JP2009240185A - Highly productive transformant of exogenous protein and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly productive yeast transformant of exogenous protein and a gene associated with a variation. <P>SOLUTION: The yeast transformant has a DNA encoding one or more exogenous proteins in which the following genes of host, one or more selected from ADA2, ADE1, ADE3, ADE5,7 etc., are inactivated. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a high production transformant of a foreign protein and use thereof.

  In recent years, as an alternative to finite petroleum resources, there is an increasing expectation for biomass derived from the photosynthesis of plants, and various attempts have been made to use biomass for energy and various materials. In order to effectively use biomass as an energy source and other raw materials, it is necessary to saccharify biomass into a carbon source that can be easily used by animals and microorganisms.

    A large amount of cellulase is required to use typical biomass such as cellulose and hemicellulose. The cost ratio of the enzyme preparation cost used for saccharifying such biomass into glucose, xylose or the like is considerably high. In order to saccharify cellulose at low cost and convert it into a useful substance, it greatly depends on how much cellulase can be reduced.

  As one solution, it is conceivable to suppress the amount of cellulase to be added by producing cellulase directly from a glucose-producing microorganism such as yeast that fermentably produces useful substances such as ethanol and organic acids from glucose. Yeast does not have a cellulase gene, and it is necessary to obtain a transformant by introducing the cellulase gene as a foreign gene. It has been reported that a mutant strain that highly produces cellulase was obtained by a mutagenesis method using a classic mutagen (Non-Patent Document 1). However, it takes a lot of time and labor to obtain such mutants.

  On the other hand, a high production mutant of a heterologous protein in yeast and a gene involved in the mutant have been disclosed (Patent Document 1). This document introduces a laccase gene, which is a heterologous gene, into a gene-disrupted strain library of Saccharomyces cerevisiae, exhaustively searches for genes that highly express laccase, and a total of 18 mutant strains are found. Genes that have been selected and promoted secretory production of 18 laccases are described.

JP 2005-58143 A Aho S., Arffman A., Korhola M., Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases., Appl. Microbiol. Biotechnol., 1996, 46 (1), 36-45.

  In the method described in Patent Document 1, in order to examine a gene-disrupted strain that highly produces a protein that does not affect growth, the substrate-decomposed substrate in the medium is cultured while the gene-disrupted strain introduced with the foreign gene is cultured on a solid medium. A high-producing mutant strain is obtained using the amount as an index, and a gene that promotes secretory production of the protein is identified. These genes include BUB2, BUB3, BUL1, END3. FYV10, IKI3, MAD1, MAD2, MAD3, NCL1, NCL1, PER1, PFK26, RPS24A, VHS2, VIK1, VRP1, YKL053W, YML094C-A and the like. Such disruption of the gene may act in the direction of promoting secretion of a heterologous protein even when the yeast is cultured in a liquid medium. On the other hand, regarding the secretion of protein, it is considered that the environments in solid culture and liquid culture are completely different. In liquid culture, it is considered that the destruction of completely different genes may be involved in the promotion of secretion.

  One object of the present invention is to provide a high-producing yeast transformant of a foreign protein, a gene involved in the high production, and use thereof. Another object of the present invention is to provide a high-yield yeast transformant of a foreign protein suitable for a production process using liquid culture, a gene involved in the high production, and use thereof. . It is still another object to provide a cellulase-producing yeast transformant, a gene involved in the mutation, and use thereof. Furthermore, an object of the present invention is to provide a yeast transformant suitable for saccharifying biomass and converting it into a useful substance, and use thereof.

  In order to solve the above-mentioned problems, the present inventors introduced a cellulase gene which is a foreign gene and a secretory protein into a gene-disrupted strain of S. cerevisiae, and these transformants are obtained by cell culture in liquid culture. Were screened for mutant strains that produced a high yield of. Furthermore, by screening the cellulase activity of the culture supernatant of the liquid culture, screening for a gene-disrupted strain that highly produces secreted protein was performed. As a result, the high production mutant screened was a disrupted strain of a gene significantly different from the conventional one. Based on these findings, the present inventors have completed the present invention. According to the present invention, the following means are provided.

According to the present invention, a DNA encoding one or more foreign proteins is retained, and the following genes of the host: ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B, RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, 7A, RSA1, SAC1, SFP1 SNF8, SPC72, SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, VMA7, VPS16, VPS25, VPS27, VPS28, VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, OPI9, YDR065W, YDR433 YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331C, YPT7, YVH1 and ZUO1
A yeast transformant in which one or more selected from is inactivated is provided.

  In the yeast transformant of the present invention, it is preferable that secretion production of the foreign protein in liquid culture is promoted more than when the gene is not inactivated. In the yeast transformant of the present invention, the genes are ARV1, DYN3, FAR8, NBP2, RMD11, RPS6A, RPL19B, RPL36A, RRD1, SNF7, SNF8, VPS3, VPS4, VPS25, VPS27, VPS28, VPS33, VPS36. 1 or 2 or more selected from the group consisting of VPS41, VPS45 and YOL138C, and the gene is RAV1, RPS6A, SNF7, SNF8, RPL36A, VPS3, VPS4, VPS25, VPS27, VPS28 , VPS33, VPS36, VPS41 and VPS45 are also preferably selected from one or more.

  In the yeast transformant of the present invention, the host is preferably Saccharomyces yeast. In the yeast transformant of the present invention, the exogenous protein preferably contains cellulase, and can be endoglucanase.

According to the present invention, a vector for expressing a foreign protein in yeast,
The following genes: ADA2, ADE1, ADE3, ADE5,7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3 RPL13B, RPL19B, RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SAC1, SFP1, SNF7, SNF8, SPC72, SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, PS4 VPS27, VPS28, VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532C, YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331U, PT1, YOR331U, PT A vector for inactivating one or more of the above is provided.

According to the present invention, a host yeast for expressing a foreign protein comprising the following genes of the host: ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23 , BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK2, MTM1, MTQ1 , NBP2, NGG1, NPR2, OPR3, OPI3, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B, RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SPC1, SPC7, SFP7 , SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, VMA7, VPS16, VPS25, VPS27, VPS28, VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532Y, YDR532Y YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331C, YPT7, YVH1 and ZUO1
A host yeast in which one or more selected from is inactivated is provided.

  According to the present invention, there is provided a method for producing a degradation product of a cellulose-containing material, the step of preparing the yeast transformant having cellulase as an exogenous protein, and a liquid medium containing the cellulose-containing material as a carbon source. And culturing the yeast transformant.

  According to the present invention, there is provided a method for producing ethanol from a cellulose-containing material, the step of preparing the yeast transformant having cellulase as an exogenous protein, and a liquid medium containing the cellulose-containing material as a carbon source. And a step of culturing the yeast transformant and subjecting it to alcohol fermentation.

  According to the present invention, there is provided a protein production method comprising the steps of preparing any one of the above yeast transformants and culturing the yeast transformant using a liquid medium. Provided.

  The present invention relates to a yeast transformant capable of producing an exogenous protein at a high rate, a host yeast for that purpose, a vector, and a production method using the yeast transformant. The present invention relates to the following 96 genes in yeast: ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6 , FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, PAT1, PER1 , PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B, RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SAC1, SFP1, SNF7, SNF8, SPC72, SPT7, SRC1, SWI2, TAF14, S1 , VMA4, VMA7, VPS16, VPS25, VPS27, VPS28, VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532C, YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL120C, YNL , YPT7, YVH1 and ZUO1 (hereinafter, this gene group is referred to as the first gene group), and the secretion of foreign proteins by inactivation Production is based on being promoted.

  The present inventors searched for a gene that promotes secretory production of exogenous secretory proteins in liquid culture using a yeast gene disruption strain library by being inactivated. A number of genes were found that were completely different from this type of gene obtained by screening on solid media. The gene found by screening in liquid culture was considered to coincide to some extent with the gene found by screening in solid culture, and the present inventors were able to reconfirm secretion promotion of secretory proteins. Of the 96 genes, only one gene overlapped with the gene obtained by screening in solid culture. Such gene differences were beyond expectations.

  It is considered that the high foreign protein strain found by the present inventors produced a high amount of heterologous secreted protein by the specific gene being disrupted in the gene disrupted strain used. In a normal state before the specific gene is destroyed, it is considered that the production amount of the foreign protein outside the cell is directly or indirectly suppressed by the gene. Among the high foreign protein production strains, some of the vacuolar protein mis-sorting (vps) gene disruption strains were included. These gene-disrupted strains are strains in which endogenous proteins normally sorted into intracellular vacuoles are missorted outside the cells and secreted outside the cells. Abnormal proteins that cannot perform their original functions even with endogenous proteins are degraded within the vacuole. Although not restricting the present invention, it is considered that a foreign protein is recognized as an abnormal protein and processed in a vacuole is secreted to the outside of the cell because the gene has been destroyed. In addition, many gene-disrupted strains related to vacuolar functions such as vma2, vma4, vma7, snf7, and snf8 have been obtained. Therefore, it is thought that the function of the vacuole contributes greatly to the high production of foreign proteins.

  According to the present invention, in the liquid culture found by the present inventors, a yeast that highly produces a heterologous secreted protein by utilizing a gene that promotes secretory production of a foreign protein by being inactivated. In addition to transformants, hosts and vectors used for such transformants are also provided. Furthermore, by using such a yeast transformant, proteins can be produced with high efficiency, and the cellulose-containing material can be efficiently decomposed and used.

  Hereinafter, various embodiments of the present invention will be described in detail. These various embodiments will be described in detail.

(Transformant)
The yeast transformant of the present invention retains DNA encoding one or more foreign proteins, and one or more selected from the first gene group of the host is inactivated. Yes. Hereinafter, the host, the heterologous secreted protein, and the gene group to be inactivated will be sequentially described.

(Host)
The host yeast of the transformant of the present invention is not particularly limited, and various known yeasts can be used. In consideration of ethanol fermentation described later, yeasts of the genus Saccharomyces cerevisiae such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe, Candida shehatae, etc. Yeast of the genus Candida, yeast of the genus Pichia such as Pichia stipitis, yeast of the genus Hansenula, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, the genus Pachysolen And yeast of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis. Of these, Saccharomyces yeasts are preferable from the viewpoint of industrial availability. Of these, Saccharomyces cerevisiae is preferable.

(DNA encoding foreign protein)
The foreign protein may be foreign to the host yeast. Therefore, even proteins derived from other yeasts belonging to the same yeast are included in the foreign protein. The foreign protein is preferably a protein derived from an organism including microorganisms other than yeast.

  The foreign protein is preferably secreted in yeast. In the present specification, the protein secretory means a protein produced as a membrane protein (including cell surface display protein) or a secreted protein in the host yeast. As a foreign protein having a secretory property, a protein that can itself be a membrane protein or a secreted protein in yeast can be used, and even if it does not have such a secretory property, a fusion in which a known secretory signal is linked. It may be a protein. Moreover, even if it has secretion itself, the secretion signal may be modified according to the host yeast. The secretion signal and the like will be described later. In addition, in this specification, the surface layer display form in cell surface layer display protein is not specifically limited. It may be retained and presented directly or indirectly on the surface of yeast cells.

  Such an exogenous secreted protein can be appropriately selected. For example, from the viewpoint of degrading cellulose-containing materials, in addition to various cellulases such as endoglucanase, cellohydrohydrolase, β-glucosidase, lignin peroxidase, manganese Examples include lignin degrading enzymes such as peroxidase and laccase. In addition, examples include cellulose binding domains (proteins) that are constituent parts of cellulose relieving proteins swollenin and expansin, cellulosome and cellulase. In addition, other biomass degrading enzymes such as xylanase and hemicellulase can also be mentioned. Any of these proteins can improve the accessibility of cellulase to cellulose.

  Preferred exogenous secreted proteins include endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 23.2.4.1, EC 3.2.1.21). By allowing these yeasts to produce a large amount of secreted yeast, the cellulose-containing material can be efficiently decomposed. Among them, it is preferable to express endoglucanase in order to decompose crystalline cellulose and efficiently reduce the molecular weight of cellulose. Note that cellulase 13 (5, 6, 7, 8, 9) of GHF (Glycoside Hydrolase family) (http://www.cazy.org/fam/acc.gh.html) is based on the similarity of amino acid sequences. , 10, 12, 44, 45, 48, 51, 61, 74). You may combine the same or different cellulases classified into a different family.

  Although it does not specifically limit as a cellulase, It is preferable that it is a cellulase with high activity itself. Examples of such cellulases include Phanerochaete bacteria, Trichoderma bacteria such as Trichoderma reesei, Fusarium bacteria, Fusarium bacteria, Tremetes bacteria, Penicillium bacteria, and Humicola bacteria. In addition to filamentous fungi such as Humicola, Acremonium, and Aspergillus, Clostridium, Pseudomonas, Cellulomonas, and Luminococcus Bacteria such as genus (Ruminococcus) and Bacillus, primordial fungi such as Sulfolobus, and more, such as Streptomyces and Thermoactinomyces Derived cellulase. Such cellulase may be artificially modified.

  The transformant of the present invention holds DNA encoding a foreign protein. This encoded DNA is retained so that it can be expressed as a protein in the host yeast. Specifically, it is linked under the control of a promoter operable in the host yeast (typically a yeast promoter) and held in a state having an appropriate terminator downstream thereof. The promoter may be a constitutive promoter or an inducible promoter. The DNA in such a state may be in a form integrated in the host chromosome, or in a form such as a 2μ plasmid held in the host nucleus or a plasmid held outside the nucleus. In general, with the introduction of such foreign DNA, a selectable marker gene that can be used in the host is also retained.

(Gene to be inactivated)
The genes that are preferably inactivated in the transformant of the present invention and the genes that have the activity of promoting the secretion production of exogenous secreted proteins by inactivation are shown in the following table.

  A total of 96 genes in the column A in Table 1 are specific genes using Cel8A (hereinafter referred to as Ctcel8A), a cellulase (endoglucanase) belonging to GHF8 of Clostridium thermocellum, as an exogenous secretory production protein. Was screened based on the endoglucanase activity of the culture supernatant obtained by introduction into inactivated yeast and liquid culture.

  These 96 genes can be divided into three groups (Table 1B column to D column) according to the degree of activity that promotes secretory production of exogenous secretory proteins by inactivation. The gene described in Table 1B column has the highest secretory production promoting activity by inactivation among 96 species, and the gene described in Table 1C column has the next highest secretory production promoting activity. The gene described in the 1D column has the next highest secretory production promoting activity.

  In addition, among the 96 genes shown in Table 1A, genes that promote secretory production of cellulase belonging to GHF8 in a nutrient rich medium by inactivation can be classified into one subgroup (Table 1E). In addition, genes that promote the secretory production of cellulase (endoglucanase) (Cccel5A) in the nutrient rich medium of Clostridium cellulovorans belonging to GHF5 by inactivation are included in one subgroup (Table 1F column). Can be classified. Genes common to GHFs 8 and 15 are genes underlined in columns E and F of Table 1. Since these genes can activate cellulases of both families, these genes can be preferably used for secretory production of these two types of cellulases.

  The gene group in Table 1 was obtained by evaluating the activity when secreting and producing endoglucanase belonging to GHF8 as an exogenous secreted protein, but is different as shown in Table 1E column and F column. It can be seen that the inactivation of the genes described in Table 1A is also effective for the culture conditions and different GHF cellulases. This supports that inactivation of the genes described in Table 1A column is widely useful for secretory production of exogenous secreted proteins beyond amino acid similarity.

  The base sequence of each gene of yeast shown in Table 1 can be obtained from the Saccharomyces genome database (http://www.yeastgenome.org/) particularly for the genus Saccharomyces such as S. cerevisiae. For these genes, including the genus Saccharomyces, the blast search site of the National Institutes of Health HP (http://www.ncbi.nlm.nih.gov/) (http: //www.ncbi.nlm. nih.gov/blast/Blast.cgi).

  In the transformant of the present invention, any one or more of the above genes are inactivated. Here, a gene is inactivated at least in a host yeast (1) a state in which transcription or translation of the gene is completely or partially suppressed, and (2) the gene It includes a state in which a mutation has been introduced and a protein that is incompletely expressed to the extent that it cannot exert its original function at all. Such an inactivated state is typically realized by introducing a mutation into a regulatory region such as a promoter of the gene or a coding region. Specifically, such inactivated form can be obtained by homologous recombination. Strains in which each yeast gene shown in Table 1 is disrupted can be obtained from Open Biosystems (USA).

  In the transformant of the present invention, the secretion production of the foreign protein in liquid culture is promoted more than when one or more genes selected from the genes shown in Table 1 are not inactivated. It can have the characteristics. That is, except that the host is a wild type (the gene shown in Table 1 is not inactivated), it is more exogenous than a transformant obtained by introducing DNA encoding an exogenous secretory protein. Secretory production of secreted proteins is promoted.

  The conditions for liquid culture are not particularly limited, and can be set as appropriate in consideration of, for example, the liquid culture process to be performed. Therefore, the medium may be a rich medium or a poor nutrient medium, and the pH may be appropriately set according to the yeast to be used, and the medium may or may not be provided with a pH buffering ability. Usually, YPD medium and SD medium, which are general yeast culture media, and media appropriately modified for these can be used.

  Whether or not the secretory production of the exogenous secretory protein is promoted can be evaluated by the secretory production amount of the exogenous secretory protein or the activity of the secretory protein. Preferably, the amount of the protein and the like is evaluated for a culture supernatant obtained by subjecting the transformant of the present invention to a wild-type transformant to be compared for a certain period of time.

  The amount of protein and the method for measuring protein activity can be appropriately selected according to the type of protein. If the protein is an enzyme or the like, the enzyme activity may be measured by a known method. For example, when the exogenous secretory protein is a cellulase such as endoglucanase, the collected culture supernatant is reacted with an appropriate cellulase substrate (carboxymethyl cellulose, cellulose phosphate, crystalline cellulose, etc.), and the amount of reaction product The enzyme activity can be evaluated by measuring the mass and the base mass. The reaction temperature, pH, and time can be appropriately set depending on the type of enzyme. In addition, methods for quantifying the amount of reducing sugar resulting from the enzyme reaction include the Somogyi method, Tauber-Kleiner method, Hanes method (titration method), Park-Johnson method, 3,5-dinitrosalicylic acid (DNS) method, TZ method (Journal of Biochemical Methods, 11 (1985) 109-115) or the like may be employed as appropriate.

  In addition, cellulase activity such as endoglucanase is supplied to a test protein having a possibility of the present invention in an evaluation region consisting of a solid phase containing cellulose such as carboxymethyl cellulose, and the cellulose in the solid phase in the region is used. The endoglucanase activity can also be evaluated based on the size of the region in which cellulose is decomposed and disappeared in the solid phase after being decomposed (halo: the region in the solid phase that is lightened or decolorized by the degradation of biomass). The halo based on the disappearance of the cellulose in the solid phase can be visually recognized, and the halo can be clearly detected by staining the cellulose with Congo Red or the like when detecting the halo. In addition, when dye-bonded cellulose (such as Cellulose Azure, manufactured by Sigma) is used as biomass, the cellulose diffuses into the solid phase due to the decomposition of the cellulose and forms a halo, so cellulolytic activity is easily detected. be able to. Similarly, halo can be easily detected by using cellulose combined with a fluorescent dye as biomass. Further, when acid-treated cellulose or the like is used as biomass, a transparent halo is formed by the decomposition of cellulose, so that the cellulolytic activity can be easily detected.

  Examples of the solid body for forming halo include a gel and a film supporting biomass. The constituent material of the gel or film is not particularly limited, and a natural or artificial polymer material can be preferably used. As such a polymer material, agarose (agar) can be preferably used.

(Production of yeast transformant)
The transformant of the present invention described above can be prepared according to the method described in Molecular Cloning 3rd edition, Current Protocols in Molecular Biology and the like. Preferably, an expression vector having a DNA encoding a foreign secretory protein is introduced into a host yeast in which one or more of the genes shown in Table 1 have been previously inactivated, It can be obtained by transformation.

  The host yeast can be obtained from Open Biosystems (USA) or can be obtained by introducing a mutation into a regulatory region or a coding region such as a promoter of a gene to be disrupted. The inactivation of a specific gene can be achieved by introducing a mutation into a specific gene or introducing another gene using a homologous recombination method, for example.

  One form of the vector for expressing the foreign protein in the yeast of the present invention includes a vector for inactivating one or more selected from the genes listed in Table 1. This vector has a homologous region with the gene to be inactivated or its vicinity, and the gene is inactivated by substituting the gene or its neighborhood with the DNA region held by the vector via this homologous region. It is to become. Such vectors for homologous recombination and their production are well known to those skilled in the art and are disclosed in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like.

  A host yeast in which two or more specific genes are inactivated can also be obtained by gene transfer by various known methods or by using an available disrupted strain.

  Another form of the vector for expressing the exogenous secretory protein in yeast has a coding region of the desired exogenous secretory protein. The protein to be expressed preferably has extracellular secretion in yeast. Such secretory property can be artificially imparted by using a secretory signal or the like in addition to the case where the intended protein originally has. For example, a coding DNA of an amino acid sequence that confers secretion can be linked to a coding region of a foreign protein. Examples of the secretion signal include a secretion signal of a glucoamylase gene of Rhizopus oryzae. Further, by using an aggregating protein or a part thereof, the protein can be secreted into a state presented on the surface of yeast. For example, there is a peptide consisting of 320 amino acid residues in the 5 'region of the SAG1 gene encoding α-agglutinin which is an aggregating protein. Polypeptides and techniques for displaying a desired protein on the cell surface are disclosed in WO01 / 79483, JP2003-235579, WO2002 / 042483, WO2003 / 016525, and JP2006. No. 136223, Fujita et al. (Fujita et al., 2004. Appl Environ Microbiol 70: 1207-1212 and Fujita et al., 2002. Appl Environ Microbiol 68: 5136-5141.), Murai et al., 1998. Appl Environ Microbiol 64: 4857 -4861.

  The vector of the present invention can include a regulatory region (promoter, terminator, etc.) together with the above-described protein-encoding DNA, as well as an appropriate expression marker gene cassette. A vector is a component other than the above coding region (chromosome by homologous recombination) depending on the transformation method and the retention form of the encoded DNA in the host cell (form introduced into the chromosome, form retained outside the chromosome, etc.). When intended to be introduced into the DNA, a region of homology with yeast chromosomal DNA is required). Moreover, the form of a vector can take various forms according to a usage form. For example, it can take the form of a DNA fragment, and can also take the form of a suitable yeast vector such as a 2 microplasmid.

  The yeast transformant of the present invention can be obtained by appropriately transforming a suitable yeast host with such a vector. For the transformation, various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and the like can be used.

(Method for producing degradation product of cellulose-containing material)
The method for producing a degradation product of a cellulose-containing material of the present invention comprises the steps of preparing a yeast transformant of the present invention that secretes and produces cellulase as an exogenous protein, and the yeast in a liquid medium containing the cellulose-containing material as a carbon source. Culturing the transformant. According to the production method of the present invention, the cellulose-containing material can be directly used by yeast without adding a cellulase for saccharifying the cellulose-containing material or suppressing the addition amount. For this reason, a cellulose containing material can be utilized efficiently.

(Preparation of transformants)
As a cellulase secreted and produced by a yeast transformant, one or a combination of two or more cellulases described above can be used. Preferably, two or more types of cellulases (for example, endoglucanase and cellobiohydrolase) can be used. Two or more types of cellulases may be secreted and produced by one transformant, but may be two or more types of transformants that secrete and produce each cellulase. In addition, various aspects of the transformant of the present invention described above can be applied to the transformant used in the production method of the present invention.

(Liquid culture)
In order to perform liquid culture of the above yeast transformant in a liquid medium, a known liquid medium can be used or appropriately modified, except that a cellulose-containing material is used as at least one kind of carbon source. The culture medium composition, temperature, pH, aeration conditions and the like can be appropriately set as necessary.

  The cellulose-containing material contains a polymer in which glucose is polymerized by β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. Examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. The cellulose may be crystalline cellulose or non-crystalline cellulose. Cellulose may be cellooligosaccharide or cellobiose, which is a partially decomposed product thereof. The cellulose-containing material may be a complex with β-glucoside, which is a glycoside, lignocellulose, which is a complex with lignin and / or hemicellulose, and pectin. The origin of the cellulose-containing material is not particularly limited. It may be derived from plants, fungi, or bacteria. Examples of the cellulose-containing material include natural fiber products such as cotton and hemp, recycled fiber products such as rayon, cupra, acetate, and lyocell, and agricultural waste such as rice straw, rice husks, and wood chips.

  Cellulose degradation products obtained by the method of the present invention include cellobiose and cellooligosaccharides in addition to glucose. The cellulose degradation product thus obtained can be used as a fermentation raw material of a useful substance, as in the case of normal glucose. In addition, as a transformant used with the manufacturing method of this invention, when it has the original alcohol fermentability of yeast, alcohol (ethanol) is manufactured by implementing a culture | cultivation process so that it may mention later. Can do. Moreover, when the transformant to be used is modified by genetic engineering to produce an organic acid such as lactic acid, the organic acid can be produced.

(Method for producing ethanol from cellulose-containing material)
The method for producing ethanol of the present invention comprises the steps of preparing a yeast transformant of the present invention that secretes and produces cellulase as an exogenous protein, and culturing the yeast transformant in a liquid medium containing a cellulose-containing material as a carbon source. And alcohol fermentation. According to the production method of the present invention, ethanol can be produced by directly using a cellulose-containing material with yeast without adding a cellulase for saccharifying the cellulose-containing material or suppressing the addition amount. For this reason, ethanol can be produced at a lower cellulase cost than in the prior art. The ethanol production method of the present invention only needs to have alcohol fermentability as a yeast transformant in the method for producing a degradation product of a cellulose-containing material of the present invention.

(Protein production method)
The method for producing a protein of the present invention can comprise a step of preparing the transformant of the present invention and a step of culturing the transformant using a liquid medium. According to the yeast transformant of the present invention, a desired foreign protein can be efficiently secreted and produced, which is advantageous for protein production. Conditions such as the composition, temperature, pH and the like of the liquid medium used in the culturing step can be appropriately set. The culture supernatant obtained in the culture step contains a protein secreted and produced by the transformant of the present invention. When the protein is an enzyme or the like, it may be purified, but the culture supernatant may be used as it is as an enzyme solution.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination operation described below was performed according to Molecular Cloning: A Laboratory Manual (T. Maniatis, et al., Cold Spring Harbor Laboratory).

Construction of cellulase expression plasmid
A signal peptide of Rhizopus Oryzae-derived glucoamylase was introduced between SacII-SpeI of a 2-micron vector for yeast expression having TDH3 promoter, CYC1 terminator and marker URA3. Clostridium thermocellum (hereinafter C. thermocellum) endoglucanase Cel8A (hereinafter Ctcel8A) or Clostridium cellulovorans (hereinafter C. cellulovorans) endoglucanase cel5A (hereinafter Cccel5A) was introduced between the downstream SphI and SpeI of this signal peptide ( FIG. 1). The prepared plasmid DNA was named pRSA436GAPcelA for Ctcel8A and pRS436GAPCccel5A for Cccel5A.

Confirmation of endoglucanase activity in yeast
The diploid yeast BY4743 (genotype: MATa / αhis3Δ1 / his3Δ1 leu2Δ0 / leu2Δ0 lys2Δ0 / LYS2 MET15 / met15Δ0 ura3Δ0 / ura3Δ0) derived from S288C strain of Saccharomyces cerevisiae was transformed by the lithium acetate method. After streaking yeast on a YPD plate and culturing at 30 ° C for 1 day, the transformant (35 µl of 60% polyethylene glycol average molecular weight 3350 + 2.5 µl of 4 M lithium acetate + 5 µl of 1 M dithiothreitol + 5 µl of 10 mg / ml sperm sperm DNA 1 μl of pRS436GAPcelA was mixed, and sterilized water was added to a total volume of 50 μl), and the yeast was cultured from the YPD plate. The transformation solution was heated at 42 ° C. for 2 hours, and spread on SD-uracil (0.67% YNB w / o aa + 2% glucose + amino acid mix (-uracil)) medium. In the amino acid mix (-uracil), 0.5 g of adenine, 2 g of tryptophan, 2 g of histidine hydrochloride, 2 g of methionine, 4 g of leucine and 2 g of lysine hydrochloride were added per liter. Whether the change in endoglucanase activity occurred when the pH of the medium was controlled was examined. Endoglucanase activity is detected in culture supernatants cultured in YPD medium (1% yeast extract, 2% peptone + 2% glucose), SD-uracil medium, and SD-uracil with 0.1 M KPO ₄ and pH buffered. It was spotted on a 2% agar medium (hereinafter, CMC medium) containing 0.1% carboxymethylcellulose and allowed to react at 40 ° C. for 1 day. The plate was stained with 0.1% CongoRed / 1 M Tris (pH 9) (hereinafter referred to as Congo red solution), and after decolorization with 1 M NaCl, an unstained halo (circle) having endoglucanase activity was confirmed (FIG. 2). The endoglucanase activity of celA decreased greatly with the decrease of culture pH. From the above, in the subsequent evaluation of endoglucanase activity, it was decided to ensure a state in which pH reduction was suppressed.

Transformation into Yeast Gene Disrupted Strain Library Plasmid pRS436GAPcelA constructed in “Example 1” was transformed into a diploid yeast gene disrupted strain library. In the method, the disrupted strain library stored in the YPD plate is inoculated into a 96-well microplate containing 25 μl of YPD, cultured at 30 ° C. for one day at rest, and then mixed with the culture solution. Mix with the transformation solution (3500 μl of 60% polyethylene glycol average molecular weight 3350 + 250 μl of 1 M dithiothreitol + 250 μl of 4 M sodium acetate + 250 μl of 10 mg / ml of sperm DNA + 250 μl of pRS436GAPcelA DNA solution) After that, heat was applied at 42 ° C. for 2 hours and spotted on the SD-uracil medium (FIG. 3).

Screening of Ctcel8A high production yeast mutant
To screen for yeast mutants that produce high Ctcel8A, the strain from the Ctcel8A-transformed plate was cultured on a 96-well microplate containing 50 μl of SD-uracil at 30 ° C for 1 day, and 5 μl of the culture supernatant was added. It was spotted on CMC medium with an 8-lane pipette, reacted at 40 ° C. for 1 day, stained with Congo red solution, and decolorized with 1 M NaCl. 226 strains having a larger halo with endoglucanase activity than other gene-disrupted strains were obtained (FIG. 4).

In order to obtain the reproducibility of 226 Ctcel8A highly active strains obtained by reconfirmation screening of Ct cel8A high-producing yeast mutants , the obtained highly active strains were shaken in SD-uracil medium one by one in a test tube. In the same manner as in Example 2, reproducibility of 147 strains was obtained as a strain having higher activity than wild type BY4743. In order to confirm the nutritional requirement of 147 Ctcel8A high-producing strains, strains that streak into SD-UMK (0.67% YNB w / o aa + 2% glucose + amino acid mix (-UMK)) medium and cannot grow are high-producing strains It became 117 shares. The SD-UMK medium amino acid mix (-UMK) was prepared by mixing 0.4 g per liter of adenine 0.5 g, tryptophan 2 g, histidine hydrochloride 2 g, and leucine 4 g into the SD medium. Since the disrupted strain library used was diploid, if the obtained highly active strain is mixed with haploid, it should be mated with haploid. To eliminate haploids, N435-1A (genotype: MATa his7 lys7 met6 arg1 gal4 MAL2 SUC), N435-2A (genotype: MATα his7 lys7 met6 arg1 gal4 MAL2 SUC) and high-producing strains on the YPD plate Streaks were mixed and cultured at 30 ° C for 1 day. Cultured plates were replicated to minimal medium (0.67% YNB w / o aa + 2% glucose). Except that 7 strains grew on the minimal medium, 110 strains were removed. In order to remove the strain with little difference in activity from the wild type (BY4743 strain) once again, the cells were cultured again in SD-uracil medium in a test tube and re-tested in the same manner as in Example 2. The reproducibility of 96 strains It could be confirmed. The 96 strains were classified as Ctcel8A highly active strains, and the activity intensity was classified into three levels: +++, ++, and + (FIGS. 5 to 7).

  These 96 strains can be said to have preferable mutations for producing and secreting foreign proteins. That is, it has been found that the genes that are disrupted in these strains are genes that are preferably inactivated in order to secrete and produce foreign proteins.

The 96 Ctcel8A high activity strains obtained by screening the activity of the Ctcel8A highly active strain on the nutrient rich medium were cultured on the YPD medium, which is the nutrient rich medium, and the activity of the culture supernatant diluted 100 times was the same as in Example 2. (After spotting on CMC medium and reacting at 40 ° C. for 1 day, the plate was stained with 0.1% CongoRed / 1 M Tris (pH 9) (hereinafter, Congo Red solution) and decolorized with 1 M NaCl.) As a result, 25 strains were more active than wild type BY4743. Ctcel8A was retransformed against these gene-disrupted strains, and 21 strains were more active than wild-type BY4743, except for two strains that showed poor growth (FIG. 8). Two strains were not reproducible. From the above, it was found that the strain obtained in this example showed high activity regardless of the composition of the medium.

Activity in Enriched Medium with Cccel5A The 23 strains obtained by retransformation in Example 6 were transformed with the plasmid pRS436GAPCccel5A (FIG. 1) described in Example 1 expressing Clostridium cellulovorans endoglucanase Cccel5A. did. When the activity of the culture supernatant cultured in YPD medium and diluted 50-fold was examined by the same method as in Example 2, 14 strains were more active than wild type BY4743 (FIG. 9). The remaining nine strains did not change in activity with BY4743. From the above, the mutant strains confirmed to have high activity in this Example are useful for secreting and producing both GHF5 and 8, that is, the inactivation of genes disrupted in these mutant strains. It was found that the conversion is effective for the production of foreign proteins.

Preparation of vectors for introduction of Ctcel8A and Cccel5A staining charges Vectors pAH-HORp-Ctcel8A and pAH-HOR7p-Cccel5A for introducing cellulase into the chromosome were prepared (FIG. 10). A sequence from -3000 bp to -2000 bp upstream of the AAP1 gene (hereinafter referred to as AAP1U) was cloned from the genomic DNA of S288C by PCR reaction. A sequence from -2000 bp to -1000 bp upstream of the AAP1 gene (hereinafter AAP1D) was cloned from the genomic DNA of S288C by PCR reaction. A hygromycin resistance gene was linked as a marker for confirming gene transfer. After extracting the genome from cultured cells of C. thermocellum (ATCC27405), C. cellulolyticum (ATCC35319), the cellulase gene C. thermocellum-derived Cel8A (ABN51508) excluding the secretory signal sequence is approximately 1.3 kb and C. cellulolyticum-derived Cel5A (AAA51444) About 1.3 kb was obtained by PCR. The obtained gene fragments were inserted downstream of the signal peptide of the Rhizopus Oryzae-derived glucoamylase in the chromosome introduction vector at the restriction enzyme KpnI-BglII for Cel8A and the KpnI-BamHI site for Cel5A.

Effect of Ctcel8A and Cccel5A Chromosome-Introduced Strains in vps3 Gene Disrupted Strains Plasmids pAH-HOR7p-Ctcel8A and pAH-HOR7p-Cccel5A prepared in Example 8 were transformed. As a transformation method, BY4743 and vps3 disrupted strains were cultured in YPD medium at 30 ° C. for 1 day, and again cultured in YPD medium for 5 hours at 30 ° C. The culture solution was collected by centrifugation, washed again with sterilized water, and then collected by centrifugation. 60% polyethylene glycol average molecular weight 3350 120μl, 4M lithium acetate 5μl, 1M dithiothreitol 10μl, and the restriction enzyme Sse8387I to make the chromosomal chromosomal transduced line DNA, add 5μl of DNA solution 50 μl was added, suspended, and reacted at 42 ° C. for 1 hour. The reaction solution was centrifuged, YPD medium was added, and recovery culture was performed overnight at 30 ° C. The culture was spread on a plate containing hygromycin B at a final concentration of 150 μg / ml in YPD and grown at 30 ° C. Colonies that became resistant to hygromycin were again single colonized. Colonies were grown again on YPD plates. After culturing at 30 ° C. for 1 day in YPD medium, the culture solution was centrifuged, and the culture supernatant was diluted x1, x2, x4, x8, x16, x32 times. 8 μl of the supernatant was spotted on CMC medium and reacted at 40 ° C. for 1 day. Staining with Congo red solution and decolorization with 1M NaCl confirmed the activity (FIG. 11). Compared to BY4743, the vps3-disrupted strain showed an increase in activity of 8 times or more in Ctcel8A and 32 times or more in Cccel5A.

  Ctcel8A and Cccel5A chromosome-introduced strains above were added to SD + complement medium (0.67% YNB w / o aa + 2% glucose + amino acid mix (complement), pH adjusted to 6 with NaOH) medium and SD + complemnent + 0.1M NaAc Culture was carried out in a medium (0.67% YNB w / o aa + 2% glucose + amino acid mix (complement) +0.1 M sodium acetate added to adjust pH to 6 with HCl). The amino acid mix was 0.5 g adenine, 2 g uracil, 2 g tryptophan, 2 g histidine hydrochloride, 2 g methionine, 4 g leucine and 2 g lysine hydrochloride and added 0.6 g per liter. After culture, 8 μl of the culture supernatant was spotted on CMC medium. The plate was reacted at 40 ° C. for 1 day, stained with Congo red solution, and decolorized with 1M NaCl (FIG. 12). When buffering so as not to lower the pH of the medium with sodium acetate (SD + complement + 0.1M NaAc medium), the activity of the vps3-disrupted strain was increased about twice as much as that of the wild type (BY4743 strain). On the other hand, in the medium not buffering pH (SD + complement), the activity of BY4743 could not be confirmed due to a decrease in the medium pH, but the activity was confirmed in the vps3-disrupted strain. Therefore, as with the YPD medium, it was confirmed that the vps3-disrupted strain can produce cellulase at a high level even in an oligotrophic medium such as SD medium.

  From the above, it was found that in the yeast in which the vps3 gene was inactivated, a high secretory production activity of the foreign protein can be obtained even if the foreign protein-encoding DNA is in the form of chromosome introduction. That is, it was found that the foreign protein can be stably secreted and produced with high activity. Moreover, it turned out that the tolerance with respect to poor nutrition and low pH is also improving rather than the wild type.

It is a figure which shows the structure of the plasmid for expressing cellulase in yeast. It is a figure which shows the evaluation result of the endoglucanase activity of Ctcel8A about the culture supernatant of various culture media. It is a figure which shows transformation to the yeast gene disruption library of a Ctcel8A expression plasmid. It is a figure which shows the screening result of the highly active strain of a Ctcel8A transformant. It is a figure which shows the high production yeast mutant strain (activity grade +++) of Ctcel8A. It is a figure which shows the high production yeast mutant strain (activity level ++) of Ctcel8A. It is a figure which shows the high production yeast mutant strain (activity grade +) of Ctcel8A. It is a figure which shows the activity of Ctcel8A in a rich medium (YPD). It is a figure which shows the activity of Cccel5A in a rich medium (YPD). It is a figure which shows the structure of the plasmid for chromosome introduction | transduction of Ctcel8A and Cccel5A. It is a figure which shows the evaluation result of the activity of the vps3 disruption strain in the cellulase chromosome introduction | transduction strain | stump | stock culture | cultivated in the rich medium. It is a figure which shows the evaluation result of the activity of the vps3 disruption strain in the cellulase chromosome introduction | transduction strain | stump | stock culture | cultivated in the oligotrophic medium.

Claims (12)

Holding the DNA encoding one or more foreign proteins, and the following genes of the host;
ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SAC1, SFP1, SNF7, SNF8, SPC72, SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, VMA7, VPS16, VPS25, 28PS VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532C, YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331C, YPT7, YV1 and Z A yeast transformant in which two or more are inactivated.   The yeast transformant according to claim 1, wherein secretory production of the exogenous protein in liquid culture is promoted more than when the gene is not inactivated.   The gene is selected from the group consisting of ARV1, DYN3, FAR8, NBP2, RMD11, RPS6A, RPL19B, RPL36A, RRD1, SNF7, SNF8, VPS3, VPS4, VPS25, VPS27, VPS28, VPS33, VPS36, VPS41, VPS45 and YOL138C. The yeast transformant of Claim 1 or 2 which is 1 type, or 2 or more types.   The gene according to claim 1 or 2, wherein the gene is one or more selected from RAV1, RPS6A, SNF7, SNF8, RPL36A, VPS3, VPS4, VPS25, VPS27, VPS28, VPS33, VPS36, VPS41 and VPS45. The yeast transformant described.   The yeast transformant according to any one of claims 1 to 4, wherein the host is Saccharomyces yeast.   The yeast transformant according to any one of claims 1 to 5, wherein the exogenous protein includes cellulase.   The yeast transformant according to claim 6, wherein the cellulase is an endoglucanase. A vector for expressing a foreign protein in yeast,
The following genes:
ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SAC1, SFP1, SNF7, SNF8, SPC72, SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, VMA7, VPS16, VPS25, 28PS VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532C, YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331C, YPT7, YV1 and Z A vector for inactivating two or more species. A host yeast for expressing a foreign protein,
The following genes in the host:
ADA2, ADE1, ADE3, ADE5, 7, ADE8, ADH1, ADO1, ARV1, ATP15, BUD19, BUD23, BUL1, BUR2, CHO2, CSG2, DYN3, ERG3, ERG6, FAR8, FSF1, FYV6, GCN5, GDH1, GND1, HMO1, HTL1, IES6, KAP120, MDM34, MMM1, MON2, MRPL20, MRPL38, MSK1, MTM1, MTQ2, NBP2, NGG1, NPR2, OPI3, OPI9, PAT1, PER1, PET309, RAV1, RMD11, ROX3, RPL13B, RPL19B RPL1B, RPL27A, RPL36A, RPL39, RPS6A, RRD1, RSA1, SAC1, SFP1, SNF7, SNF8, SPC72, SPT7, SRC1, SWI3, TAF14, TPS2, URM1, VMA2, VMA4, VMA7, VPS16, VPS25, 28PS VPS3, VPS33, VPS36, VPS4, VPS41, VPS45, VRP1, YDR065W, YDR433W, YDR532C, YGL218W, YGR102C, YGR272C, YKL118W, YNL120C, YNL228W, YNL296W, YOL138C, YOR331C, YPT7, YV1 and Z A host yeast in which two or more are inactivated. A method for producing a degradation product of a cellulose-containing material,
Preparing a yeast transformant according to claim 6 or 7,
Culturing the yeast transformant in a liquid medium containing the cellulose-containing material as a carbon source;
A manufacturing method comprising: A method for producing ethanol from a cellulose-containing material,
Preparing a yeast transformant according to claim 6 or 7,
Culturing the yeast transformant in a liquid medium containing the cellulose-containing material as a carbon source and subjecting it to alcohol fermentation;
A manufacturing method comprising: A method for producing a protein comprising:
Preparing a yeast transformant according to claims 1-7;
Culturing the yeast transformant using a liquid medium;
A manufacturing method comprising: