WO2010005044A1 - Levure transgénique et méthode de production d’éthanol - Google Patents

Levure transgénique et méthode de production d’éthanol Download PDF

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WO2010005044A1
WO2010005044A1 PCT/JP2009/062502 JP2009062502W WO2010005044A1 WO 2010005044 A1 WO2010005044 A1 WO 2010005044A1 JP 2009062502 W JP2009062502 W JP 2009062502W WO 2010005044 A1 WO2010005044 A1 WO 2010005044A1
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gene
strain
xylose
yeast
glucosidase
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PCT/JP2009/062502
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Japanese (ja)
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齋藤聡志
高橋和志
宮田佳代
鈴木詠美子
近藤昭彦
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トヨタ自動車株式会社
国立大学法人神戸大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a recombinant yeast introduced with a predetermined gene and a method for producing ethanol using the recombinant yeast.
  • Woody biomass is effectively used as a raw material for useful alcohols such as ethanol and organic acids.
  • Woody biomass is mainly composed of cellulose, hemicellulose and lignin.
  • cellulose or hemicellulose is hydrolyzed (saccharified) into constituent monosaccharides, and the monosaccharides are converted to ethanol by fermentation.
  • Cellulose is mainly composed of glucose
  • hemicellulose is mainly composed of arabinose and xylose. Accordingly, when ethanol is produced using woody biomass, it is desirable that not only glucose but also xylose be effectively used as a fermentation substrate.
  • Patent Documents 1 and 2 and Non-Patent Document 1 disclose a technique in which xylose is used as a substrate using yeast imparted with xylose metabolic ability.
  • yeast with the ability to metabolize xylose has the ability to ferment ethanol using glucose as a substrate, when glucose and xylose are contained in the medium, glucose metabolism preferentially progresses and xylose metabolism is delayed. There was a problem such as.
  • Non-Patent Document 1 discloses a recombinant yeast capable of fermenting xylose and cellooligosaccharide.
  • the recombinant yeast disclosed in Non-Patent Document 1 introduces a xylose reductase gene and a xylitol dehydrogenase gene derived from Pichia stipitis and a xylulokinase gene derived from S. cerevisiae and a ⁇ -glucosidase gene derived from Aspegillus aculeatus on the cell surface.
  • Recombinant yeast introduced in the form presented.
  • an object of the present invention is to provide a recombinant yeast having an excellent metabolic rate of xylose even in the presence of a coexisting sugar, and a method for producing ethanol using the recombinant yeast.
  • the present invention includes the following.
  • Recombinant yeast in which 2 or more copies of cell surface display ⁇ -glucosidase gene and 2 or more copies of xylose metabolism-related gene have been introduced into the genome.
  • a recombinant yeast in which a cell surface display ⁇ -glucosidase gene and two or more copies of a xylose metabolism-related gene are introduced into the genome, and the xylose metabolism rate is 2.0 g / h / L or more.
  • xylose metabolism-related gene is a xylose reductase gene, a xylitol dehydrogenase gene, or a xylulokinase gene.
  • the cell surface-presenting ⁇ -glucosidase gene is introduced in such a manner that its expression is controlled by the expression control region of the endogenous hyperosmotic response 7 (HOR7) gene (1) to (3) The recombinant yeast according to any one of the above.
  • HOR7 hyperosmotic response 7
  • a method for producing ethanol comprising culturing the recombinant yeast according to any one of (1) to (8) in a xylose-containing medium and recovering ethanol from the xylose-containing medium.
  • Japanese Patent Application No. 2007-200978 a technique that allows a multicopy gene to be introduced into a genome simply by using yeast having homothallic properties.
  • the ability to metabolize ⁇ -glucosidase and xylose can be improved by introducing a cell surface display-type ⁇ -glucosidase gene and a xylose metabolism-related gene into the yeast disclosed in this prior application.
  • the xylose metabolism rate can be improved in ethanol fermentation using a medium containing xylose. Therefore, by using the recombinant yeast according to the present invention, for example, ethanol fermentation using woody biomass can be carried out more efficiently and with excellent productivity.
  • ethanol can be recovered more efficiently and in high yield by fermentation using xylose contained in the medium as a substrate.
  • xylose and glucose are contained in the medium, ethanol can be efficiently produced without a delay in the xylose metabolism rate.
  • FIG. 3 is a diagram showing a step of producing a plasmid pRS404-NotI-s.s-BGL1-3′half-a-agglutinin-SphI-TRP1-NotI. It is a figure which shows the process of producing plasmid pUC19-AscI-HOR7promoter-NotI-SalI, pUC19-AscI-PDC1promoter-NotI-SalI, and pUC19-AscI-TDH3promoter-NotI-SalI. It is a figure which shows the process of producing plasmid pABGL-HOR7P, pABGL-PDC1P, and pABGL-TDH3P.
  • the recombinant yeast according to the present invention has a cell surface display-type ⁇ -glucosidase gene and two or more copies of xylose metabolism-related genes introduced into the genome, and has a high xylose metabolism rate (eg, 2.0 g / h / L or more). Yeast.
  • the cell surface-presenting ⁇ -glucosidase gene is a gene obtained by fusing a ⁇ -glucosidase gene and a cell surface-localized protein gene so that the expressed ⁇ -glucosidase is displayed on the surface of the cell.
  • the cell surface localized protein refers to a protein that is fixed on the cell surface layer of yeast and is present on the cell surface layer.
  • ⁇ - or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned.
  • a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal on the C-terminal side.
  • the cell surface localized protein is different from the secreted protein in that it is transported by being immobilized on the cell membrane via a GPI anchor.
  • the GPI anchor attachment recognition signal sequence is selectively cleaved, and is bound to the GPI anchor at the newly protruding C-terminal portion and fixed to the cell membrane.
  • the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC).
  • PI-PLC phosphatidylinositol-dependent phospholipase C
  • the ⁇ -glucosidase gene encoding ⁇ -glucosidase displayed on the cell surface is not particularly limited, and examples thereof include ⁇ -glucosidase gene derived from Aspergillus aculeatus (Murai et al., Appl. Environ. Microbiol. 64: 4857-4861) be able to.
  • ⁇ -glucosidase gene a ⁇ -glucosidase gene derived from Aspergillus oryzae, a ⁇ -glucosidase gene derived from Clostridium cellulovorans, a ⁇ -glucosidase gene derived from Saccharomycopsis fibligera, and the like can be used.
  • the xylose metabolism-related gene is a xylose reductase gene encoding xylose reductase that converts xylose into xylitol, a xylitol dehydrogenase gene encoding xylitol dehydrogenase that converts xylitol into xylulose, and xylulose by phosphorylating xylulose 5-phosphate It is meant to include a xylulokinase gene encoding xylulokinase which produces Xylulose 5-phosphate produced by xylulokinase enters the pentose phosphate pathway and is metabolized.
  • the xylose metabolism-related gene introduced into the yeast genome is not particularly limited, and examples thereof include a xylose reductase gene and a xylitol dehydrogenase gene derived from Pichia stipitis, and a xylulokinase gene derived from Saccharomyces cerevisiae (Eliasson A. et al. Appl. Environ. Microbiol, 66: 3381-3386 and Toivari MN et al., Metab. Eng. 3: 236-249).
  • a xylose reductase gene derived from Candida ⁇ ⁇ ⁇ tropicalis or Candida prapsilosis can be used as the xylose reductase gene.
  • a xylitol dehydrogenase gene a xylitol dehydrogenase gene derived from Candida tropicalis or Candida prapsilosis can be used.
  • a xylulokinase gene a xylulokinase gene derived from Pichia stipitis can also be used.
  • a xylose isomerase gene derived from Streptomyces murinus cluster or the like can also be used.
  • the method for introducing two or more copies of each gene is not particularly limited. For example, a method using the homothallic property of yeast having a plurality of selection markers can be applied.
  • the yeast having homothallic properties is synonymous with homothallic yeast.
  • yeast having homothallic properties sex transition by endonuclease encoded by HO gene occurs, and daughter cells of different sex bud from haploid mother cells.
  • haploid yeast there are two types of sex (mating type), a cell and ⁇ cell, and a cell and ⁇ cell do not join.
  • a cell and ⁇ cell secrete a specific sex hormone (pheromone) called a factor and ⁇ factor, respectively, and stop normal proliferation when they are detected by receptors on the other pheromone cell membrane in close proximity to each other Then start joining.
  • pheromone specific sex hormone
  • yeast having homothallicity forms a life cycle in which daughter cells sprouting from a haploid mother cell join with the mother cell to form a diploid.
  • haploid yeast having homothallic properties changes to diploid yeast having exactly the same genotype except for the MAT genes (MATa gene and MAT ⁇ gene) that determine the mating type. become.
  • diploid yeast forms spores including meiosis under nutrient starvation conditions such as depletion of nitrogen sources in the medium.
  • Yeast spores have two a cells and two ⁇ cells in a sac-like structure called ascosm. The spore germinates when the nutritional starvation condition is resolved, and begins to multiply by budding again as a haploid.
  • the yeast having homothallic properties is not particularly limited, and any yeast can be used.
  • yeast having homothallic properties include, but are not limited to, Saccharomyces cerevisiae ⁇ OC-2 strain (NBRC2260).
  • Other yeasts with homothallic properties include alcoholic yeasts (Taken 396, NBRC0216) (Source: “Characteristics of Alcoholic Yeast”, Sakekenkai Bulletin, No37, p18-22 (1998.8)), isolated in Brazil and Okinawa Ethanol producing yeast (Source: “Genetic properties of wild strains of Saccharomyces cerevisiae isolated in Brazil and Okinawa” Journal of Japanese Agricultural Chemical Society, Vol.65, No.4, p759-762 (1991.4)) and 180 (Source: Alcohol The screening of yeast having a strong fermenting ability ”, Journal of Japan Brewing Association, Vol.82, No.6, p439-443 (1987.6)).
  • yeast having homothallic properties can be used as a yeast having homothallic properties by introducing the HO gene so that it can be expressed. That is, in the present invention, the yeast having homothallic properties is meant to include yeast into which the HO gene has been introduced so as to be expressed.
  • the Saccharomyces cerevisiae OC-2 strain is preferable because it is a strain that has been confirmed to be safe and has been used in the winemaking scene. Further, the Saccharomyces cerevisiae OC-2 strain is preferable because it has excellent promoter activity under high sugar concentration conditions, as shown in the Examples described later. In particular, the Saccharomyces cerevisiae OC-2 strain is preferable because of its excellent promoter activity of the pyruvate decarboxylase gene (PDC1) under high sugar concentration conditions.
  • PDC1 pyruvate decarboxylase gene
  • the transformation yeast according to the present invention is provided with a plurality of selection markers.
  • the selection marker uses a phenotype that is observed by distinguishing the growth state under a predetermined condition from the growth state under other conditions by defunctionalizing a predetermined gene in the host. is there.
  • the defunctionalization of a gene means a deletion of the gene, transcriptional suppression of the gene, translational suppression of the gene, mutation of the gene itself, and the like. In higher polyploids, genes in all chromosomes are disabled.
  • examples of the selection marker include auxotrophy required by defunctionalizing genes involved in biosynthesis of nutrient components essential for growth.
  • uracil requirement given by defunctionalizing URA3 gene in yeast As auxotrophy, uracil requirement given by defunctionalizing URA3 gene in yeast, histidine requirement given by defunctionalizing HIS3 gene in yeast, and deactivation of TRP1 gene
  • the tryptophan requirement given in (1) can be mentioned.
  • the genes to be disabled are not limited to URA3 gene, HIS3 gene and TRP1 gene, and examples thereof include Leu2 gene, Ade2 gene, Lys2 gene and the like.
  • yeast having a single selection marker is prepared.
  • the yeast has homothallic properties as described above.
  • Saccharomyces cerevisiae OC-2T strain disclosed in Applied and Environmental microbiology, May 2005, p. 2789-2792 can be used.
  • This Saccharomyces cerevisiae OC-2T strain is a strain lacking a pair of TRP1 genes in the Saccharomyces cerevisiae OC-2 strain known as wine yeast, and is a homothallic yeast provided with tryptophan auxotrophy as a selection marker It is.
  • the Saccharomyces cerevisiae OC-2U strain disclosed in the same report can also be used.
  • the Saccharomyces cerevisiae OC-2U strain is a strain lacking the pair of URA3 genes in the Saccharomyces cerevisiae OC-2 strain, and is a homothallic yeast provided with uracil auxotrophy as a selection marker.
  • FIG. 1 schematically shows a method for imparting histidine auxotrophy as a second selection marker to homothallic yeast (OC-2U strain) to which uracil auxotrophy is imparted.
  • a DNA fragment for deleting the host HIS3 gene is prepared. This DNA fragment has a structure in which the upstream region of the HIS3 gene in the host, the URA3 gene, and the downstream region of the HIS3 gene in the host are connected in this order.
  • a strain from which the introduced URA3 gene has been deleted is selected.
  • This can be positively selected by culturing uracil-requiring yeast in a medium containing 5-fluoroorotic acid (referred to as 5-FOA). That is, when a yeast having a uracil biosynthesis system is cultured in the presence of 5-FOA, a uracil analog is synthesized using 5-FOA as a substrate, and the yeast having a uracil biosynthesis system is lethal. Therefore, a strain grown by culturing uracil-requiring yeast in a medium containing 5-FOA can be identified as having the URA3 gene dropped from the chromosome. As shown in FIG. 1 (c), the chromosomal structure of the yeast in which the URA3 gene has been dropped from the chromosome is a deletion of only the HIS3 gene in one chromosome.
  • yeast having the chromosomal structure shown in FIG. 1 (c) is cultured in a spore formation medium, and spore is formed through meiosis.
  • the spore formation medium is not particularly limited, and a medium having a conventionally known composition can be used.
  • the spores (ascombs) are separated from the culture medium. In the separated ascension, there are two spores (haploids) containing any one of the pair of chromosomes shown in FIG. 1 (c) and spores (haploids) containing the other chromosome. Two will be included.
  • the spores are cultured in a germination medium, whereby individual spores are grown in the life cycle of vegetative growth.
  • a daughter cell sprouting from a haploid mother cell shows a life cycle in which the daughter cell joins with the mother cell to become a diploid, so that the MAT gene that determines the mating type (
  • the diploid yeast has exactly the same genotype except for the MATa gene and the MAT ⁇ gene. Therefore, it is possible to obtain two types of yeasts having a chromosome structure as shown in FIG. 1 (d) by germinating homothallic yeast having the chromosome structure shown in FIG. 1 (c) after sporulation. it can.
  • a homothallic yeast to which histidine auxotrophy is imparted as a second selection marker can be produced as a strain that grows on a uracil and histidine-containing medium but does not grow on a uracil and histidine-free medium.
  • the third selection marker can be given by repeating the above-described method. That is, by adopting the above-described method, a plurality of selection markers can be sequentially given to yeast having homothallic properties.
  • the above-described method is a method that effectively utilizes a characteristic life cycle such as homothallic property and a phenomenon such as loss of the URA3 gene.
  • a system that uses the URA3 gene as a DNA fragment for deleting the HIS3 gene and uses 5-FOA to determine the loss of the URA3 gene has been described.
  • This gene is not limited to the HIS3 gene, but the marker recycling marker is preferably the URA3 gene. This is because 5-FOA that enables selective acquisition of uracil-requiring strains is used.
  • the above-mentioned DNA fragment for deleting the HIS3 gene is also disclosed in Rinji Akada et al. (Yeast, Volume 23, Issue 5, Page 399-405) for the purpose of use in the marker recycling method.
  • a promoter of glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), a promoter of 3-phosphoglycerate kinase gene (PGK1), a promoter of hyperosmotic response 7 gene (HOR7), and the like can be used.
  • TDH3 glyceraldehyde 3-phosphate dehydrogenase gene
  • PGK1 3-phosphoglycerate kinase gene
  • HOR7 hyperosmotic response 7 gene
  • the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene.
  • the cell surface display-type ⁇ -glucosidase gene may be introduced into the genome of yeast having homothallic properties together with a promoter controlling its expression and other expression control regions.
  • the cell surface display-type ⁇ -glucosidase gene may be introduced so that expression is controlled by a promoter of a gene originally present in the genome of yeast having homothallic properties or other expression control regions.
  • the cell surface display-type ⁇ -glucosidase gene is introduced downstream of the expression control region containing the promoter of the hyperosmotic response 7 (HOR7) gene in yeast having homothallic properties, that is, the expression is controlled by the expression control region. It is preferable to do.
  • HOR7 hyperosmotic response 7
  • the yeast that presents ⁇ -glucosidase on the cell surface is a so-called arming yeast.
  • Arming yeast is a new type that does not exist in normal yeast by fusing cell surface localized proteins with various functional proteins (enzymes, antigens, antibodies, reporter proteins, etc.) and peptides and displaying them on the cell surface.
  • the cell surface layer is a periplasm which is a cell membrane, a cell wall, and a space between them, and a yeast cell that uses these layers and satisfies the above requirements is called an arming yeast. That is, a yeast that presents ⁇ -glucosidase on the cell surface layer can be created based on published patent publications (JP-A-11-290078, WO02 / 085935) that details the creation of arming yeast.
  • xylose metabolism-related genes described above it is sufficient that two or more copies of each gene are incorporated in the yeast genome so that they can be expressed. That is, it may be introduced together with a promoter that controls the expression of a gene related to xylose metabolism and other expression control regions.
  • promoters for genes related to xylose metabolism various promoters listed above can be used as appropriate.
  • any conventionally known method known as a yeast transformation method can be applied.
  • electroporation method “Meth. Enzym., 194, p182 (1990)”
  • spheroplast method Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”
  • acetic acid Lithium Method J. Bacteriology, 153, p163 (1983)
  • Proc. Natl. Acad. Sci. USA, 75 p1929 (1978) Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual The method can be implemented, but is not limited thereto.
  • an expression vector having either one of the genes is selected from a plurality of selection vectors assigned to the yeast. Introduce using one of the markers.
  • the selection marker to be used is auxotrophic
  • a gene that eliminates auxotrophy as well as the target gene is introduced into the expression vector, and a strain can be selected using the loss of auxotrophy as an index.
  • the ascomb of the strain is isolated and haploid spores are cultured in a germination medium.
  • a daughter cell sprouting from a haploid mother cell shows a life cycle in which the daughter cell joins with the mother cell to become a diploid, and therefore, the MAT gene that determines the mating type (
  • the diploid yeast has exactly the same genotype except for the MATa gene and the MAT ⁇ gene.
  • the target gene is introduced into all chromosomes, it is introduced into the chromosome in multiple copies by one transformation.
  • heterothallic yeast when heterothallic yeast is used, only one copy of the target gene can be introduced by one transformation.
  • an expression vector having either one of the cell surface display ⁇ -glucosidase gene and the xylose metabolism-related gene is selected from a plurality of selection markers given to yeast, excluding those used in the previous treatment. Introduce using markers.
  • the other gene can also be introduced into the chromosome in multiple copies by a single transformation.
  • a yeast having homothallic properties into which a plurality of selection markers are introduced a plurality of target genes can be introduced according to the number of selection markers by a simple technique. Therefore, if the selection marker remains, the cell surface display ⁇ -glucosidase gene and the xylose metabolism-related gene can be further introduced, and further high expression of these genes can be achieved.
  • the recombinant yeast prepared as described above has ⁇ -glucosidase presented on the cell surface, exhibits excellent ⁇ -glucosidase activity, and exhibits excellent xylose metabolism rate.
  • ⁇ -glucosidase activity can be defined based on the amount of p-nitrophenol generated (PNPG activity).
  • the xylose metabolism rate can be calculated from the amount of xylose consumed and the culture time by quantifying the amount of xylose in the medium by, for example, HPLC.
  • ⁇ Ethanol production> By using the recombinant yeast described above, ethanol fermentation using a sugar such as xylose as a substrate can be performed.
  • the above-mentioned recombinant yeast is suitable for ethanol production using woody biomass containing xylose as a constituent sugar because the xylose metabolism rate in fermentation using xylose as a substrate is greatly improved.
  • cellulose or hemicellulose is hydrolyzed (saccharified) to constituent monosaccharides.
  • the woody biomass Prior to saccharification, the woody biomass may be subjected to a conventionally known pretreatment. Although it does not specifically limit as pre-processing, For example, the process which decomposes
  • saccharified woody biomass is collected to collect monosaccharides constituting cellulose and hemicellulose.
  • the saccharification method is not particularly limited, and a conventionally known method can be used without any limitation.
  • Examples of the saccharification method include a sulfuric acid method using dilute sulfuric acid or concentrated sulfuric acid, and an enzymatic method using cellulase or hemicellulase.
  • ethanol fermentation is performed using the above-described recombinant yeast.
  • the temperature is preferably 25 to 35 ° C. and the pH is preferably 4 to 6.
  • the above-described recombinant yeast has a particularly improved xylose metabolism rate in a medium containing a sugar component (coexisting sugar) other than xylose of glucose sugar.
  • a sugar component coexisting sugar
  • the sugar component derived from the woody biomass contains a lot of coexisting sugars such as xylose and glucose other than xylose. Therefore, it can be said that the above-mentioned recombinant yeast is particularly effective when ethanol is produced from woody biomass with high efficiency.
  • ethanol is collected from the medium.
  • the method for recovering ethanol is not particularly limited, and any conventionally known method can be applied.
  • a liquid layer containing ethanol and a solid layer containing recombinant yeast and solid components are separated by solid-liquid separation operation.
  • ethanol contained in the liquid layer is separated and purified by a distillation method, whereby high purity ethanol can be recovered.
  • the purity of ethanol can be adjusted as appropriate according to the intended use of ethanol.
  • Example 1 In this example, a second Saccharomyces cerevisiae OC-2U strain (Saito et al., Journal of Ferment. Bioeng. 81: 98-103 (1996)), which imparted uracil requirement to the Saccharomyces cerevisiae OC-2 strain, was used as the host. As a selection marker, histidine requirement was further given (see FIGS. 1 (a) to (d)).
  • the HIS3 disruption DNA fragment described in Akada et al. Yeast 2006 23: 399-405 (PCR fragment that consisted of a URA3 marker attached to a 40 base repeated-generating sequence franked by the HIS3 targeteing sequence at both ends.)
  • the OC-2U strain was transformed by Frozen EZ transformation kitII: Zymo Research, and colonies that grew on the SD plate were isolated.
  • the SD medium cannot grow uracil auxotrophic strains, but can be used to grow uracil auxotrophic strains, that is, the loss of uracil auxotrophy caused by the expression of the URA3 gene contained in the HIS3 disrupted DNA fragment The transformant was selected.
  • the obtained colony was made into a single colony on an SD plate, and then cultured in a YPD liquid medium at 30 ° C. for 1 day, and 0.2 ml of the culture solution was spread on a FOA plate.
  • the composition of the FOA plate is Bacto yeast nitro base 0.67%, glucose 2%, uracil 50 ⁇ g / ml, and 5-FOA 0.1%.
  • the colonies grown on this FOA plate are strains in which the URA3 gene contained in the introduced gene fragment has been lost and the original uracil requirement of the OC-2U strain has been restored.
  • the obtained ascomb contained 3 or 4 spores. Since the OC-2U strain originally has homothallic properties having the HO gene that converts the sex of yeast, haploid spores are diploidized with vegetative cells after meiosis. For this reason, 2 spores out of the 4 spores of the transformed strain were isolated as a strain having both uracil requirement and histidine requirement because the URA3 gene was lost and the HIS3 gene was destroyed and uracil requirement was restored. The remaining two strains have normal HIS3 gene and have only uracil requirement.
  • Example 2 In this example, it was verified that a strain derived from the Saccharomyces cerevisiae OC-2 strain was excellent in productivity in substance production. That is, when a heterologous gene is introduced by transformation for the purpose of high substance productivity, it is necessary to utilize a high expression promoter. Promoters include constitutive promoters and inducible promoters. When using either promoter, it is necessary to select a promoter with high promoter expression at a high sugar concentration when high productivity is desired. is there. Therefore, for the YPH500 strain (laboratory yeast) and the OC-2T strain (Saito et al., Journal of Ferment. Bioeng.
  • the TDH1 promoter commonly used in yeast genetic recombination technology.
  • the promoter activities of the TDH3 promoter and the PDC1 promoter were examined by comparing the expression levels of genes transcribed by the respective promoters.
  • YPD liquid culture (shinto) was performed on the YPH500 strain and the OC-2T strain at two levels of 2% or 10% glucose.
  • the obtained cultured cells are sampled, cDNA is prepared using 1ststrand cDNA synthesis kit for RT-PCR manufactured by Roche, and the expression levels of TDH1, TDH3, and PDC1 genes are measured using Roche Light cycler 330 using the following primers: Quantitative PCR was performed.
  • the primer sequences used are shown in Table 1.
  • FIG. 2 (a) shows the expression levels of the TDH1 gene, the TDH3 gene and the PDC1 gene depending on the glucose concentration of the YPH500 strain
  • FIG. 2 (b) the OC-2T strain.
  • the YPH500 strain which is a laboratory yeast
  • the expression levels of the TDH3 gene and the PDC1 gene used in the experiment all decreased.
  • the OC-2T strain conversely, when the glucose concentration was increased from 2% to 10%, the expression levels of all of the TDH1 gene, TDH3 gene, and PDC1 gene tended to increase.
  • YPH500 strain was originally used as a laboratory yeast, a strain capable of exhibiting sufficient performance in a normal 2% glucose-containing YPD medium has been selected.
  • the two strains are yeasts that have been used for wine brewing in Japan and so on, so it is thought that they support the ability of the strains to easily exert their performance in a medium with a high sugar concentration.
  • Example 3 the spore formation rate of the OC-2HU strain prepared in Example 1 was confirmed. It is preferable that the spore formation rate is 0.1% or more because it is easy to perform the operation of separating the ascomy using a micromanipulator, as will be described in Examples below.
  • the OC-2HU strain prepared in Example 1 was streaked on a YPD plate containing 2% glucose and cultured at 30 ° C. for 1 day, and then the cultured cells were transferred to a Sherman plate, which is a sporulation medium, at 25 ° C. After culturing for 3 to 4 days, sporulation was confirmed with a microscope. Table 2 shows the sporulation rate of the OC-2HU strain. The spore formation rate was calculated according to the following formula.
  • Example 1 From the results of this example, it was clarified that the OC-2HU strain prepared in Example 1 can perform ascending separation using a micromanipulator on the second day from the start of culture.
  • Example 4 the Saccharomyces cerevisiae OC-2HU strain prepared in Example 1 was used as a host, and tryptophan requirement was further imparted as a third selection marker.
  • a TRP1 disruption fragment was prepared using the primers shown below to prepare an OC-2HUT (triple marker) strain.
  • 1stPCR is performed using TRP1-998 and TRP1-28r
  • 2ndPCR is performed using TRP1-URA3 and TRP1-40r
  • the DNA fragment amplified by 1stPCR and the DNA fragment amplified by 2ndPCR are used as templates.
  • TRP1-998 and TRP1-40r were used for annealing PCR to obtain a TRP1-disrupted fragment.
  • genomic DNA of OC-2T strain Saito et al., Journal of Ferment. Bioeng. 81: 98-103 (1996)
  • 2ned PCR pRS406 manufactured by Stratagene having the URA3 gene was used as a template.
  • the temperature conditions in 1st PCR and 2nd PCR are maintained at 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 20 seconds, 50 ° C for 30 seconds and 68 ° C for 1 minute, and then maintained at 68 ° C for 2 minutes.
  • the condition was Temperature conditions in annealing-PCR are maintained at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 20 seconds, 50 ° C for 30 seconds and 68 ° C for 2 minutes, and then maintained at 68 ° C for 2 minutes. Condition.
  • the TRP1-disrupted fragment obtained as a result of annealing PCR contains the URA3 gene and has a structure in which homologous recombination regions are added to both ends.
  • the recombinant yeast introduced with the TRP1-disrupted fragment is cultured in a medium containing 5-FOA, the URA3 gene will be lost.
  • the obtained TRP1 disruption fragment was transformed in the same manner as in Example 1 using the OC-2HU strain (histidine-requiring yeast and uracil-requiring yeast) shown in Example 1 as a host, and uracil-requiring A strain complementary to was isolated.
  • an appropriate amount of the obtained strain was smeared on a plate containing 5-FOA (hereinafter referred to as FOA plate), and then colonies that appeared were isolated. Since the FOA plate can positively select uracil-requiring strains, the strain contained in the isolated colony is considered to be a strain in which the URA3 gene has been removed from the transformed TRP1-disrupted fragment.
  • spore formation was performed on a Sherman plate, which is a spore formation medium, and after spore formation was confirmed, spore separation was performed using a micromanipulator under a microscope.
  • the resulting four spores have a 2: 2 strain that has a TRP1 disruption fragment homologously on the chromosome and a strain that has a normal TRP1 gene homology. For this reason, if m tryptophan auxotrophic strain is isolate
  • the obtained spores were SD plate, SD plate + uracil, SD plate + histidine, SD plate + tryptophan, SD plate + uracil + histidine, SD plate + uracil + tryptophan, SD plate + tryptophan + histidine, SD plate + Cultivation was performed on 8 types of plates of uracil + histidine + tryptophan.
  • the homothallic yeast having the uracil requirement, the histidine requirement and the tryptophan requirement prepared in this example was named Saccharomyces cerevisiae OC-2HUT strain.
  • Example 5 In this example, the OC-2HUT strain prepared in Example 4 was used as a yeast for transformation, and 4 copies of the surface-presented BGL gene were introduced as the target gene.
  • the obtained colonies were cultured on a YPD plate at 30 ° C. for 1 day, and then the spore-forming medium (sherman) was applied to the spore-forming medium (sherman), followed by plate culture at 25 ° C. Then, four spores contained in the ascending were separated using a Narishige micromanipulator. As a result, the strain into which 2 copies of pIBG13 have been introduced and the strain from which pIBG13 has been dropped are separated into 2: 2. Therefore, the obtained spore was cultured in SD medium at 30 ° C. for 2 days, and a strain in which histidine requirement disappeared was identified. The obtained strain was a strain into which 2 copies of pIBG13 had been introduced and was named OC-2ABGL2.
  • pIBG13 is composed of pIHCS (Yasuya Fujita et al. “Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes” mesAppl Environ Microbiol. 68 (Oc10ber) ) And pBG211 (Murai, T et al. "Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing ⁇ -glucosidase and carboxymethylcellulase from Aspergillus aculeatusAspergillus aculeatus.” Appl. Environ. . Specifically, a BGL1 fragment (2.5 kbp) having NcoI / XhoI sites at both ends was amplified by PCR using pBG211 as a template and the following pair of primer sets.
  • the obtained colonies were cultured on a YPD plate at 30 ° C. for 1 day, and then the cells were applied to a sherman plate, which is a spore-forming medium, and the plate was cultured at 25 ° C. The formation was confirmed, and the 4 spores contained in the ascomy were separated using a Narishige micromanipulator.
  • the obtained spores were cultured in SD medium for 2 days at 30 ° C., and a strain in which tryptophan requirement disappeared was identified.
  • the identified strain was a strain in which 2 copies of pIBG13 and 2 copies of pIWBGL1 were introduced, and a total of 4 copies of the arming BGL gene fragment were introduced into the genome, and was named OC-2ABGL4.
  • PIWBGL1 was constructed from pRS404 (ATCC Number: 87875) and pIBG13 as shown in FIG.
  • pRS404 ATCC Number: 87875
  • pIBG13 pIBG13
  • GIBDH-promoter-BGL1 gene-3'half-of-a-agglutinin gene was excised by treating NotI with pIBG13 and ligated with pRS404 after NotI treatment and alkaline phosphatase treatment. This constructed pIWBGL1.
  • the xylose reductase gene, the xylitol dehydrogenase gene and the xylulokinase gene are each of the GAPDH promoter. It is a plasmid introduced so that it can be expressed under control.
  • the obtained colonies were cultured on a YPD plate at 30 ° C. for 1 day, and then the cells were applied to a sherman plate, which is a spore-forming medium, and the plate was cultured at 25 ° C. The formation was confirmed, and the 4 spores contained in the ascomy were separated using a micromanipulator manufactured by Narishige. The obtained spore was cultured in SD medium at 30 ° C. for 2 days, and a strain in which uracil requirement was lost was identified.
  • the identified strain is a strain into which 2 copies of pIBG13, 2 copies of pIWBGL1 and 2 copies of pIUX1X2Xk have been introduced, and a total of 4 copies of arming BGL gene fragments and 2 copies of xylose metabolism-related genes have been introduced into the genome. , Named OC-2ABGL4Xyl2.
  • PNPG p-nitrophenyl- ⁇ -D-glucoside
  • the reagents used for the measurement are as follows.
  • a reaction mixture (1.0 ml of 0.1 M acetate buffer and 0.5 ml of PNPG aqueous solution) was prepared in a test tube, and pre-warmed at 37 ° C. for about 5 minutes. Next, 0.5 ml of the diluted culture solution was added to start the reaction. Next, after reaction at 37 ° C. for exactly 15 minutes, 2 ml of Na 2 CO 3 solution was added to stop the reaction.
  • the absorbance at 400 nm in the reaction solution was measured.
  • the reaction solution was allowed to stand at 37 ° C. for 15 minutes, 2 ml of Na 2 CO 3 solution was added and mixed, and then 0.5 ml of enzyme solution was added and adjusted.
  • the PNPG activity was not detected in the parent strain (OC-2HUT strain) into which the arming BGL gene was not introduced, whereas the PNPG activity increased as the number of arming BGL genes increased. Remarkably improved.
  • Example 6 the OC-2ABGL4Xyl2 strain prepared in Example 5 (3) was evaluated for the effect on the xylose metabolism rate due to the difference in coexisting sugars.
  • YP medium three types of media containing (1) glucose 9% + xylose 6%, (2) cellobiose 9% + xylose 6%, and (3) xylose 6% were prepared. These three types of medium were inoculated with OC-2ABGL4Xyl2 strain, respectively, and cultured under conditions of 30 ° C. and 60 rpm.
  • the xylose concentration contained in the culture solution was measured over time for 24 hours from the start of the culture.
  • the xylose concentration was measured by HPLC. The measurement results are shown in FIG.
  • the xylose metabolism of the OC-2ABGL4Xyl2 strain was only 1.83% when the medium contained only xylose.
  • the xylose metabolism was increased to 4.34% and 4.92%, respectively.
  • the amount of xylose metabolism is shown as a value (%) obtained by subtracting the xylose concentration contained in the medium at 24 hours from the start of cultivation from the xylose concentration contained in the medium at the start of cultivation.
  • the OC-2ABGL4Xyl2 strain prepared in Example 3 has a feature that the xylose metabolism rate is further improved particularly when a coexisting sugar is present. Furthermore, as a coexisting sugar, cellobiose was found to be more preferable than glucose from the viewpoint of xylose metabolism rate.
  • Example 7 In this example, using the OC-2ABGL4Xyl0032 and OC-2ABGL2Xyl2 strains prepared in Example 5 (3), the influence on the xylose metabolism rate due to the difference in the copy number of the BGL gene was evaluated. First, based on the YP medium, a medium containing cellobiose 9% + xylose 6% was prepared. This medium was inoculated with the OC-2ABGL4Xyl2 strain or OC-2ABGL2Xyl2 strain and cultured under conditions of 30 ° C. and 60 rpm.
  • the OC-2ABGL2Xyl2 strain had a xylose metabolism of 3.38% 24 hours after the start of culture
  • the OC-2ABGL4Xyl2 strain had a xylose metabolism of 4.92% 24 hours after the start of culture.
  • the xylose metabolism is shown as a value (%) obtained by subtracting the xylose concentration contained in the medium at 24 hours from the start of cultivation from the xylose concentration contained in the medium at the start of cultivation.
  • Example 8 the xylose metabolic rate was evaluated using the OC-2ABGL4Xyl2 strain prepared in Example 5 (3). First, based on the YP medium, a medium containing cellobiose 9% + xylose 6% was prepared. This medium was inoculated with OC-2ABGL4Xyl2 strain and cultured under conditions of 30 ° C. and 120 rpm.
  • the xylose concentration, cellobiose concentration and ethanol concentration contained in the culture solution were measured over time for 24 hours from the start of the culture.
  • the xylose concentration was measured in the same manner as in Example 4.
  • Cellobiose concentration was measured by HPLC.
  • the ethanol concentration was measured using an enzyme sensor (manufactured by Oji Scientific Instruments, model number BF4). The measurement results are shown in FIG.
  • the OC-2ABGL4Xyl2 strain was able to almost completely metabolize cellobiose 8.2% and xylose 5% within 24 hours from the start of culture, and produce 5.8% ethanol.
  • the results shown in FIG. 8 revealed that the OC-2ABGL4Xyl2 strain had a xylose metabolic rate of 2.0 g / h / L. From this result, it became clear that by using OC-2ABGL4Xyl2 strain, ethanol can be efficiently produced by effectively using xylose in ethanol production using woody biomass.
  • Example 9 an expression control region that controls the expression of the BGL gene was examined in order to further improve ⁇ -glucosidase activity.
  • the glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), pyruvate decarboxylase gene (PDC1), or the hyperosmotic response 7 gene endogenous to the OC-2HUT strain prepared in Example 4 was used.
  • An arming BGL gene was introduced downstream of the expression control region of (HOR7), and ⁇ -glucosidase activity was examined.
  • Plasmid pABGL-HOR7P for introducing the arming BGL gene was constructed downstream of the control region (see FIG. 9). The primers used are shown in Table 4 below.
  • PCR was performed using the plasmid pIBG13 shown in FIG. 3 as a template and primers (1) and (2).
  • the obtained PCR fragment was treated with the restriction enzyme BssHII.
  • the obtained DNA fragment was ligated with pRS403 (Stratagene) treated with the restriction enzyme BssHII to prepare plasmid pRS404-NotI-s.s-BGL1-3′half-a-agglutinin-SphI.
  • PCR was performed using the genomic DNA of S. cerevisiae as a template and primers (3) and (4), and the resulting PCR fragment was treated with the restriction enzyme SphI.
  • the obtained DNA fragment was ligated with plasmid pRS404-NotI-ss-BGL1-3'half-a-agglutinin-SphI treated with restriction enzyme SphI, and plasmid pRS404-NotI-ss-BGL1-3'half-a -agglutinin-SphI-TRP1-NotI was prepared (FIG. 10).
  • a DNA fragment containing the HOR7 promoter region was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (5) and (8).
  • a DNA fragment containing the PDC1 promoter region was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (6) and (9).
  • a DNA fragment containing the TDH3 promoter region was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (7) and (10).
  • the DNA fragment containing the HOR7 promoter region, the DNA fragment containing the PDC1 promoter region, and the DNA fragment containing the TDH3 promoter region were treated with restriction enzymes EcoRI and SalI, respectively. Then, the obtained three kinds of DNA fragments were ligated with pUC19 treated with restriction enzymes EcoRI and SalI, and three kinds of plasmids pUC19-AscI-HOR7promoter-NotI-SalI, pUC19-AscI-PDC1promoter-NotI-SalI PUC19-AscI-TDH3promoter-NotI-SalI was prepared (FIG. 11).
  • a DNA fragment containing a part of the coding region of the HOR7 gene was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (11) and (14).
  • a DNA fragment containing a part of the coding region of the PDC1 gene was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (12) and (15).
  • a DNA fragment containing a part of the coding region of the TDH3 gene was amplified by PCR using the genomic DNA of S. cerevisiae as a template and primers (13) and (16).
  • the obtained DNA fragment containing a part of the coding region of PDC1 gene (NotI-SalI fragment) was ligated with plasmid pUC19-AscI-PDC1promoter-NotI-SalI treated with restriction enzymes NotI and SalI, and plasmid pUC19 -AscI-PDC1promoter-NotI-truncated-AscI was prepared.
  • the obtained DNA fragment containing a part of the coding region of the TDH3 gene (NotI-SalI fragment) and the plasmid pUC19-AscI-TDH3promoter-NotI-SalI treated with the restriction enzymes NotI and SalI were ligated, and the plasmid pUC19 -AscI-TDH3promoter-NotI-truncated-AscI was produced (FIG. 11).
  • the plasmid pRS404-NotI-ss-BGL1-3′half a-agglutinin-SphI-TRP1-NotI shown in FIG. 10 is treated with NotI, and contains the BGL gene, 3′half a-agglutinin and TRP1 gene. A fragment (NotI fragment) was excised.
  • the obtained DNA fragment and three kinds of plasmids pUC19-AscI-HOR7promoter-NotI-truncated-AscI, pUC19-AscI-PDC1promoter-NotI-truncated-AscI and pUC19-AscI-TDH3promoter-NotI treated with restriction enzyme NotI -truncated-AscI was ligated to prepare plasmids pABGL-HOR7P, pABGL-PDC1P, and pABGL-TDH3P (FIG. 12).
  • plasmids pABGL-HOR7P, pABGL-PDC1P and pABGL-TDH3P are treated with the restriction enzyme AscI, so that the cell surface-displayed ⁇ -glucosidase gene and the TRP1 gene, which is a selection marker, contain a homologous recombination region to the chromosome. It can be cut out as a DNA fragment.
  • each of these plasmids pABGL-HOR7P, pABGL-PDC1P, and pABGL-TDH3P contains a pair of DNA fragments so that homologous recombination occurs in a desired region.
  • the plasmid pABGL-TDH3P includes an approximately 300 bp region (SEQ ID NO: 9) located upstream of the TDH3 gene and an approximately 300 bp region (SEQ ID NO: 10) contained in the coding region of the TDH3 gene. .
  • the plasmid pABGL-PDC1P includes an approximately 300 bp region (SEQ ID NO: 11) located upstream of the PDC1 gene and an approximately 300 bp region (SEQ ID NO: 12) contained in the coding region of the PDC1 gene.
  • the plasmid pABGL-HOR7P contains an approximately 300 bp region (SEQ ID NO: 13) located upstream of the HOR7 gene and an approximately 300 bp region (SEQ ID NO: 14) contained in the coding region of the HOR7 gene.
  • each plasmid was treated with the restriction enzyme AscI, and then transformed with Frozen-EZ Yeast Transformation II (Zymo Research). SD + histidine + uracil plate was used as the selection plate. Colonies obtained by transformation were isolated using the same plate to eliminate false positive strains.
  • the obtained colonies were cultured on a YPD plate at 30 ° C. for 2 days, and then the spore formation medium (sherman) was applied to the spore-forming medium (sherman), followed by plate culture at 25 ° C. and spore formation on the third day of culture. Then, four spores contained in the ascending were separated using a Narishige micromanipulator. As a result, a strain into which 2 copies of the above plasmid have been introduced and a strain from which pIBG13 has been removed are separated into 2: 2. Therefore, the obtained spores were cultured in SD medium at 30 ° C. for 2 days, and a strain in which the tryptophan requirement disappeared was identified.
  • the strain introduced with 2 copies of plasmid pABGL-TDH3P was named OC-2ATDH37PBGL2, the strain introduced with 2 copies of plasmid pABGL-PDC1P was named OC-2APDC1PBGL2, and 2 copies of plasmid pABGL-HOR7P were introduced. This strain was named OC-2AHOR7PBGL2.
  • the PNPG activity of the three transformed yeasts OC-2ATDH37PBGL2, OC-2APDC1PBGL2, and OC-2AHOR7PBGL2 obtained was measured according to the method described in Example 5 (4). The results are shown in FIG. As can be seen from FIG. 13, ⁇ -glucosidase activity caused by the introduced arming BGL gene could be confirmed in all transformed yeasts. In particular, OC-2AHOR7PBGL2 was found to exhibit ⁇ -glucosidase activity superior to that of other transformed yeasts. From these results, it was found that introducing the arming BGL gene so that the expression is controlled by the expression control region of the HOR7 gene is very excellent in improving the ⁇ -glucosidase activity.
  • Example 10 the transformed yeasts OC-2APDC1PBGL2 and OC-2AHOR7PBGL2 prepared in Example 9 were used for the xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene using pIUX1X2Xk used in Example 5 (3). It was introduced so as to allow expression under the control of the GAPDH promoter. PIUX1X2Xk is disclosed in S. Katahira et al., Appl. Microbiol. Biotechnol.
  • the xylose reductase gene, the xylitol dehydrogenase gene and the xylulokinase gene are each of the GAPDH promoter. It is a plasmid introduced so that it can be expressed under control.
  • transformation was performed using a plasmid obtained by linearizing pIUX1X2Xk (URA3 marker) with the restriction enzyme PstI using Frozen-EZ Yeast Transformation II (Zymo Research).
  • the selection plate used was an SD (G) + histidine plate. Colonies obtained by transformation were separated on the same plate and false positive strains were excluded.
  • Example 5 the transformed yeast in which 2 copies of pIUX1X2Xk were introduced into the transformed yeasts OC-2APDC1PBGL2 and OC-2AHOR7PBGL2 produced in Example 9 in the same manner as at the time.
  • OC-2APDC1PBGL2XYL2 and OC-2AHOR7PBGL2XYL2 Were named OC-2APDC1PBGL2XYL2 and OC-2AHOR7PBGL2XYL2, respectively.
  • FIG. 14 shows pIUX1X2Xk for the TDH3PBGL2 strain, TDH3PBGL4 strain, TDH3PBGL4 strain, TDH3PBGL6 strain, and OC-2HUT strain introduced with 2, 4 or 6 copies of arming BGL gene in the OC-2HUT strain or OC-2HUT strain.
  • the PNPG activity measured for the introduced OC-2HTx2 strain is also shown.
  • the strain into which the arming BGL gene was introduced so that the expression was controlled by the expression control region of the HOR7 gene showed ⁇ -glucosidase activity superior to the strain into which 6 copies of the arming BGL gene were introduced.
  • the expression was controlled by the expression control region of the PDC1 gene.
  • the arming is controlled so that the expression is controlled by the expression control region of the HOR7 gene.
  • the ⁇ -glucosidase activity was further improved.
  • the strain that introduced the arming BGL gene so that its expression was controlled by the expression control region of the HOR7 gene and introduced a gene related to xylose metabolism was superior to the predicted range. It was found to exhibit ⁇ -glucosidase activity.

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Abstract

Cette invention concerne une levure transgénique dont la vitesse de métabolisme du xylose est excellente même en présence d’un sucre coexistant. L’invention concerne aussi une méthode de production d’éthanol en utilisant la levure transgénique. La levure transgénique comporte deux copies ou plus d’un gène de la β-glucosidase à marqueur antigénique et deux copies ou plus d’un gène associé au métabolisme du xylose introduites dans son génome, ou comporte un gène de la β-glucosidase à marqueur antigénique et deux copies ou plus d’un gène associé au métabolisme du xylose introduits dans son génome, et possède une activité β-glucosidase (c’est-à-dire une valeur de 0,02 U/OD 660) de 1 ou plus.
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