose, 1.5% agarose, 100mg/L clone NAT (Werner BioAgent)) or YPDbsd selective agar plate (1% yeast extract (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical), 2% glucose, 1.5% agarose, 500 mg/L Blasticidin S (Wako Jun) Pharmaceutical company)) or YPDZeo selective agar plate (1% yeast extract (Becton Dickinson), 2% polypeptone (Nihon Pharmaceutical), 2% glucose, 1.5% agarose, 100mg/L Zeocin (trademark) (InvivoGen) (manufactured)), and select strains that grow in static culture at 30 ° C for 3 days, and the genes consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9 are further inactivated. Anti-lysozyme single-chain antibody-expressing yeast, BGL1-expressing yeast, and mUkG1-expressing yeast were obtained.

Table 5 summarizes the obtained transformed yeast by transformed yeast ID, expression vector used, and gene inactivation vector used.

<Example 5: Confirmation of gene inactivation>
The chromosomal DNA of the transformed yeast obtained in Example 4 was prepared using Kaneka Simple DNA Extraction Kit version 2 (manufactured by Kaneka). Using the prepared chromosomal DNA mixture as a template, gene inactivation was confirmed by PCR. Table 6 shows the IDs of the gene-inactivating vectors used for transformation, and the numbers and sequences of the primers (Fw and Re) used to amplify the nucleic acid fragments.

Confirmation of gene inactivation was performed by sequence analysis of the fragment length of the amplified nucleic acid fragment and the internal sequence. As a result, it was confirmed that the desired gene was inactivated in all of the transformed yeast obtained in Example 4.

<Example 6: Culture of transformed yeast>
2 ml of BMGY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nihon 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), After shaking culture at 170 rpm for 24 hours, a preculture was obtained. Subsequently, the cell concentration of the pre-culture solution was measured at OD600, and the initial OD600 was 0.5. , 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 0.4mg/l Biotin, 100mM potassium phosphate (pH6.0), 2% Methanol). After shaking culture at 30°C, 170 rpm for 48 hours, the culture supernatant was collected by centrifugation (12000 rpm, 5 minutes, 4°C).

2 ml of BMGY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nihon Pharmaceutical), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 0.4mg/l Biotin, 100mM Potassium phosphate (pH 6.0, 2% Glycerol) was inoculated, and shake culture was carried out at 30° C., 170 rpm for 24 hours to obtain a preculture solution. 2 ml of BMMY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nihon Pharmaceutical), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 200 μl of the pre-culture solution was transferred to 0.4 mg/l Biotin, 100 mM potassium phosphate (pH 6.0), 2% Methanol), shake cultured at 30° C., 170 rpm for 72 hours, and then centrifuged (12000 rpm, 5 minutes, 4°C) to collect the culture supernatant.

2 ml of BMGY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nihon 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), After shaking culture at 170 rpm for 24 hours, a preculture was obtained. 2 ml of BMGY medium (1% yeast extract bacto (manufactured by Becton Dickinson), 2% polypeptone (manufactured by Nihon Pharmaceutical), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (manufactured by Becton Dickinson), 1% Ammonium Sulfate, 0.4mg/l Biotin, 100mM potassium phosphate (pH6.0), 2% Glycerol) was transferred to 200μl of the pre-culture solution, cultured with shaking at 30°C, 170rpm for 72 hours, and then centrifuged (12000rpm, 5 minutes, 4°C) to collect the culture supernatant.

<Example 7: Measurement of secretion amount of anti-lysodium single-chain antibody by ELISA>
The expression level of anti-lysozyme single chain antibody secretion into the culture supernatant obtained in Example 6 was determined by the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method according to the following method.

Immobilization buffer ( ⁸ g/L sodium chloride, 0.2 g/L potassium chloride, 1.15 g/L sodium monohydrogen phosphate (anhydrous), 50 μl of lysotium (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 0.2 g/L potassium dihydrogen phosphate (anhydrous), 1 mM magnesium chloride) to 1 μM/mL was added to each well and incubated overnight at 4°C. . After incubation, the solution in the wells was removed, blocked with 200 μl of Immunoblock (manufactured by Sumitomo Dainippon Pharma Co., Ltd.), and allowed to stand at room temperature for 1 hour. Wash three times with PBST buffer (8g/L sodium chloride, 0.2g/L potassium chloride, 1.15g/L sodium monohydrogen phosphate (anhydrous), 0.2g/L potassium dihydrogen phosphate (anhydrous), 0.1% Tween20) After that, 50 μl of serially diluted standard anti-lysodium single chain antibody and diluted culture supernatant were added to each well and reacted at room temperature for 1 hour. After washing three times with PBST buffer, add 50 μl of secondary antibody solution (secondary antibody: Anti-6X His tag antibody (HRP) (manufactured by Abcam)) diluted 120,000-fold with PBST buffer and keep at room temperature. React for 1 hour. After washing three 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 stopping the reaction by adding 50 μl of TMB Stop Solution (manufactured by KPL), the absorbance at 450 nm was measured with a microplate reader (Envison; manufactured by PerkinElmer). Quantification of the anti-lysodium single-chain antibody in the culture supernatant was performed using a standard anti-lysodium single-chain antibody calibration curve. Tables 7 to 9 show the amount of anti-lysozyme single-chain antibody secretion expression obtained by this method and the concentration of each cell (OD660).

Table 7 shows the amount of anti-lysodium single-chain antibody secreted in the culture supernatant obtained by culturing the anti-lysodium single-chain antibody-expressing yeast (AOXscFv_1-7) obtained in Example 4 in 25 ml of BMMY medium as in Example 6. shows the results of Table 8 shows the anti-lysodium single-chain antibody of the culture supernatant obtained by culturing the anti-lysodium single-chain antibody-expressing yeast (AOXscFv_1-5, AOXscFv_8-10) obtained in Example 4 in 2 ml of BMMY medium as in Example 6. shows the results of the secretion expression level of Table 9 shows the secretion expression level of the anti-lysodium single-chain antibody in the culture supernatant obtained by culturing the anti-lysodium single-chain antibody-expressing yeast (GAPscFv_1-5) obtained in Example 4 in 2 ml of BMGY medium as in Example 6. shows the results of

As a result, strains (AOX1scFv_2-7) in which the gene consisting of the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11 were inactivated, expressed anti-lysozyme antibody secretion. Amounts increased approximately 1.2- to 2-fold compared to the non-inactivated strain (AOX1scFv_1) (Table 7). Strains (AOX1scFv_8-10) in which the genes consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 were further inactivated, the anti-lysozyme single chain increased as the number of inactivated genes increased. Antibody secretion expression increased (Table 8). In addition, the strain (AOX1scFv_10) in which all the genes consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 were inactivated compared to the non-inactivated strain (AOX1scFv_1), secreted The expression level increased about 3-fold (Table 8). In the strain expressing the anti-lysozyme single-chain antibody under the control of the GAP promoter, the nucleotides shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 were compared with the non-inactivated strain (GAPscFv_1). In the strains (GAPscFv_2-5) in which the gene consisting of the sequence was inactivated, the amount of secretory expression of the anti-lysozyme single chain antibody was increased in the same manner as above (Table 9).

<Example 8: Measurement of secretion amount of BGL1 by colorimetric analysis using pNPG as a chromogenic substrate>
The expression level of BGL1 secreted into the culture supernatant obtained in Example 6 was determined by a colorimetric analysis method using pNitrophenyl-β-D-glucopyranoside (pNPG) as a chromogenic substrate by the method shown below.

After adding 97.5 μl of substrate solution (50 mM citrate buffer (pH 5.0), 2 mM pNPG) to each well of a 96-well plate (assay plate (medium binding capacity) (manufactured by IWAKI)), add 2.5 μl of the culture supernatant. In addition, the mixture was reacted at 25°C for 10 minutes. After 100 μl of 3M sodium carbonate aqueous solution was added to stop the reaction, the absorbance at 405 nm was measured with a microplate reader (Envision; manufactured by PerkinElmer). Table 10 shows the absorbance and each cell concentration (OD660) obtained by this method. Each absorbance was corrected with the absorbance of the wild-type strain without the BGL1 gene.

As a result, the strains (AOXBGL1_2-5) in which the genes consisting of the base sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 were inactivated were compared with the non-inactivated strain (AOX1scFv_1). , the absorbance increased with each inactivated gene. Absorbance is a measure of the enzymatic activity of BGL1, and since all yeasts use the same sequence of the BGL1 gene, an increase in absorbance indicates an increase in secreted expression of BGL1.

<Example 9: Measurement of secretion amount of mUkG1 by fluorescence intensity measurement of culture supernatant>
The expression level of mUkG1 secreted into the culture supernatant obtained in Example 6 was determined by fluorescence analysis of the culture supernatant by the method shown below.
150 μl of the culture supernatant obtained in Example 6 was added to each well of a black 96-well plate (Microfluoro 1 Black (manufactured by IWAKI)), and fluorescence intensity (excitation wavelength 485 nm fluorescence wavelength 510 nm) was measured. Table 11 shows the relative fluorescence intensity and each cell concentration (OD660) obtained by this method. The relative fluorescence intensity was corrected by the fluorescence intensity of the wild-type strain without the mUkG1 gene, and calculated with the fluorescence intensity of the mUkG1-expressing yeast strain (AOXmUkG1_1) as 1.

As a result, strains (AOXmUkG1_2-3) in which the genes consisting of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 were inactivated showed an increase in relative fluorescence intensity compared to the non-inactivated strain (AOXmUkG1_1). was Furthermore, the strain (AOXmUkG1_4) in which all the genes consisting of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 were inactivated, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 The relative fluorescence intensity was higher than that of the inactivated strain (AOXmUkG1_2 or 3), and was twice that of the non-inactivated strain (AOXmUkG1_1). For the green fluorescent protein mUkG1, the number of protein molecules correlates with the fluorescence intensity, so an increase in fluorescence intensity indicates an increase in the amount of secretory expression.

Claims (8)

any of the following genes (a) to (d) are inactivated,
A vector containing a nucleotide sequence encoding a protein of interest,
Host cells that are yeast, bacterial, fungal, or insect cells:
(a) a gene comprising the base sequence shown in SEQ ID NO: 3;
(b) under stringent conditions for a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3, which encodes a protein having a function equivalent to that encoded by the gene containing the nucleotide sequence shown in SEQ ID NO: 3; a gene comprising a base sequence of hybridizing nucleic acids;
(c) contains a nucleotide sequence that encodes a protein having the same function as the protein encoded by the gene containing the nucleotide sequence shown in SEQ ID NO:3 and has 90 % or more sequence identity with the nucleotide sequence shown in SEQ ID NO:3 gene,
(d) encodes a protein having a function equivalent to the protein encoded by the gene containing the nucleotide sequence shown in SEQ ID NO: 3, and encodes an amino acid sequence having 90 % or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; gene to The production amount of the target protein is increased compared to before the inactivation of the gene,
A host cell according to claim 1 . A nucleotide sequence having 90% or more sequence identity with any of the genes (a) to (d), and 90% or more sequence identity with the promoter of any of the genes (a) to (d) and/ or obtained by transforming with a vector containing a nucleotide sequence having 90% or more sequence identity with the terminator of any of the genes (a) to (d). A host cell as described in . The host cell according to any one of claims 1 to 3 , wherein the yeast is methanol-assimilating yeast, fission yeast, or budding yeast. 5. The host cell according to claim 4 , wherein the methanol-assimilating yeast is Komagataella yeast or Ogataea yeast. A method for producing a target protein, comprising the steps of culturing the host cell according to any one of claims 1 to 5 and recovering the target protein from the culture medium. 7. The method of claim 6 , wherein the protein of interest is a heterologous protein. 8. The method according to claim 6 or 7 , wherein the culturing step uses a culture medium containing one or more carbon sources selected from the group consisting of glucose, glycerol and methanol.