JP2010035556A - Transgenic yeast and method for production of ethanol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transgenic yeast which has an excellent xylose metabolism rate even in the presence of a co-existing sugar, and to provide a method for producing ethanol by utilizing the transgenic yeast. <P>SOLUTION: The transgenic yeast has two or more copies of a cell-surface-presenting β-glucosidase gene and two or more copies of a xylose-metabolism-related gene introduced into the genome thereof or has a cell-surface-presenting β-glucosidase gene and two or more copies of a xylose-metabolism-related gene introduced into the genome thereof, and has β-glucosidase activity of 0.02 U/OD660=1 or greater. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a recombinant yeast introduced with a predetermined gene and a method for producing ethanol using the recombinant yeast.

  In view of global warming, great expectations are placed on alternative petroleum resources such as ethanol produced from woody biomass. That is, 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. In order to produce a liquid fuel such as ethanol from woody biomass, cellulose or hemicellulose is hydrolyzed (saccharified) into constituent monosaccharides, and the monosaccharides are converted to ethanol by fermentation. Cellulose is mainly composed of glucose, and 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.

  However, normal yeasts used for ethanol production can only use 6 monosaccharide glucose as a substrate. Therefore, 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. However, since 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 β-glucosidase gene derived from Aspegillus aculeatus on the cell surface. Recombinant yeast introduced in the form presented. However, even in the recombinant yeast disclosed in Non-Patent Document 1, the metabolic delay of xylose has not been eliminated, and the above-described problems have not been solved.

Special table 2000-509988 Special table 2008-506383

S. Katahira et al., Appl. Microbiol. Biotechnol. (2006) 72: 1136-1143

  Therefore, in view of the above-described circumstances, 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.

  As a result of intensive studies by the present inventors in order to achieve the above object, a novel finding that, in a yeast into which a cell surface-presenting β-glucosidase gene has been introduced together with a xylose metabolism-related gene, if the β-glucosidase activity is high, the xylose metabolism rate is improved. As a result, the present invention has been completed.

The present invention includes the following.
(1) A recombinant yeast in which two or more copies of a cell surface-presenting β-glucosidase gene and two or more copies of a xylose metabolism-related gene have been introduced into the genome.

  (2) A recombinant yeast in which a cell surface display-type β-glucosidase gene and two or more copies of a xylose metabolism-related gene are introduced into the genome, and β-glucosidase activity is 0.02U / OD660 = 1 or more.

  (3) A recombinant yeast in which a cell surface display β-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.

  (4) The recombinant yeast according to any one of (1) to (3), wherein the xylose metabolism-related gene is a xylose reductase gene, a xylitol dehydrogenase gene, or a xylulokinase gene.

  (5) The recombinant yeast according to any one of (1) to (3), wherein 4 copies of the surface display β-glucosidase gene are introduced into the genome.

  (6) The recombinant yeast according to any one of (1) to (3), wherein the gene is introduced using a higher-ploidy yeast as a host.

  (7) The cell surface-presenting β-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.

  (8) The recombinant yeast according to any one of (1) to (3), wherein the above gene is introduced using Saccharomyces cerevisiae OC-2 strain (NBRC2260) as a host.

  (9) A method for producing ethanol, comprising a step of culturing the recombinant yeast according to any one of (1) to (8) in a xylose-containing medium and a step of recovering ethanol from the xylose-containing medium.

  (10) The method for producing ethanol according to (9), wherein the xylose-containing medium further contains 6 monosaccharides.

  (11) The method for producing ethanol according to (10), wherein the six monosaccharides are glucose and / or cellobiose.

  In addition, the inventors of the present application disclosed in the previous application (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. For example, the ability to metabolize β-glucosidase and xylose can be improved by introducing a cell surface display-type β-glucosidase gene and a xylose metabolism-related gene into the yeast disclosed in this prior application.

  According to the recombinant yeast according to the present invention, 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.

  Moreover, according to the manufacturing method of ethanol which concerns on this invention, ethanol can be collect | recovered more efficiently and with a high yield by fermentation which used the xylose contained in a culture medium as a substrate. In particular, even when xylose and glucose are contained in the medium, ethanol can be efficiently produced without a delay in the xylose metabolism rate.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-180434, which is the basis of the priority of the present application.

It is a figure which shows typically the process at the time of constructing the yeast for transformation which has several selection markers which have homothallic property by applying this invention. It is a characteristic view which shows the result of having measured the relationship between the sugar concentration in a culture medium, and the expression level of TDH1 gene, TDH3 gene, and PDC1 gene about YPH500 strain and OC-2T strain. It is a figure explaining the flow which builds pIBG13 from pIHCS and pBG211. It is a figure explaining the flow which builds pIWBGL1 from pRS404 and pBlue-BGL1. It is a characteristic view which shows the result of having measured PNPG activity about OC-2ABGL4 strain, OC-2ABGL2 strain, and OC-2HUT. It is a characteristic view which shows the result of having measured the xylose metabolism amount by the presence and kind of coexisting sugar about OC-2ABGL4Xyl2 strain. It is a characteristic view which shows the result of having measured the xylose metabolic rate about OC-2ABGL4Xyl2 strain and OC-2ABGL2Xyl2 strain. It is a characteristic view which shows the result of having measured the xylose density | concentration, the cellobiose density | concentration, and the ethanol density | concentration in a culture process about OC-2ABGL4Xyl2 strain | stump | stock. It is a figure which shows the structure of plasmid pABGL-TDH3P, pABGL-PDC1P, and pABGL-HOR7P. FIG. 3 is a view 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. It is a characteristic figure which shows the result of having measured PNPG activity about transformed yeast OC-2ATDH3PBGL2, OC-2APDC1PBGL2, and OC-2AHOR7PBGL2. Transformed yeast OC-2APDC1PBGL2XYL2, OC-2AHOR7PBGL2XYL2, OC-2HUT strain, TDH3PBGL2 strain, TDH3PBGL4 strain, TDH3PBGL6 strain, OC-2HUT strain introduced OC-2HTUT, OC-2HUT strain into which OC-2HUT was introduced It is a characteristic view which shows the result of having measured the PNPG activity about the strain | stump | stock.

Hereinafter, the present invention will be described in more detail with reference to the drawings and examples.
<Recombinant yeast>
In the recombinant yeast according to the present invention, a cell surface-presenting β-glucosidase gene and two or more copies of a xylose metabolism-related gene are introduced into the genome, and the activity of β-glucosidase displayed on the cell surface is high (for example, 0.02 U / OD660 = 1 or higher) Yeast. In addition, the recombinant yeast according to the present invention has a cell surface display-type β-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.

  Here, the cell surface-displaying β-glucosidase gene is a gene obtained by fusing a β-glucosidase gene and a cell surface-localized protein gene so that the expressed β-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. For example, α- or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned. In general, 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. Although it has the same secretory protein in that it has a secretion signal, 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. When the cell surface localized protein passes through the cell membrane, 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. Thereafter, the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Next, the protein separated from the cell membrane is incorporated into the cell wall, fixed to the cell surface layer, and localized on the cell surface layer (see, for example, JP-A-2006-174767).

  The β-glucosidase gene encoding β-glucosidase displayed on the cell surface is not particularly limited, and examples thereof include β-glucosidase gene derived from Aspergillus aculeatus (Murai et al., Appl. Environ. Microbiol. 64: 4857-4861). be able to. In addition, as the β-glucosidase gene, a β-glucosidase gene derived from Aspergillus oryzae, a β-glucosidase gene derived from Clostridium cellulovorans, a β-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 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). In addition, a xylose reductase gene derived from Candida tropicalis or Candida prapsilosis can be used as the xylose reductase gene. As the xylitol dehydrogenase gene, a xylitol dehydrogenase gene derived from Candida tropicalis or Candida prapsilosis can be used. A xylulokinase gene derived from Pichia stipitis can also be used as the xylulokinase gene. A xylose isomerase gene derived from Streptomyces murinus cluster or the like can also be used.

  When introducing the above-mentioned genes, introduce 2 or more copies of each gene, or introduce 2 or more copies of xylose metabolism-related genes and the β-glucosidase activity of the cell surface display β-glucosidase gene is 0.02U / OD660 = 1. It introduces so that it may become the above. 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.

  Yeast having homothallic properties is synonymous with homothallic yeast. In yeast having homothallic properties, sex transition by endonuclease encoded by HO gene occurs, and daughter cells of different sex bud from haploid mother cells. In haploid yeast, there are two types of sex (mating type), a cell and α cell, and a cell and α cell do not join. a cell and α cell secrete a specific sex hormone (pheromone) called a factor and α 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. Cells extend in the direction of each other, and each cell membrane, followed by the nucleus, fuses into a diploid cell. Therefore, 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. According to this life cycle, haploid yeast having homothallic properties changes to diploid yeast having exactly the same genotype except for the MAT genes (MATa gene and MATα gene) that determine the mating type. become. In addition, 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 α 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. Examples of 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)). In addition, even a yeast exhibiting a heterothallic phenotype 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.

  Of these, 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 conditions of high sugar concentration, as shown in the Examples described later. In particular, the Saccharomyces cerevisiae OC-2 strain is preferable because it has an excellent promoter activity of the pyruvate decarboxylase gene (PDC1) under high sugar concentration conditions.

  The transformation yeast according to the present invention is provided with a plurality of selection markers. Specifically, 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. Here, 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. More specifically, examples of the selection marker include auxotrophy required by defunctionalizing genes involved in biosynthesis of nutrient components essential for growth. 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. By defunctionalizing the Leu2 gene, leucine requirement can be imparted, by defunctionalizing the Ade2 gene, adenine requirement can be imparted, and by lysing the Lys2 gene, lysine requirement Sex can be imparted.

  In order to give a plurality of selection markers, first, yeast having a single selection marker is prepared. In addition, the yeast has homothallic properties as described above. As the homothallic yeast having such a single selection marker, for example, the 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 that has been given tryptophan auxotrophy as a selection marker. It is. The Saccharomyces cerevisiae OC-2U strain disclosed in the same report can also be used. This 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.

  Next, the second selection marker is introduced as follows. As a second selection marker, for example, when auxotrophy for different nutrient components is given, a gene involved in biosynthesis of the nutrient component is deleted from all chromosomes. As an example, 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. First, 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. Next, when this DNA fragment is introduced into homothallic yeast imparted with uracil auxotrophy, as shown in FIG. 1 (a), in the upstream region and the downstream region contained in the introduced DNA fragment, Homologous recombination occurs between them. When the DNA fragment is introduced into the chromosome, uracil requirement is lost due to the expression of the URA3 gene. For this reason, the yeast transformed with the DNA fragment can be grown in a uracil-free medium, and the loss of uracil requirement can be identified as an index. At this time, as shown in FIG. 1 (b), the chromosomal structure of yeast transformed with the DNA fragment is inserted into the URA3 gene so as to replace the HIS3 gene in one of the chromosomes. The HIS3 gene in the other chromosome remains and the yeast at this stage does not show auxotrophy.

  Next, a strain from which the introduced URA3 gene has been eliminated 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.

  Next, yeast having the chromosomal structure shown in FIG. 1 (c) is cultured in a spore formation medium to form spores via meiosis. The spore formation medium is not particularly limited, and a medium having a conventionally known composition can be used. Next, using a device such as a micromanipulator, for example, 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. Next, the spores are cultured in a germination medium, whereby individual spores are grown in the life cycle of vegetative growth. At this time, in the yeast having homothallic properties, 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 MATα 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. One diploid yeast shows only uracil auxotrophy, and the other diploid yeast is given histidine auxotrophy in addition to uracil auxotrophy. Therefore, 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.

  It goes without saying that 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. In the above explanation, 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 a paper by Rinji Akada et al. (Yeast, Volume 23, Issue 5, Pages 399-405) for the purpose of use in the marker recycling method.

  From the above description, it is understood that two or more copies of the above-described cell surface display β-glucosidase gene and xylose metabolism-related gene can be introduced by utilizing the homothallic property of yeast having a plurality of selection markers. In addition, it is not always necessary to introduce two or more copies of the above-described cell surface-presenting β-glucosidase gene, the activity of β-glucosidase displayed on the cell surface is 0.02 U / OD660 = 1 or more, or the xylose metabolic rate is 2.0 g / You may introduce | transduce so that it may become more than h / L. That is, if the cell surface display β-glucosidase gene introduced into the yeast host is highly expressed, the activity of the β-glucosidase is 0.02 U / OD660 = 1 or more or the xylose metabolism rate is 2.0 g / h / L or more. Can be controlled. For example, by making the promoter of the cell-presenting β-glucosidase gene a high-expression promoter, the activity of the β-glucosidase can be 0.02U / OD660 = 1 or higher or the xylose metabolic rate can be 2.0 g / h / L or higher. . As the promoter, for example, 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. Of these, the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene.

  That is, the cell surface-presenting β-glucosidase gene may be introduced into the genome of yeast having homothallic properties together with a promoter controlling the expression and other expression control regions. Alternatively, the cell surface display-type β-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. In particular, the cell surface display-type β-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. When the expression of the cell surface-presenting β-glucosidase gene is controlled by the expression control region of the HOR7 gene, higher β-glucosidase activity can be achieved.

  The yeast that presents β-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. A yeast cell having a function or having an originally enhanced function. 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 β-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.

  On the other hand, as for the xylose metabolism-related genes described above, it is sufficient that two or more copies of each gene are incorporated into 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. As promoters for genes related to xylose metabolism, various promoters listed above can be used as appropriate.

  As a method for introducing the cell surface-displaying β-glucosidase gene and the xylose metabolism-related gene, any conventionally known method known as a yeast transformation method can be applied. Specifically, for example, 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, etc. The method can be implemented, but is not limited thereto.

  Here, when introducing a cell surface display β-glucosidase gene and a xylose metabolism-related gene into yeast having homothallic properties, first, 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. When 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. Thereafter, the ascomb of the strain is isolated and haploid spores are cultured in a germination medium. As a result, in yeast having homothallic properties, 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 MATα gene. Thus, since the target gene is introduced into all chromosomes, it is introduced into the chromosome in multiple copies by one transformation. On the other hand, when heterothallic yeast is used, only one copy of the target gene can be introduced by one transformation.

  Next, an expression vector having either one of the cell surface display β-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. Thus, in the same manner as the mechanism described above, the other gene can also be introduced into the chromosome in multiple copies by a single transformation.

  Thus, by using 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 β-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 produced as described above has β-glucosidase presented on the cell surface, exhibits excellent β-glucosidase activity, and exhibits excellent xylose metabolism rate. Here, since β-glucosidase generates p-nitrophenol and D-glucose using PNPG as a substrate, β-glucosidase activity can be defined based on the amount of p-nitrophenol generated (PNPG activity). The amount of enzyme that produces 1 micromole of p-nitrophenol per minute under the specified conditions is defined as 1 U, and the cell concentration measured at OD660nm is a given value (for example, OD660 = 1) per enzyme amount. The activity value (U / OD660 = 1) can be measured. 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. In particular, 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.

  In order to produce ethanol using woody biomass, first, cellulose or hemicellulose is hydrolyzed (saccharified) to constituent monosaccharides. 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 | disassembles a lignin with microorganisms, the grinding | pulverization process of wood type biomass, etc. can be mentioned.

  Next, monosaccharides constituting cellulose and hemicellulose are collected by saccharifying woody biomass. 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. By saccharification treatment, monosaccharides derived from components constituting woody biomass can be obtained, but disaccharides other than monosaccharides and oligosaccharides may be collected simultaneously. That is, it is not necessary to positively remove disaccharides and oligosaccharides other than monosaccharides.

  Next, ethanol fermentation is performed using the above-described recombinant yeast. In ethanol fermentation, it is preferable to culture the recombinant yeast described above under optimum conditions. As the culture conditions for the recombinant yeast, for example, it is preferable that the temperature is 25 to 35 ° C. and the pH is 4 to 6. Moreover, you may stir and shake in the case of culture | cultivation.

  In particular, since the above-described recombinant yeast is used in this ethanol fermentation, the metabolic rate of xylose contained in the medium is improved. In contrast, in the recombinant yeast disclosed in S. Katahira et al., Appl. Microbiol. Biotechnol. (2006) 72: 1136-1143, the cell surface display β-glucosidase gene and xylose metabolism-related gene are 1 Since only copies are introduced, the metabolic rate of xylose remains low.

  In addition, the above-described recombinant yeast has a particularly improved xylose metabolism rate in a medium containing a sugar component (coexisting sugar) other than glucose sugar xylose. As described above, 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.

  Next, ethanol is collected from the medium. The method for recovering ethanol is not particularly limited, and any conventionally known method can be applied. For example, after the above-described ethanol fermentation is completed, a liquid layer containing ethanol and a solid layer containing recombinant yeast and solid components are separated by solid-liquid separation operation. Thereafter, 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.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
[Example 1]
In this example, a second Saccharomyces cerevisiae OC-2U strain (Saito et al., Journal of Ferment. Bioeng. 81: 98-103 (1996)), which has been given uracil requirement to the Saccharomyces cerevisiae OC-2 strain, is used as the second one. As a selection marker, histidine requirement was further imparted (see FIGS. 1A to 1D).

  First, the HIS3-disrupted 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 kit II: 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.

  Next, the obtained colony was made into a single colony using 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 plated on a FOA plate. Here, the composition of the FOA plate is Bacto yeast nitrogen base 0.67%, glucose 2%, uracil 50 μg / 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.

  Next, colonies that grew on the FOA plate were pre-cultured on the YPD plate at 30 ° C. for 1 day. Thereafter, the Sherman plate, which is a spore formation medium, was inoculated and cultured at 25 ° C. for 3 days to confirm spore formation. Then, spore separation was performed using a micromanipulator manufactured by Narishige Co., Ltd.

  The obtained ascomb contained 3 to 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.

  Therefore, the obtained 4 spores were cultured on four plates of SD plate, SD plate + uracil (20 mg / L), SD plate + histidine (20 mg / L) and SD plate + uracil + histidine, respectively. As a result, we succeeded in isolating strains that grow only on SD + uracil + histidine plates. That is, Saccharomyces cerevisiae OC-2U strain having histidine requirement in addition to uracil requirement has been successfully produced. The homothallic yeast having both uracil requirement and histidine requirement prepared in this example was named Saccharomyces cerevisiae OC-2HU strain.

[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. is there. Therefore, for the YPH500 strain (laboratory yeast) and the OC-2T strain (Saito et al., Journal of Ferment. Bioeng. 81: 98-103 (1996)), 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.

  First, 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 and cDNA is prepared using Roche's 1ststrand cDNA synthesis kit for RT-PCR, and the expression levels of TDH1, TDH3, and PDC1 genes are determined using the following primers using Roche Light cycler 330. Quantitative PCR was performed. The primer sequences used are shown in Table 1.

  2 (a) shows the expression levels of the TDH1 gene, TDH3 gene and PDC1 gene depending on the glucose concentration of the YPH500 strain and FIG. 2 (b) shows the OC-2T strain. When the glucose concentration was increased from 2% to 10%, the YPH500 strain, which is a laboratory yeast, showed a tendency that the expression levels of the TDH3 gene and the PDC1 gene used in the experiment all decreased. In contrast, in 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. From this result, since the 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.

  From the above results and discussion, it can be said that the OC-2HU strain prepared in Example 1 is useful as a practical yeast because it is excellent in substance productivity.

Example 3
In this example, 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.
Spore formation rate = (spore count) × 100 / (measured cell count)

  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 about 2 days after the start of culture.

Example 4
In this example, tryptophan requirement was further imparted as a third selection marker using the Saccharomyces cerevisiae OC-2HU strain prepared in Example 1 as a host. In this example, in order to confer tryptophan requirement, a TRP1 disruption fragment was prepared using the primers shown below to prepare an OC-2HUT (triple marker) strain.

  Specifically, 1stPCR is performed using TRP1-998 and TRP1-28r, 2ndPCR is performed using TRP1-URA3 and TRP1-40r, and the DNA fragment amplified by 1stPCR and the DNA fragment amplified by 2ndPCR are used as templates. Then, TRP1-998 and TRP1-40r were used for annealing PCR to obtain a TRP1-disrupted fragment. In 1st PCR, genomic DNA of OC-2T strain (Saito et al., Journal of Ferment. Bioeng. 81: 98-103 (1996)) was used as a template. In 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. When 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. Next, 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.

  Furthermore, 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 | separated using SD plate, the triple marker strain | stump | stock which has tryptophan requirement, histidine requirement, and uracil requirement can be acquired.

  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. As a result, we succeeded in isolating strains that grow only on SD + uracil + histidine + tryptophan plates. That is, the present inventors succeeded in producing a Saccharomyces cerevisiae OC-2HU strain imparted with tryptophan requirement in addition to uracil requirement and histidine requirement. The homothallic yeast having the uracil requirement, the histidine requirement and the tryptophan requirement produced 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.
(1) Preparation of arming BGL 2-copy-introduced strain First, the arming BGL-DNA pIBG13 (HIS3 marker) constructed as described below was linearized with the restriction enzyme NdeI in the OC-2HUT strain prepared in Example 4. The resulting plasmid was transformed by Frozen-EZ Yeast Transformation II (Zymo Research). SD + tryptophan + uracil plate was used as the selection plate. Colonies obtained by transformation were isolated using the same plate to eliminate false positive strains.

  Next, 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.

In addition, as shown in FIG. 3, pIBG13 is pIHCS (Yasuya Fujita et al. “Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes” Appl Environ Microbiol. 2002 October; 68 (10): 5136-5141 And pBG211 (see Murai, T et al. “Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing β-glucosidase and carboxymethylcellulase from Aspergillus aculeatus Aspergillus aculeatus.” Appl. Environ. Microbiol. 1998 64: 4857-4861) . 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.
5'-GATCTCCATGGCTGATGAACTGGCGTTCTCTCCTCCTTTC-3 '(SEQ ID NO: 7)
5'-TGGCGCTCGAGCCTTGCACCTTCGGGAGCGCCGCGTGAAG-3 '(SEQ ID NO: 8)
The amplified BGL1 fragment was treated with NcoI / XhoI. Also, pIBG13 was constructed by treating pIHCS with NcoI / XhoI and ligating it with the BGL1 fragment after NcoI / XhoI treatment.

(2) Preparation of an arming BGL4 copy-introduced strain Using the OC-2ABGL2 prepared in (1) above, a pIWBGL1 (TRP1 marker) constructed as described below was linearized with a restriction enzyme Bst1107I using a frozen- Transformation was performed by EZ Yeast Transformation II (Zymo Research). SD + uracil plate was used as the selection plate. Colonies obtained by transformation were separated on the same plate and false positive strains were excluded.

  Next, 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: 87515) and pIBG13 as shown in FIG. When constructing pIWBGL1, first, pRS404 was treated with NotI, followed by alkaline phosphatase treatment. Then, pIBG13 was digested with NotI to excise the GAPDH promoter-BGL1 gene-3'half of a-agglutinin gene and ligated with pRS404 after NotI treatment and alkaline phosphatase treatment. This constructed pIWBGL1.

(3) Preparation of an arming BGL4 copy and a xylose metabolism-related gene 2 copy-introduced strain Using the OC-2ABGL4 prepared in (2) above, pIUX1X2Xk (URA3 marker) was linearized with the restriction enzyme NcoI. Transformation was performed by EZ Yeast Transformation II (Zymo Research). SD plates were used as selection plates. Colonies obtained by transformation were separated on the same plate and false positive strains were excluded. PIUX1X2Xk is disclosed in S. Katahira et al., Appl. Microbiol. Biotechnol. (2006) 72: 1136-1143, and 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.

  Next, 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.

  According to the method described above, a strain was prepared by introducing two copies of xylose metabolism-related gene into OC-2ABGL2 prepared in (1) (OC-2ABGL2Xyl2).

(4) Measurement of p-nitrophenyl-β-D-glucoside (PNPG) activity Next, it is the BGL gene product in OC-2ABGL2 prepared in (1) above and OC-2ABGL4 prepared in (2) above β-glucosidase activity was evaluated. Specifically, since β-glucosidase generates p-nitrophenol and D-glucose using PNPG as a substrate, the amount of p-nitrophenol was measured (PNPG activity) and evaluated. The amount of enzyme that produces 1 micromole of p-nitrophenol per minute under the following conditions was defined as 1U.

The reagents used for the measurement are as follows.
・ 0.1M acetate buffer pH5.0 (25 ℃)
・ 20mM PNPG aqueous solution (603mg PNPG dissolved in 100ml distilled water)
・ 0.2MN _a2 CO ₃ solution (21.2 g of anhydrous sodium carbonate dissolved in distilled water)

The measurement procedure was as follows. First, 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. Then, after exactly allowed to react for 15 minutes at 37 ° C., added N _a2 CO ₃ solution 2 ml, the reaction was stopped. Next, the absorbance at 400 nm in the reaction solution was measured. In the blind test, 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.

  In the present example, the following was prepared as a diluted culture solution. That is, first, the culture composition was adjusted so that OD660 = 20 in the YPD medium, and OC-2ABGL2 prepared in (1) above or (2) above in a medium supplemented with 5% cellobiose, 1% yeast extract and 2% peptone. OC-2ABGL4 prepared in the above was inoculated to a total volume of 20 ml, and cultured under conditions of 30 ° C. and 60 rpm. On the first day and the second day of the culture, 1 ml of the medium was collected, diluted 50 times, and used for this experiment. The results are shown in FIG.

  As is clear from FIG. 5, 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
In this example, 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. First, based on the 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.

  Then, 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.

  As shown in FIG. 6, the xylose metabolism of the OC-2ABGL4Xyl2 strain was only 1.83% when the medium contained only xylose. In contrast, when the medium contained coexisting sugars such as glucose and cellobiose in addition to xylose, the xylose metabolism was improved to 4.34% and 4.92%, respectively. Here, 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.

  From this result, it was clarified that the OC-2ABGL4Xyl2 strain prepared in Example 3 has a characteristic 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.

  Then, 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 in the same manner as in Example 4. The measurement results are shown in FIG.

  As shown in FIG. 7, the OC-2ABGL2Xyl2 strain had a xylose metabolism of 3.38% 24 hours after the start of culture, whereas the OC-2ABGL4Xyl2 strain had a xylose metabolism of 4.92% 24 hours after the start of culture. there were. Here, 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.

  From this result, it was clarified that when 4 copies of the surface-presenting BGL gene were introduced, the xylose metabolic rate was improved by about 50% or more compared to the strain into which 2 copies of the surface-presenting BGL gene were introduced. .

Example 8
In this example, 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.

  Then, 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.

  As shown in FIG. 8, 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
In this example, an expression control region that controls the expression of the BGL gene was examined in order to further improve β-glucosidase activity. Specifically, in this example, 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 β-glucosidase activity was examined.

  First, expression of plasmid pABGL-TDH3P for introducing arming BGL gene downstream of the expression control region of TDH3 gene, expression of plasmid pABGL-PDC1P and HOR7 gene for introducing arming BGL gene downstream of the expression control region of PDC1 gene 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.

  First, 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. Then, the obtained DNA fragment was ligated with pRS403 (Stratagene) treated with restriction enzyme BssHII to prepare plasmid pRS404-NotI-s.s-BGL1-3′half a-agglutinin-SphI. Further, 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. Then, 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).

  On the other hand, 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). In addition, 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). Further, 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).

  Next, 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). Further, 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). Furthermore, 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). These three types of DNA fragments were treated with restriction enzymes NotI and SalI, respectively. Then, the obtained DNA fragment containing a part of the coding region of HOR7 gene (NotI-SalI fragment) and plasmid pUC19-AscI-HOR7promoter-NotI-SalI treated with restriction enzymes NotI and SalI were ligated, and plasmid pUC19 -AscI-HOR7promoter-NotI-truncated-AscI was prepared. Further, 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. Further, the obtained DNA fragment containing a part of the coding region of the TDH3 gene (NotI-SalI fragment) was ligated with plasmid pUC19-AscI-TDH3promoter-NotI-SalI treated with restriction enzymes NotI and SalI, and plasmid pUC19 -AscI-TDH3promoter-NotI-truncated-AscI was produced (FIG. 11).

  Finally, the plasmid pRS404-NotI-ss-BGL1-3′half a-agglutinin-SphI-TRP1-NotI shown in FIG. 10 is treated with NotI, and DNA containing 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). These plasmids pABGL-HOR7P, pABGL-PDC1P and pABGL-TDH3P are treated with the restriction enzyme AscI, so that the cell surface-displayed β-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.

  These plasmids pABGL-HOR7P, pABGL-PDC1P, and pABGL-TDH3P each contain a pair of DNA fragments so that homologous recombination occurs in a desired region. Specifically, 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. Similarly, 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.

  These plasmids pABGL-HOR7P, pABGL-PDC1P and pABGL-TDH3P constructed as described above were transformed into the OC-2HUT strain prepared in Example 4. First, 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.

  Next, 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. 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 obtained three transformed yeasts OC-2ATDH37PBGL2, OC-2APDC1PBGL2, and OC-2AHOR7PBGL2 was measured according to the method described in Example 5 (4). The results are shown in FIG. As can be seen from FIG. 13, β-glucosidase activity caused by the introduced arming BGL gene could be confirmed in all transformed yeasts. In particular, OC-2AHOR7PBGL2 was found to exhibit β-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 β-glucosidase activity.

Example 10
In this example, 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 that expression was possible under the control of the GAPDH promoter. PIUX1X2Xk is disclosed in S. Katahira et al., Appl. Microbiol. Biotechnol. (2006) 72: 1136-1143, and 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.

  First, a plasmid obtained by linearizing pIUX1X2Xk (URA3 marker) with the restriction enzyme PstI was transformed 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.

  Next, according to the method described in Example 5 (3), 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. Were named OC-2APDC1PBGL2XYL2 and OC-2AHOR7PBGL2XYL2, respectively.

  The PNPG activity of the obtained two transformed yeasts OC-2APDC1PBGL2XYL2 and OC-2AHOR7PBGL2XYL2 was measured according to the method described in Example 5 (4). The results are shown in FIG. For comparison, 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.

  As can be seen from FIG. 14, 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 β-glucosidase activity superior to the strain into which 6 copies of the arming BGL gene were introduced. . In addition, as can be seen from Table 5, in the strain into which the arming BGL gene was introduced and the xylose metabolism-related gene was introduced so that the expression was controlled by the expression control region of the PDC1 gene, the expression was controlled by the expression control region of the PDC1 gene. Thus, the arming BGL gene was introduced, and β-glucosidase activity equivalent to that of the strain was shown. On the other hand, in the strain in which the armored BGL gene is introduced so that the expression is controlled by the expression control region of the HOR7 gene and the xylose metabolism-related gene is introduced, the arming is controlled so that the expression is controlled by the expression control region of the HOR7 gene. Compared with the strain into which the BGL gene was introduced, the β-glucosidase activity was further improved. Although the detailed mechanism is unknown from this result, 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 β-glucosidase activity.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (11)

  A recombinant yeast in which two or more copies of a cell surface-displaying β-glucosidase gene and two or more copies of a xylose metabolism-related gene have been introduced into the genome.   A recombinant yeast in which a cell surface display β-glucosidase gene and two or more copies of a xylose metabolism-related gene are introduced into the genome, and β-glucosidase activity is 0.02U / OD660 = 1 or more.   A recombinant yeast in which a cell surface display β-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.   The recombinant yeast according to any one of claims 1 to 3, wherein the xylose metabolism-related gene is a xylose reductase gene, a xylitol dehydrogenase gene, or a xylulokinase gene.   The recombinant yeast according to any one of claims 1 to 3, wherein 4 copies of the surface-presenting β-glucosidase gene are introduced into the genome.   The recombinant yeast according to any one of claims 1 to 3, wherein the gene is introduced using a higher-ploidy yeast as a host.   4. The cell surface display β-glucosidase gene is introduced in such a manner that its expression is controlled by an expression control region of an endogenous hyperosmotic response 7 (HOR7) gene. Recombinant yeast.   The recombinant yeast according to any one of claims 1 to 3, wherein the gene is introduced using Saccharomyces cerevisiae OC-2 strain (NBRC2260) as a host. Culturing the recombinant yeast according to any one of claims 1 to 8 in a xylose-containing medium;
And a step of recovering ethanol from the xylose-containing medium.   The method for producing ethanol according to claim 9, wherein the xylose-containing medium further contains 6 monosaccharides.   The method for producing ethanol according to claim 10, wherein the six monosaccharides are glucose and / or cellobiose.

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