Yeast that suppresses xylitol accumulation

Abstract

To provide a technique of inhibiting xylitol accumulation in ethanol production from xylose.SOLUTION: Used is transgenic yeast having the ability of producing ethanol from xylose, wherein a gene encoding pyruvate decarboxylase is introduced so as to allow expression and / or a gene encoding pyruvate dehydrogenase is attenuated.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a yeast in which xylitol accumulation during ethanol production from xylose is suppressed. [Background technology]

[0002] In recent years, the use of bioalcohol as an alternative fuel to petroleum has been attracting attention from the viewpoint of reducing CO2 emissions. In particular, bioalcohol made from plants and trees has been actively studied. Since xylose accounts for about one-third of the sugar obtained from cellulosic biomass such as plants and trees, various studies have been conducted on xylose-utilizing yeasts used in the production of bioalcohol.

[0003] Yeasts such as Saccharomyces cerevisiae are mainly used for bioethanol production. Saccharomyces cerevisiae has a high ethanol production ability from hexoses such as glucose and mannose, and has a high tolerance to ethanol. However, Saccharomyces cerevisiae cannot utilize pentoses such as xylose.

[0004] Scheffersomyces stipitis is known as a yeast capable of assimilating xylose. Saccharomyces cerevisiae contains a group of genes corresponding to the group of genes for assimilating xylose possessed by Scheffersomyces stipitis, but it is believed that most of these genes in Saccharomyces cerevisiae are not expressed, or even if they are expressed, the amount is extremely small. Therefore, efforts are being made to improve Saccharomyces cerevisiae by introducing genes derived from xylose-assimilating yeast (Patent Document 1).

[0005] However, even if these genes are introduced to impart ethanol production ability from xylose, it is known that a large amount of the intermediate metabolite xylitol is produced and excreted outside the cell. The metabolism stops at xylitol, which is undesirable because it reduces the ethanol yield. Furthermore, the production of by-products such as xylitol causes problems such as inefficient use of raw materials, and there are also concerns about the impact on yeast growth.

[0006] In the past, attempts have been made to inhibit xylitol production in yeast strains endowed with the ability to utilize xylose, but the effects have been insufficient (Non-Patent Documents 1 and 2).

[0007] Therefore, there remains a need for the development of a yeast in which xylitol accumulation during ethanol production from xylose is suppressed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2016 / 060171 [Non-patent literature]

[0009] [Non-Patent Document 1] Guo-Chang Zhang et al., Applied and Environmental Microbiology (2012) Vol. 78, No. 4, p.1081-1086 [Non-Patent Document 2] Hou et al. BMC Biotechnology (2014) 14:13 Summary of the Invention

[0010] The present invention provides a new technical means for suppressing xylitol accumulation during ethanol production from xylose.

[0011] The present inventors have now found that by expressibly introducing a gene encoding pyruvate decarboxylase and / or attenuating a gene encoding pyruvate dehydrogenase, accumulation of xylitol can be effectively suppressed. The present invention is based on these findings.

[0012] According to the present invention, the following inventions are provided. [1] A transformed yeast capable of producing ethanol from xylose, in which a gene encoding pyruvate decarboxylase has been introduced in an expressible manner and / or a gene encoding pyruvate dehydrogenase has been attenuated. [2] The yeast according to [1], wherein the parent yeast that has not been subjected to the introduction and / or attenuation has at least the ability to produce pyruvic acid from xylose. [3] The transformed yeast described in [2], wherein the parent yeast has at least one xylose assimilation-related gene selected from the group consisting of a gene encoding a xylose reductase, a gene encoding a xylulose phosphorylase, a gene encoding a xylitol dehydrogenase, a gene encoding a transaldolase, a gene encoding a transketolase, and a gene encoding an alcohol dehydrogenase introduced in advance in an expressible manner. [4] The transformed yeast according to [3], wherein the xylose assimilation-related genes are a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase. [5] The transformed yeast according to any one of [1] to [4], wherein at least one gene selected from the group consisting of the gene encoding pyruvate decarboxylase, the gene encoding pyruvate dehydrogenase, and the xylose assimilation-related genes is an endogenous gene of the parent yeast that has not been introduced and / or attenuated. [6] The transformed yeast according to any one of [1] to [5], wherein at least one gene selected from the gene encoding pyruvate decarboxylase and the gene related to xylose assimilation is inserted in an expressible manner on a chromosome. [7] The transformed yeast according to any one of [1] to [6], wherein the gene encoding pyruvate decarboxylase is PDC1. [8] The transformed yeast according to any one of [1] to [7], wherein the gene encoding pyruvate dehydrogenase is PDA1. [9] The transformed yeast according to any one of [3] to [8], wherein the gene encoding xylose reductase is GRE3.

[10] The transformed yeast according to any one of [3] to [9], wherein the gene encoding xylulose phosphorylase is XKS1.

[11] The transformed yeast according to any one of [3] to

[10] , wherein the gene encoding xylitol dehydrogenase is SOR1.

[12] The transformed yeast according to any one of [3] to

[11] , wherein the gene encoding transaldolase is TAL1.

[13] The transformed yeast according to any one of [3] to

[12] , wherein the gene encoding transketolase is TKL1.

[14] The transformed yeast according to any one of [3] to

[13] , wherein the gene encoding alcohol dehydrogenase is ADH1.

[15] The transformed yeast according to any one of [2] to

[14] , wherein the parent yeast is a yeast belonging to the genus Saccharomyces.

[16] The transformed yeast according to any one of [2] to

[15] , wherein the parent yeast is a yeast belonging to the species Saccharomyces cerevisiae.

[17] A method for producing ethanol, comprising culturing the transformed yeast according to any one of [1] to

[16] in a glucose- and xylose-containing medium and collecting ethanol from the resulting culture.

[0013] According to the present invention, the accumulation of xylitol during ethanol production from xylose can be effectively suppressed. [Brief description of the drawings]

[0014] [Figure 1] 1 is a graph showing the results of xylitol accumulation after 48 hours of culture. [Diagram 2] 1 is a graph showing the results of xylose consumption rate after 24 hours of culture. [Diagram 3] 1 is a graph showing the results of glycerol production after 48 hours of culture. Description of the Invention

[0015] One of the characteristics of the present invention is a transformed yeast having the ability to produce ethanol from xylose, in which a gene encoding pyruvate decarboxylase has been introduced in an expressible manner and / or a gene encoding pyruvate dehydrogenase has been attenuated.

[0016] In the xylose metabolic pathway of yeast, xylose is first reduced to xylitol by xylose reductase, and then dehydrogenated to xylulose by xylitol dehydrogenase. The resulting xylulose is converted to xylulose 5-phosphate by xylulokinase, which is then sent to the pentose phosphate pathway for metabolism. Glyceraldehyde triphosphate is then produced by a reaction in the pentose phosphate pathway. Pyruvic acid is then produced from glyceraldehyde triphosphate. Pyruvic acid is then decarboxylated to acetaldehyde by pyruvate decarboxylase, and ethanol is then produced from acetaldehyde by alcohol dehydrogenase. On the other hand, there is another pathway for pyruvic acid metabolism. Pyruvic acid is oxidatively decarboxylated to produce acetyl CoA by a complex containing pyruvate dehydrogenase, and then the pyruvic acid proceeds to the TCA cycle.

[0017] An example of a gene to be introduced in an expressible manner in the transformed yeast of the present invention is a gene encoding the pyruvate decarboxylase. An example of a gene to be attenuated in the transformed yeast of the present invention is a gene encoding pyruvate dehydrogenase.

[0018] In the present invention, a "xylose utilization gene" is a gene encoding an enzyme involved in the utilization of xylose. In the present invention, the xylose utilization genes previously introduced into the parent yeast in an expressible manner are at least one, preferably three, of a gene encoding a xylose reductase, a gene encoding a xylitol dehydrogenase, and a gene encoding a xylulose phosphorylase. In the present invention, the gene encoding a xylitol dehydrogenase is preferably a gene encoding a sorbitol dehydrogenase.

[0019] In the present invention, at least one, preferably three, of the genes encoding transaldolase, transketolase, and alcohol dehydrogenase may be introduced into the parent yeast in advance so as to be expressible. These three genes include genes encoding enzymes related to the pentose phosphate pathway and / or the ethanol production pathway. In the present invention, the genes encoding transaldolase, transketolase, and alcohol dehydrogenase in addition to the above xylose utilization genes are referred to as xylose utilization-related genes.

[0020] In another embodiment of the present invention, it is preferable that the genes introduced into the yeast are genes derived from the parent yeast. That is, in another embodiment of the present invention, one of the features is that the expression of the endogenous enzyme gene inherent to the yeast is activated, and the activity of the enzyme contained in the yeast itself is enhanced.

[0021] Among yeasts, there are some that do not have the ability to assimilate pentoses such as xylose because the xylose assimilation enzymes are essentially not functioning, i.e., in a dormant state. For example, yeasts belonging to the genus Saccharomyces cannot use xylose to produce ethanol, even though they have genes encoding the xylose assimilation enzymes.

[0022] By introducing a xylose assimilation gene derived from the yeast itself or by substituting the promoter, the activity of the endogenous gene for xylose assimilation can be increased and xylose assimilation ability can be imparted to such yeast that is considered to have no ethanol production ability. Furthermore, by introducing not only the xylose assimilation gene derived from the yeast itself but also the genes for enzymes related to the pentose phosphate pathway and / or the ethanol production pathway derived from the yeast itself (a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase) or by substituting the promoters of these genes, the efficiency of ethanol production from xylose can be further improved. Therefore, the parent yeast of the present invention may be a yeast into which a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase have been previously introduced into a yeast having a xylose assimilation gene inserted on the chromosome.

[0023] <Transformed yeast> The transformed yeast of the present invention has a gene encoding pyruvate decarboxylase introduced so as to be capable of expression and / or a gene encoding pyruvate dehydrogenase attenuated, and can be a transformed yeast having the ability to produce ethanol from xylose. A preferred transformed yeast of the present invention is a transformed yeast having a gene encoding pyruvate decarboxylase introduced so as to be capable of expression and / or a gene encoding pyruvate dehydrogenase attenuated, and further having at least one gene selected from the group consisting of a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase introduced so as to be capable of expression. A more preferred transformed yeast of the present invention is a transformed yeast in which a gene encoding pyruvate decarboxylase has been introduced in an expressible manner and / or a gene encoding pyruvate dehydrogenase has been attenuated, and in which a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase have been further introduced in an expressible manner.

[0024] (1) Parent yeast In the present invention, the parent yeast to be transformed by introduction of a gene encoding pyruvate decarboxylase or transformation with the gene and / or transformation by attenuation of a gene encoding pyruvate dehydrogenase, i.e., a parent yeast to which a gene encoding pyruvate decarboxylase has not been introduced and a gene encoding pyruvate dehydrogenase has not been attenuated (hereinafter, also simply referred to as a parent yeast), may be a yeast having at least the ability to produce pyruvic acid from xylose. In addition, the parent yeast may be a yeast having a part or all of the enzyme reaction cascade leading to ethanol production and having the ability to produce ethanol from xylose, so long as the accumulation of xylitol can be suppressed by the transformation in the present invention. Therefore, according to one embodiment of the present invention, the parent yeast may be a yeast further having the ability to produce ethanol from pyruvic acid. In addition, according to one embodiment of the present invention, the parent yeast may be a yeast having the ability to produce ethanol from xylose. Such a parent yeast may be a wild type or a genetically modified yeast, but a genetically modified yeast is preferable. As the genetically modified yeast, yeast into which a xylose assimilation-related gene has been introduced so as to be expressible or yeast transformed with a xylose assimilation-related gene is preferred. The yeast used for the gene introduction or transformation of the xylose assimilation-related gene is preferably a yeast that does not have the ability to assimilate pentose such as xylose. The yeast may have no pentose assimilation ability before gene introduction or transformation, and may have the ability to assimilate hexose such as glucose. "Pentose assimilation ability" refers to the ability to grow using pentose such as xylose as a carbon source. Yeast having pentose assimilation ability can grow in a medium containing only pentose as a carbon source, so pentose assimilation ability can be confirmed by measuring the degree of growth of the yeast in a medium containing only pentose as a carbon source at a wavelength of 600 nm or 660 nm.

[0025] The parent yeast of the present invention may also be a yeast that has been genetically modified in advance. In the present invention, a parent yeast that has been genetically modified in advance by introduction of a xylose assimilation-related gene or transformation with the gene can be selected for the purpose of improving ethanol productivity. Examples of such parent yeast include yeast that has been imparted with xylose assimilation ability and yeast that has been activated with xylose assimilation ability.

[0026] In the present invention, the parent yeast is not particularly limited, but may be, for example, yeast belonging to the genus Saccharomyces. Examples of yeast belonging to the genus Saccharomyces include Saccharomyces cerevisiae, such as laboratory yeast strains. In addition, in the present invention, not only haploid but also diploid yeast can be used as the yeast to be subjected to gene introduction or transformation. Diploid yeast is excellent as a practical yeast, and examples thereof include brewing yeast such as baker's yeast, sake yeast, shochu yeast, and wine yeast.

[0027] In the present invention, the parent yeast is preferably a brewing yeast having tolerance to ethanol, and examples of such yeast include, but are not limited to, yeast belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae).

[0028] Therefore, in the present invention, the parent yeast is preferably a yeast belonging to the genus Saccharomyces, more preferably Saccharomyces cerevisiae.

[0029] (2) Transgene or modified gene In the present invention, a gene encoding pyruvate decarboxylase is introduced into a parent yeast in an expressible manner, and / or the gene encoding pyruvate dehydrogenase in the parent yeast is modified so as to be attenuated.

[0030] Furthermore, in the present invention, as described above, it is preferable that the parent yeast is transfected or transformed with a xylose utilization gene. The xylose utilization gene includes at least one of a gene encoding a xylose reductase, a gene encoding a xylitol dehydrogenase, and a gene encoding a xylulose phosphorylase, and preferably three genes, and among them, the xylitol dehydrogenase is preferably a sorbitol dehydrogenase. More specifically, the gene encoding a xylose reductase, the gene encoding a xylitol dehydrogenase, and the gene encoding a xylulose phosphorylase are preferably GRE3 (aldo-keto reductase gene 3), SOR1 (sorbitol dehydrogenase gene 1), and XKS1 (xylulose phosphorylase gene 1), respectively.

[0031] In addition, at least one of xylose utilization-related genes, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase, is preferably introduced into the parent yeast, and more preferably three genes are introduced. More specifically, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase are preferably TAL1 (transaldolase gene 1), TKL1 (transketolase gene 1), and ADH1 (alcohol dehydrogenase gene 1), respectively.

[0032] In the present invention, the nucleic acid used to attenuate the gene encoding pyruvate decarboxylase and the gene encoding pyruvate dehydrogenase may be either a foreign gene or an endogenous gene, but is preferably an endogenous gene. Furthermore, the xylose assimilation-related genes, such as the xylose assimilation gene, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase, may be either a foreign gene or an endogenous gene, but is preferably an endogenous gene. The term "endogenous gene" refers to a gene possessed by the yeast into which the gene is inserted, a gene derived from the yeast into which the gene is inserted, or a gene derived from the same yeast as the yeast into which the gene is inserted. Therefore, a yeast into which an endogenous gene has been introduced does not fall under the category of a recombinant organism. Therefore, yeast into which an endogenous gene has been introduced does not require diffusion prevention measures to prevent leakage of bacterial cells into the outside world during ethanol production, and is advantageous in that ethanol can be produced at low cost.

[0033] (The gene encoding pyruvate decarboxylase) PDC1, PDC5, and PDC6 are known as genes encoding pyruvate decarboxylase in yeast. Therefore, in the present invention, for example, PDC1, PDC5, and PDC6 can be used as genes encoding pyruvate decarboxylase. In this specification, PDC1 is described as an example of a gene encoding pyruvate decarboxylase, but PDC5 and PDC6 can be similarly used in the present invention by applying the description of PDC1 in this specification.

[0034] Those skilled in the art can obtain the nucleotide sequence information of PDC1, PDC5, and PDC6 from known databases such as Genbank. The accession numbers for the sequence information of each gene in Saccharomyces cerevisiae are shown below. PDC1:NM_001181931, PDC5:NM_001182021, PDC6:NM_001181216

[0035] In the present invention, PDC1 (pyruvate decarboxylase gene 1) is a gene containing a base sequence encoding pyruvate decarboxylase, for example, DNA derived from Saccharomyces cerevisiae consisting of the base sequence shown in SEQ ID NO: 31, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 32.

[0036] Those skilled in the art can obtain amino acid sequence information of PDC1 and the like from known databases such as Genbank. For example, the accession number for sequence information of PDC1 in Saccharomyces cerevisiae is NP_013145.

[0037] In the present invention, PDC1 can be obtained, for example, by designing primers based on the nucleotide sequence shown in SEQ ID NO:31 and using gene amplification techniques from a yeast library or a genomic library.

[0038] PDC1 used in the present invention may include DNA encoding a mutant of PDC1 protein. The DNA encoding a mutant of PDC1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 31 and encodes a protein having pyruvate decarboxylase activity. The pyruvate decarboxylase activity will be described later.

[0039] DNA encoding a mutant of the PDC1 protein can be obtained from a cDNA library and a genomic library by known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting using the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 31 or a fragment thereof as a probe. For the method of constructing the library, see "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Press (2012)) and the like. Alternatively, commercially available cDNA libraries and genomic libraries may be used.

[0040] Here, stringent conditions include post-hybridization washing conditions such as "2xSSC, 0.1% SDS, 42°C" and "1xSSC, 0.1% SDS, 37°C," and more stringent conditions include, for example, "1xSSC, 0.1% SDS, 65°C" and "0.5xSSC, 0.1% SDS, 50°C."

[0041] Hybridization can be carried out by known methods. For hybridization methods, see, for example, "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Laboratory Press (2012)) and "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997)).

[0042] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 31. The value showing the identity can be calculated using a known program such as BLAST.

[0043] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 31 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 31 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 31; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 31 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 31; and (iv) DNA in which these mutations are combined and which encodes a protein having pyruvate decarboxylase activity.

[0044] In the present invention, the base sequence can be confirmed by sequencing using a conventional method, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0045] In the present invention, for example, PDC1 (pyruvate decarboxylase gene 1) derived from Saccharomyces cerevisiae also includes DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 32. In the present invention, PDC1 (pyruvate decarboxylase gene 1) also includes DNA encoding the PDC1 protein derived from Saccharomyces cerevisiae or a mutant thereof.

[0046] Furthermore, in the present invention, PDC1 protein mutants include proteins comprising an amino acid sequence having at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% identity (homology) to the amino acid sequence shown in SEQ ID NO: 32. According to one embodiment of the present invention, the PDC1 protein mutant is a protein comprising an amino acid sequence having any of the above identities and has pyruvate decarboxylase activity.

[0047] Furthermore, examples of PDC1 protein mutants include: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 32 are deleted; (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 32 are substituted with other amino acids; (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 32; and (iv) a protein in which these mutations are combined and which has pyruvate decarboxylase activity.

[0048] Here, "pyruvate decarboxylase activity" means the activity of decarboxylating pyruvate to acetaldehyde. In the present invention, the PDC1 protein mutant is not particularly limited in the level of its activity as long as it has pyruvate decarboxylase activity, but it may have, for example, about 10% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 32. The pyruvate decarboxylase activity of a protein can be measured by a known method.

[0049] (The gene encoding pyruvate dehydrogenase) PDA1 and PDA2 are known as genes encoding pyruvate dehydrogenase in yeast. Therefore, in the present invention, for example, PDA1 and PDA2 can be used as genes encoding pyruvate dehydrogenase. In this specification, PDA1 is described as an example of a gene encoding pyruvate dehydrogenase, but PDA2 can be similarly used in the present invention by applying the description of PDA1 in this specification.

[0050] Those skilled in the art can obtain the nucleotide sequence information of PDA1 and PDA2 from known databases such as Genbank. The accession numbers for the sequence information of each gene in Saccharomyces cerevisiae are shown below. PDA1:NM_001179068, PDA2:NM_001182909

[0051] In the present invention, PDA1 (pyruvate dehydrogenase gene 1) is a gene containing a base sequence encoding pyruvate dehydrogenase, and is, for example, a DNA consisting of the base sequence shown in SEQ ID NO: 33 derived from Saccharomyces cerevisiae, or a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 34. Pyruvate dehydrogenase encoded by PDA1 is also called pyruvate dehydrogenase, and can form a complex together with dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase.

[0052] Those skilled in the art can obtain amino acid sequence information of PDA1 and the like from known databases such as Genbank. For example, the accession number for sequence information of PDA1 in Saccharomyces cerevisiae is NP_011105.

[0053] The PDA1 used in the present invention may include DNA encoding a mutant of the PDA1 protein. The DNA encoding a mutant of the PDA1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the DNA having the base sequence shown in SEQ ID NO: 33 derived from Saccharomyces cerevisiae and encodes a protein having pyruvate dehydrogenase activity.

[0054] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 33. The value showing the identity can be calculated using a known program such as BLAST.

[0055] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 33 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 33 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 33; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 33 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 33; and (iv) DNA in which these mutations are combined and which encodes a protein having pyruvate dehydrogenase activity.

[0056] In the present invention, for example, PDA1 (pyruvate dehydrogenase gene 1) derived from Saccharomyces cerevisiae also includes DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 34. In the present invention, PDA1 (pyruvate dehydrogenase gene 1) also includes DNA encoding the PDA1 protein derived from Saccharomyces cerevisiae or a mutant thereof.

[0057] In the present invention, the PDA1 protein mutant includes, for example, a protein comprising an amino acid sequence having at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% identity (homology) with the amino acid sequence shown in SEQ ID NO: 34. According to one embodiment of the present invention, the PDA1 protein mutant is a protein comprising an amino acid sequence having any of the above identities and has pyruvate dehydrogenase activity.

[0058] Furthermore, examples of mutants of the PDA1 protein include: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 34 are deleted; (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 34 are replaced with other amino acids; (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 34; and (iv) a protein in which these mutations are combined and which has pyruvate dehydrogenase activity.

[0059] Here, "pyruvate dehydrogenase activity" refers to the activity of converting pyruvate to acetyl-CoA (acetyl coenzyme A) in the presence of NAD+ (or NADP+). In the present invention, the PDA1 protein mutant is not particularly limited in the level of activity as long as it has pyruvate dehydrogenase activity, but it may have, for example, about 10% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 34. The pyruvate dehydrogenase activity of a protein can be measured by a known method.

[0060] The above-mentioned hybridizing DNA and hybridization conditions, etc., are similarly applicable to the explanation for PDC1. Furthermore, in the present invention, PDA1 can be obtained or produced by the same method as that described for PDC1.

[0061] (The gene encoding xylose reductase) GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w are known as genes encoding xylose reductase in yeast. Therefore, in the present invention, for example, GRE3, YJR096w, YPR1, GCY1, ARA1 or YDR124w can be used as a gene encoding xylose reductase. In this specification, GRE3 is described as an example of a gene encoding xylose reductase, but YJR096w, YPR1, GCY1, ARA1 and YDR124w can be used in the present invention in the same manner as described in this specification for GRE3.

[0062] Those skilled in the art can obtain the nucleotide sequence information of GRE3, YJR096w, YPR1, GCY1, ARA1, and YDR124w from known databases such as Genbank. The accession numbers for the sequence information of each gene in Saccharomyces cerevisiae are shown below. GRE3:NM_001179234, YJR096w:NM_001181754, YPR1:NM_001180676, GCY1:NM_001183539, ARA1: NM_001178497, YDR124w: NM_001180432.

[0063] In the present invention, GRE3 (aldo-keto reductase gene 3) is a gene containing a nucleotide sequence encoding an aldo-keto reductase, and is, for example, a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 35 derived from Saccharomyces cerevisiae, or a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 36. The protein encoded by GRE3 is known to function as a xylose reductase in yeast. In addition, the GRE3 protein is a protein having amino acid sequence identity (homology) with XYL1 (xylose reductase (XR)) of Shephasomyces stipitis.

[0064] Those skilled in the art can obtain amino acid sequence information of GRE3 and the like from known databases such as Genbank. For example, the accession number for sequence information of GRE3 in Saccharomyces cerevisiae is NP_011972.

[0065] In the present invention, GRE3 can be obtained, for example, by designing a primer based on the nucleotide sequence shown in SEQ ID NO:35 and using a gene amplification technique from a yeast library or a genome library.

[0066] The GRE3 used in the present invention may include DNA encoding a mutant of the GRE3 protein. The DNA encoding a mutant of the GRE3 protein includes, for example, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 35, and encodes a protein having xylose reductase activity. The xylose reductase activity will be described later.

[0067] The DNA encoding the mutant GRE3 protein can be obtained from a cDNA library and a genomic library by known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting using the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 35 or a fragment thereof as a probe. For the method of constructing the library, see "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Press (2012)) and the like. Commercially available cDNA libraries and genomic libraries may also be used.

[0068] Here, stringent conditions include post-hybridization washing conditions such as "2xSSC, 0.1% SDS, 42°C" and "1xSSC, 0.1% SDS, 37°C," and more stringent conditions include, for example, "1xSSC, 0.1% SDS, 65°C" and "0.5xSSC, 0.1% SDS, 50°C."

[0069] Hybridization can be carried out by known methods. For hybridization methods, see, for example, "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Laboratory Press (2012)) and "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997)).

[0070] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 35. The value showing the identity can be calculated using a known program such as BLAST.

[0071] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 35 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 35 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 35; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 35 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 35; and (iv) DNA in which these mutations are combined and which encodes a protein having xylose reductase activity.

[0072] In the present invention, the base sequence can be confirmed by sequencing using a conventional method, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0073] In the present invention, for example, GRE3 (aldo-keto reductase gene 3) derived from Saccharomyces cerevisiae also includes DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 36. In the present invention, GRE3 (aldo-keto reductase gene 3) also includes DNA encoding the GRE3 protein derived from Saccharomyces cerevisiae or a mutant thereof.

[0074] Examples of GRE3 protein mutants include: (i) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 36 are deleted; (ii) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 36 are substituted with other amino acids; (iii) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 36; and (iv) a protein in which these mutations are combined and which has xylose reductase activity.

[0075] Here, "xylose reductase activity" means the activity of converting xylose to xylitol in the presence of NAD+ (or NADP+). In the present invention, the level of activity of the GRE3 protein mutant is not particularly limited as long as it has xylose reductase activity, but it is sufficient that the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence shown in SEQ ID NO: 36, for example. The xylose reductase activity of the protein can be measured by a known method.

[0076] (The gene encoding xylitol dehydrogenase) SOR1, SOR2 and YLR070c are known as genes encoding xylitol dehydrogenase in yeast. Therefore, in the present invention, for example, SOR1, SOR2 or YLR070c can be used as a gene encoding xylitol dehydrogenase, but SOR1 is preferred. In this specification, SOR1 is described as an example of a gene encoding xylitol dehydrogenase, but the description of SOR1 in this specification can be applied to SOR2 and YLR070c. Note that SOR1 and SOR2 have 99.9% identity in gene sequence with each other.

[0077] Those skilled in the art can obtain the nucleotide sequence information of SOR1, SOR2, ​​and YLR070c from known databases such as Genbank. The accession numbers for the sequence information of each gene in Saccharomyces cerevisiae are shown below. SOR1:NM_001181817, SOR2:NM_001180306, YLR070c: NM_001181957.

[0078] In the present invention, SOR1 (sorbitol dehydrogenase gene 1) is a gene containing a base sequence encoding sorbitol dehydrogenase, for example, DNA consisting of the base sequence shown in SEQ ID NO: 37 derived from Saccharomyces cerevisiae, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 38. The protein encoded by SOR1 is known to function as xylitol dehydrogenase in yeast. In addition, the SOR1 protein is a protein having amino acid sequence identity (homology) (53%) with XYL2 (xylitol dehydrogenase (XDH)) of Shephasomyces stipitis.

[0079] Those skilled in the art can obtain amino acid sequence information of SOR1 and the like from known databases such as Genbank. For example, the accession number for sequence information of SOR1 in Saccharomyces cerevisiae is NP_012693.

[0080] The SOR1 used in the present invention may include DNA encoding a mutant of the SOR1 protein. The DNA encoding the mutant of the SOR1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the DNA having the base sequence shown in SEQ ID NO: 37 derived from Saccharomyces cerevisiae and encodes a protein having xylitol dehydrogenase activity.

[0081] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 37. The value showing the identity can be calculated using a known program such as BLAST.

[0082] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 37 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 37 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 37; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 37 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 37; and (iv) DNA in which these mutations are combined and which encodes a protein having xylitol dehydrogenase activity.

[0083] Furthermore, the SOR1 used in the present invention may be a DNA encoding the following mutants of the SOR1 protein: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 38 are deleted, (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 38 are replaced with other amino acids, (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 38, and (iv) a protein in which these mutations are combined and which has xylitol dehydrogenase activity.

[0084] Here, "xylitol dehydrogenase activity" means the activity of dehydrogenating xylitol to xylulose. In the present invention, the SOR1 protein mutant is not particularly limited in the level of activity as long as it has xylitol dehydrogenase activity, but it may have, for example, about 10% or more of the activity of the protein consisting of the amino acid sequence shown in SEQ ID NO: 38. The xylitol dehydrogenase activity of a protein can be measured by a known method.

[0085] The above-mentioned explanations are similarly applicable to the hybridizing DNA and hybridization conditions, etc. In the present invention, SOR1 can be obtained or produced by the same method as that described for GRE3.

[0086] (The gene encoding xylulose phosphorylase) In the present invention, for example, XKS1 (xylulose kinase gene 1) can be used as a gene encoding xylulose kinase. Those skilled in the art can obtain the base sequence information of XKS1 from known databases such as Genbank. For example, the accession number of XKS1 of Saccharomyces cerevisiae is NM_001181323.

[0087] In the present invention, XKS1 (xylulose phosphorylase gene 1) is a gene containing a base sequence encoding xylulose phosphorylase, for example, DNA consisting of the base sequence shown in SEQ ID NO: 39 derived from Saccharomyces cerevisiae, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 40.

[0088] Those skilled in the art can obtain the amino acid sequence information of XKS1 from known databases such as Genbank. For example, the accession number for the sequence information of XKS1 in Saccharomyces cerevisiae is NP_011710.

[0089] XKS1 used in the present invention may include DNA encoding a mutant of XKS1 protein. The DNA encoding a mutant of XKS1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence shown in SEQ ID NO: 39 derived from Saccharomyces cerevisiae and encodes a protein having xylulose phosphorylase activity.

[0090] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 39. The value showing the identity can be calculated using a known program such as BLAST.

[0091] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 39 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 39 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 39; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 39 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 39; and (iv) DNA in which these mutations are combined and which encodes a protein having xylulose phosphorylase activity.

[0092] Furthermore, the XKS1 used in the present invention may be a DNA encoding the following mutants of the XKS1 protein: (i) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 40 are deleted, (ii) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 40 are substituted with other amino acids, (iii) a protein in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 40, and (iv) a protein in which these mutations are combined and which has xylulose phosphorylation enzyme activity.

[0093] Here, "xylulose phosphorylation enzyme activity" means the activity of phosphorylating xylulose. In the present invention, the XKS1 protein mutant is not particularly limited in the level of activity as long as it has xylulose phosphorylation enzyme activity, but it may have, for example, about 10% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 40. The xylulose phosphorylation enzyme activity of a protein can be measured by a known method.

[0094] The above explanations are similarly applicable to the hybridizing DNA and hybridization conditions, etc. In the present invention, XKS1 can be obtained or produced by the same method as that described for GRE3.

[0095] (The gene encoding transaldolase) In the present invention, for example, TAL1 (transaldolase gene 1) or TAL2 (transaldolase gene 2) can be used as a gene encoding transaldolase. Those skilled in the art can obtain the base sequence information of TAL1 and TAL2 from known databases such as Genbank. For example, the accession numbers of TAL1 and TAL2 of Saccharomyces cerevisiae are NM_001182243 and NM_001181172, respectively. In this specification, TAL1 will be described as an example of a gene encoding transaldolase, but the description of TAL1 in this specification can be similarly applied to TAL2.

[0096] In the present invention, TAL1 (transaldolase gene 1) is a gene containing a base sequence encoding transaldolase, for example, DNA derived from Saccharomyces cerevisiae consisting of the base sequence shown in SEQ ID NO: 41, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 42.

[0097] Those skilled in the art can obtain amino acid sequence information of TAL1 and TAL2 from known databases such as Genbank. For example, the accession number for sequence information of TAL1 in Saccharomyces cerevisiae is NP_013458, and the accession number for TAL2 is NP_011557.

[0098] TAL1 used in the present invention may include DNA encoding a mutant of TAL1 protein, for example, DNA encoding a protein that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence shown in SEQ ID NO: 41 derived from Saccharomyces cerevisiae and has transaldolase activity.

[0099] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 41. The value showing the identity can be calculated using a known program such as BLAST.

[0100] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 41 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 41 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 41; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 41 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 41; and (iv) DNA in which these mutations are combined and which encodes a protein having transaldolase activity.

[0101] Furthermore, the TAL1 used in the present invention may be a DNA encoding the following mutants of the TAL1 protein: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 42 are deleted, (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 42 are replaced with other amino acids, (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 42, and (iv) a protein in which these mutations are combined and which has transaldolase activity.

[0102] Here, "transaldolase activity" refers to the activity of catalyzing the reaction of sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate ⇔ erythrose-4-phosphate + fructose-6-phosphate. In the present invention, the level of activity of a TAL1 protein mutant is not particularly limited as long as it has transaldolase activity, but it is sufficient that the mutant has, for example, about 10% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 42. The transaldolase activity of a protein can be measured by a known method.

[0103] The above-mentioned explanations are similarly applicable to the hybridizing DNA and hybridization conditions, etc. In the present invention, TAL1 can be obtained or produced by the same method as that described for GRE3.

[0104] (The gene encoding transketolase) In the present invention, for example, TKL1 (transketolase gene 1) or TKL2 (transketolase gene 2) can be used as a gene encoding transketolase. Those skilled in the art can obtain the base sequence information of TKL1 and TKL2 from known databases such as Genbank. For example, the accession numbers of TKL1 and TKL2 of Saccharomyces cerevisiae are NM_001184171 and NM_001178465, respectively. In this specification, TKL1 is described as an example of a gene encoding transketolase, but the description of TKL1 in this specification can be similarly applied to TKL2.

[0105] In the present invention, TKL1 (transketolase gene 1) is a gene containing a base sequence encoding transketolase, for example, DNA derived from Saccharomyces cerevisiae consisting of the base sequence shown in SEQ ID NO: 43, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 44.

[0106] Those skilled in the art can obtain amino acid sequence information for TKL1 and TKL2 from known databases such as Genbank. For example, the accession number for sequence information of TKL1 in Saccharomyces cerevisiae is NP_015399, and the accession number for TKL2 is NP_009675.

[0107] The TKL1 used in the present invention may include DNA encoding a mutant TKL1 protein. The DNA encoding a mutant TKL1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence shown in SEQ ID NO: 43 derived from Saccharomyces cerevisiae and encodes a protein having transketolase activity.

[0108] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 43. The value showing the identity can be calculated using a known program such as BLAST.

[0109] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 43 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 43 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 43; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 43 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 43; and (iv) DNA in which these mutations are combined and which encodes a protein having transketolase activity.

[0110] Furthermore, the TKL1 used in the present invention may be a DNA encoding the following mutants of the TKL1 protein: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 44 are deleted, (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 44 are substituted with other amino acids, (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 44, and (iv) a protein in which these mutations are combined and which has transketolase activity.

[0111] Here, "transketolase activity" means activity to catalyze the reaction of sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate ⇔ xylulose-5-phosphate + ribose-5-phosphate. In the present invention, the level of activity of a TKL1 protein mutant is not particularly limited as long as it has transketolase activity, but it may have, for example, about 10% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 44. The transketolase activity of a protein can be measured by a known method.

[0112] The above-mentioned explanations are similarly applicable to the hybridizing DNA and hybridization conditions, etc. In the present invention, TKL1 can be obtained or produced by the same method as that described for GRE3.

[0113] (the gene encoding alcohol dehydrogenase) In the present invention, for example, ADH1 (alcohol dehydrogenase gene 1) can be used as a gene encoding alcohol dehydrogenase. Those skilled in the art can obtain the nucleotide sequence information of ADH1 from known databases such as Genbank. For example, the accession number of ADH1 of Saccharomyces cerevisiae is NM_001183340.

[0114] In the present invention, ADH1 (alcohol dehydrogenase gene 1) is a gene containing a base sequence encoding alcohol dehydrogenase, for example, DNA consisting of the base sequence shown in SEQ ID NO: 45 derived from Saccharomyces cerevisiae, or DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 46.

[0115] Those skilled in the art can obtain the amino acid sequence information of ADH1 from known databases such as Genbank. For example, the accession number for the sequence information of ADH1 in Saccharomyces cerevisiae is NP_014555.

[0116] The ADH1 used in the present invention may include DNA encoding a mutant of the ADH1 protein. The DNA encoding a mutant of the ADH1 protein includes, for example, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence shown in SEQ ID NO: 45 derived from Saccharomyces cerevisiae and encodes a protein having alcohol dehydrogenase activity.

[0117] In the present invention, DNA that hybridizes under stringent conditions includes, for example, DNA containing a base sequence having an identity (homology) of at least 50% or more, preferably 70% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, even more preferably 99% or more, even more preferably 99.7% or more, and particularly preferably 99.9% to the base sequence shown in SEQ ID NO: 45. The value showing the identity can be calculated using a known program such as BLAST.

[0118] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 45 includes, for example, DNA containing a base sequence in which one or several nucleic acids in the base sequence shown in SEQ ID NO: 45 have been mutated by deletion, substitution, addition or the like. Examples of such DNA include: (i) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are deleted from the base sequence shown in SEQ ID NO: 45; (ii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases in the base sequence shown in SEQ ID NO: 45 are substituted with other bases; (iii) DNA in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) bases are added to the base sequence shown in SEQ ID NO: 45; and (iv) DNA in which these mutations are combined and which encodes a protein having alcohol dehydrogenase activity.

[0119] Furthermore, the ADH1 used in the present invention may be a DNA encoding the following mutants of the ADH1 protein: (i) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 46 are deleted, (ii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids in the amino acid sequence shown in SEQ ID NO: 46 are substituted with other amino acids, (iii) a protein in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2) amino acids are added to the amino acid sequence shown in SEQ ID NO: 46, and (iv) a protein in which these mutations are combined and which has alcohol dehydrogenase activity.

[0120] Here, "alcohol dehydrogenase activity" refers to the activity of oxidizing alcohol to aldehyde. In the present invention, the level of activity of the ADH1 protein mutant is not particularly limited as long as it has alcohol dehydrogenase activity, but it is sufficient that the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence shown in SEQ ID NO: 46, for example. The alcohol dehydrogenase activity of a protein can be measured by a known method.

[0121] The above-mentioned explanations can be similarly applied to the DNA contained in the hybridizing DNA and the hybridization conditions, etc. In the present invention, ADH1 can be obtained or produced by the same method as that described for GRE3.

[0122] Furthermore, in the present invention, it is preferable to introduce the above six types of genes, i.e., the xylose assimilation gene, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase, into yeast of the same species as the origin of the genes in advance, so that the enzyme activity of the yeast itself as a parent yeast is improved compared to before the genes are introduced. Furthermore, when producing a non-recombinant yeast, the above six types of genes introduced into the parent yeast in advance must be endogenous genes.

[0123] (Characteristics of transformed yeast) The transformed yeast of the present invention can preferably have a xylose consumption rate equivalent to that of the parent yeast. Here, equivalent means that there is no significant difference when a t-test is performed. Therefore, the transformed yeast of the present invention can preferably have a xylose consumption rate approximately equivalent to that of the parent yeast, despite the suppression of accumulation of xylitol. When cultured in a glucose- and xylose-containing medium, the xylose consumption rate is, for example, 0.1 to 1.0 g / L / h, preferably 0.2 to 0.8 g / L / h, and more preferably 0.3 to 0.5 g / L / h. The culture conditions for determining the xylose consumption rate are, after inoculation to an OD600 of 15, culture in a glucose- and xylose-containing medium at 30°C and 140 rpm for 24 hours. More specific conditions are as described in Example 7. OD600 (absorbance at a wavelength of 600 nm) can be measured using a commercially available spectrophotometer.

[0124] It is preferable that the transformed yeast of the present invention has suppressed accumulation of xylitol compared to the parent yeast. Such transformed yeast can reduce the amount of accumulated xylitol compared to the parent yeast cultured under the same culture conditions. When cultured in a glucose- and xylose-containing medium, the amount of accumulated xylitol is, for example, 0.1 to 1.0 g / L, preferably 0.2 to 0.8 g / L, more preferably 0.3 to 0.5 g / L, and even more preferably 0.3 to 0.4 g / L. Such an amount of accumulated xylitol can be specified using the amount of accumulation in Example 7 as an index. In addition, the ratio of the amount of accumulated xylitol of the transformed yeast of the present invention to the amount of accumulated xylitol of the parent yeast under the same culture conditions (the amount of accumulated xylitol of the transformed yeast of the present invention: the amount of accumulated xylitol of the parent yeast) is, for example, 0.4 to 0.9:1, preferably 0.5 to 0.8:1, more preferably 0.6 to 0.8:1, and even more preferably 0.6 to 0.7:1. Here, "the same culture conditions" refers to culturing in a glucose and xylose-containing medium at 30°C and 140 rpm for 48 hours after inoculation to an OD600 of 15. More specific conditions are as in Example 7.

[0125] Usually, yeast that consumes xylose and produces ethanol tends to have a suppressed production of glycerol. However, the transformed yeast of the present invention consumes xylose and produces ethanol at the same level as the parent strain, but the amount of glycerol produced can be preferably made equal to that of the parent yeast. Here, "equal" means that there is no significant difference when a t-test is performed. The amount of glycerol produced is, for example, 3 to 18 g / L, preferably 5 to 15 g / L, and more preferably 7 to 9 g / L, when cultured in a glucose and xylose-containing medium. The culture conditions are that the yeast is inoculated to an OD600 of 15, and then cultured in a glucose and xylose-containing medium at 30°C and 140 rpm for 48 hours. More specific conditions are as in Example 7.

[0126] The transformed yeast of the present invention preferably has a growth rate equivalent to that of the parent yeast. Such a growth rate can be achieved by maintaining the viable cell count OD600 at 20 to 50 after 48 to 72 hours of culture in a glucose- and xylose-containing medium, preferably at 30 to 40. The culture conditions are as follows: after inoculation to give an OD600 of 10 to 20, the yeast is cultured in a glucose- and xylose-containing medium at 30°C and 140 rpm for 48 hours. More specific conditions are as in Example 7.

[0127] <Introduction of genes into yeast or modification of yeast genes> In the present invention, the transformed yeast of the present invention can be prepared by expressibly introducing a gene encoding pyruvate decarboxylase into a parent yeast and / or modifying the parent yeast so that the gene encoding pyruvate dehydrogenase is attenuated. Furthermore, in the present invention, the parent yeast can be prepared by expressibly introducing at least one selected from the group consisting of a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase. Preferably, in the present invention, the parent yeast can be prepared by expressibly introducing a xylose assimilation gene (e.g., three genes, a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, and a gene encoding xylitol dehydrogenase), as well as a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase. The transformed yeast of the present invention can also be prepared by replacing the promoters of these genes with promoters that increase the expression levels of the genes.

[0128] The transformed yeast of the present invention is, for example, yeast into which PDC1 has been introduced so as to be expressible and / or into which PDA1 has been attenuated. The transformed yeast of the present invention is preferably yeast into which PDC1 has been introduced so as to be expressible and / or into which PDA1 has been attenuated, and into which at least one selected from the group consisting of GRE3, SOR1, XKS1, TAL1, TKL1 and ADH1 has been further introduced so as to be expressible. The transformed yeast of the present invention is more preferably yeast into which PDC1 has been introduced so as to be expressible and / or into which PDA1 has been attenuated, and into which GRE3, SOR1, XKS1, TAL1, TKL1 and ADH1 have further been introduced so as to be expressible. In the present invention, "expressibly (introduced or inserted)" means that the introduced or inserted gene is introduced or inserted in such a form that it can be expressed in the transformant under predetermined conditions.

[0129] <Attenuation of the gene encoding pyruvate dehydrogenase> "The gene encoding pyruvate dehydrogenase is attenuated" and "modified to attenuate the gene encoding pyruvate dehydrogenase" mean that the pyruvate dehydrogenase is modified so that it does not function normally compared to unmodified yeast, such as parent yeast or wild-type yeast. For example, the modification of the pyruvate dehydrogenase gene by genetic recombination includes cases where the number of pyruvate dehydrogenase molecules per cell is reduced compared to parent yeast or wild-type yeast, or where no pyruvate dehydrogenase molecules are produced at all, or where the activity of pyruvate dehydrogenase per molecule is reduced or lost. The number of pyruvate dehydrogenase molecules can be reduced by reducing the expression level of the gene encoding pyruvate dehydrogenase. The reduction in the expression level of the gene encoding pyruvate dehydrogenase includes a reduction in the transcription level of pyruvate dehydrogenase mRNA and a reduction in the translation level of pyruvate dehydrogenase mRNA. Furthermore, completely preventing the production of pyruvate dehydrogenase molecules, reducing the number of pyruvate dehydrogenase molecules, or reducing or eliminating the activity per molecule of pyruvate dehydrogenase can be achieved by disrupting the gene encoding pyruvate dehydrogenase. When the yeast is diploid, the gene may be attenuated on one chromosome, and the other chromosome may not be attenuated.

[0130] Specifically, modification for attenuating the gene encoding pyruvate dehydrogenase can be achieved by deleting a part or the whole of the coding region of the gene encoding pyruvate dehydrogenase on the chromosome, or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. In addition, the expression level of the gene can also be reduced by modifying a non-translated region other than the expression regulatory sequence. Furthermore, the entire gene encoding pyruvate dehydrogenase, including the sequences before and after the gene encoding pyruvate dehydrogenase on the chromosome, may be deleted. In addition, attenuation can also be achieved by introducing an amino acid substitution (missense mutation) into the region encoding pyruvate dehydrogenase on the chromosome by genetic recombination, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation with one or two bases added or deleted.

[0131] Furthermore, it is preferable to reduce the activity of pyruvate dehydrogenase in yeast by deleting part or all of the expression regulatory sequences, such as the promoter region, or the coding region or non-coding region, of the gene encoding pyruvate dehydrogenase on the chromosome, or by inserting another nucleic acid sequence into these regions.

[0132] The expression regulatory sequence is preferably modified by one or more bases, more preferably two or more bases, and particularly preferably three or more bases. When deleting a coding region, the region to be deleted may be any of the N-terminal region, the internal region, and the C-terminal region, or may be the entire coding region, so long as the function of the pyruvate dehydrogenase produced is reduced or deleted. In general, the longer the region to be deleted, the more reliably the gene encoding pyruvate dehydrogenase can be attenuated.

[0133] When inserting another nucleic acid sequence into the coding region, the position of insertion may be any region of the gene encoding pyruvate dehydrogenase, but the longer the inserted sequence, the more reliably the gene encoding pyruvate dehydrogenase can be attenuated. The other nucleic acid sequence is not particularly limited as long as it reduces or eliminates the function of pyruvate dehydrogenase, and examples of such other nucleic acid sequences include antibiotic resistance genes and genes necessary for nucleic acid synthesis, such as uracil synthase.

[0134] The gene encoding pyruvate dehydrogenase on the chromosome can be modified as described above, for example, as follows. First, a partial sequence of the gene encoding pyruvate dehydrogenase is deleted to produce an attenuated gene modified so that pyruvate dehydrogenase is not normally produced, or another nucleic acid sequence such as an antibiotic resistance gene or a gene required for nucleic acid synthesis is inserted into the coding region to produce an attenuated gene modified so that pyruvate dehydrogenase that functions normally is not produced. Then, the attenuated gene obtained and the gene encoding pyruvate dehydrogenase on the chromosome of the parent yeast are subjected to homologous recombination to replace the gene encoding pyruvate dehydrogenase on the chromosome with the attenuated gene. Here, the parent yeast is preferably a yeast that cannot synthesize the antibiotic resistance gene or the gene required for nucleic acid synthesis. Such gene disruption by gene replacement using homologous recombination has already been established, and includes a method using a plasmid containing a temperature-sensitive replication origin, a conjugatively transferable plasmid, a method using a suicide vector that does not have a replication origin in the host, and a method of introducing a fusion gene fragment (DNA fragment) containing an auxotrophy complementary gene or a drug marker into yeast using the lithium acetate method or the like (for example, U.S. Pat. No. 6,303,383, or Japanese Patent Laid-Open Publication No. 05-007491).

[0135] The decrease in the transcription level of the gene encoding pyruvate dehydrogenase can be confirmed by comparing the amount of mRNA of the gene encoding pyruvate dehydrogenase with that of the parent yeast, wild-type yeast, or unmodified yeast. Methods for evaluating the amount of mRNA include northern hybridization and RT-PCR.

[0136] The reduction in the amount of the protein encoded by the gene encoding pyruvate dehydrogenase can be confirmed by Western blotting using an antibody.

[0137] <Introduction of a gene encoding pyruvate decarboxylase or a gene related to xylose utilization> In the present invention, the gene encoding pyruvate decarboxylase is preferably inserted into the yeast chromosome so as to be expressible. In addition, in the present invention, the gene encoding xylose reductase, the gene encoding xylulose phosphorylase, and the gene encoding xylitol dehydrogenase are preferably inserted into the yeast chromosome so as to be expressible. In addition, in the present invention, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase are preferably inserted into the yeast chromosome so as to be expressible. That is, in the present invention, the introduction of genes into yeast preferably includes the insertion of genes into the yeast chromosome. The transformed yeast of the present invention is preferably a yeast in which a gene encoding pyruvate decarboxylase is inserted into the chromosome so as to be expressible, and / or a gene encoding pyruvate dehydrogenase is attenuated, and further, at least one selected from the group consisting of a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase is inserted into the chromosome so as to be expressible. The transformed yeast of the present invention is more preferably a yeast in which a gene encoding pyruvate decarboxylase has been expressibly inserted onto the chromosome and / or a gene encoding pyruvate dehydrogenase has been attenuated, and further in which a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase have been expressibly inserted onto the chromosome. Also, the transformed yeast of the present invention is preferably a yeast in which PDC1 has been expressibly inserted onto the chromosome and / or PDA1 has been attenuated, and further in which at least one selected from the group consisting of GRE3, SOR1, XKS1, TAL1, TKL1, and ADH1 has been expressibly inserted onto the chromosome.The transformed yeast of the present invention is more preferably a yeast in which PDC1 has been expressibly inserted onto the chromosome and / or PDA1 has been attenuated, and further in which GRE3, SOR1, XKS1, TAL1, TKL1 and ADH1 have been expressibly inserted onto the chromosome.

[0138] In the present invention, the number of each gene to be inserted into a chromosome is not limited, and may be, for example, one or more. The order of the genes to be inserted into a chromosome is not particularly limited. The position of the chromosome to which the gene is inserted is not particularly limited, but is preferably a site that does not function in yeast, and examples thereof include the XYL2 site (Genbank accession number Z73242), the HXT13 site, the HXT17 site, and the AUR1 site. It is also possible to insert the gene into a site on a chromosome that does not code for a gene. An example of a site on a chromosome that does not code for a gene is the δ sequence, which is one of the Ty elements. It is known that multiple δ sequences (about 100 copies) exist on the chromosome of yeast. The position and sequence information of the δ sequence on the yeast chromosome are publicly known (for example, Science 265, 2077 (1994)). For example, by introducing a plasmid into yeast in which a xylose assimilation-related gene has been inserted into the middle of the δ sequence, one or more copies of the gene can be inserted into a desired position on the chromosome. In addition to the delta sequence, it can also be inserted into the sigma and tau sequences, which are also Ty elements. It can also be inserted into the ribosomal gene site of NTS2 or the like.

[0139] When genes are inserted onto a chromosome, each gene may be inserted onto the chromosome individually, or an expression cassette containing one gene under the control of a promoter or an expression cassette in which two or more genes are linked in tandem under the control of a promoter may be prepared and inserted onto the chromosome. When genes are linked in tandem, the order of gene arrangement and the number of genes to be linked are not particularly limited, and any conceivable combination may be used.

[0140] A plasmid or an expression cassette can be used to introduce a gene into yeast. The plasmid or expression cassette can contain one or more of the enzyme genes used in the present invention. For example, a gene encoding pyruvate decarboxylase can be contained in a plasmid or expression cassette. Also, for example, three genes, a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, and a gene encoding xylitol dehydrogenase, can be contained in one plasmid or expression cassette. Also, for example, three genes, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase, can be contained in one plasmid or expression cassette. When multiple genes are contained in one plasmid or expression cassette, the order and number of each gene on the plasmid are not particularly limited. The present invention includes a plasmid or expression cassette containing these genes. Examples of such a plasmid or expression cassette include a plasmid or expression cassette in which PDC1 is linked under the control of a promoter, a plasmid or expression cassette in which GRE3, SOR1 and XLS1 are linked under the control of a promoter, a plasmid or expression cassette in which TAL1 and TKL1 are linked under the control of a promoter, or a plasmid or expression cassette in which TAL1, TKL1 and ADH1 are linked under the control of a promoter. A plurality of genes are linked so that, for example, when introduced into yeast, particularly when inserted into a chromosome, each gene can be expressed. If necessary when linking two genes to prepare a fusion gene, a linker sequence, a restriction enzyme site, or the like may be appropriately added. These operations can be performed using conventional genetic engineering techniques well known in the art. By using these plasmids or expression cassettes, three types of xylose assimilation genes and / or a gene encoding transaldolase, a gene encoding transketolase and / or a gene encoding alcohol dehydrogenase may be introduced onto the yeast chromosome together with a gene encoding pyruvate decarboxylase.

[0141] The plasmid used in the present invention can be prepared by inserting the above gene into a yeast expression vector in an expressible manner. The gene can be inserted into the vector by ligase reaction, topoisomerase reaction, etc. For example, a method can be adopted in which purified DNA is cleaved with an appropriate restriction enzyme, and the obtained DNA fragment is inserted into an appropriate restriction enzyme site or multicloning site in the vector to link it to the vector.

[0142] The plasmid used in the present invention is not particularly limited in terms of the origin of the base vector, and for example, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, etc. can be used. For example, commercially available vectors such as pGADT7, pAUR135, and pUC18 can also be used. When the transformed yeast of the present invention is prepared using a plasmid derived from a parent yeast, the transformed yeast of the present invention does not fall under the category of a genetically modified organism.

[0143] The plasmid of the present invention may contain a multicloning site, a promoter, an enhancer, a terminator, a selection marker cassette, etc., so long as it can express the target gene. Furthermore, if necessary when inserting DNA, a linker or a restriction enzyme site may be added as appropriate. These manipulations can be performed using conventional genetic manipulation techniques well known in the art.

[0144] The promoter can be incorporated upstream of the target gene. The promoter is not particularly limited as long as it can appropriately express the target protein in the transformant, and examples of the promoter that can be used include PGK promoter, ADH promoter, TDH promoter, ENO promoter, CIT promoter, TEF promoter, CDC promoter, GPM promoter, and PDC promoter.

[0145] A terminator can be incorporated downstream of the gene of interest. For example, a PGK terminator, a CIT terminator, a TEF terminator, a CDC terminator, a GPM terminator, or a PDC terminator can be used. In the present invention, it is preferable to use a PGK promoter and / or a PGK terminator in order to efficiently express a gene of interest in yeast.

[0146] Examples of the selection marker include drug resistance genes such as ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, hygromycin resistance gene, etc., dihydrofolate reductase gene, leucine synthase gene, uracil synthase gene, etc. When the vector contains an amino acid synthesis gene cassette such as leucine, histidine, tryptophan, or the like, or a uracil synthesis gene cassette, the transformed yeast can be selected by culturing the yeast in a medium not containing the amino acid or uracil.

[0147] A transformed yeast can be produced by introducing the plasmid or expression cassette of the present invention into the yeast of the present invention that is the target for gene introduction.

[0148] The method for introducing the plasmid of the present invention into yeast is not particularly limited, and examples thereof include known methods such as the lithium acetate method, electroporation, calcium phosphate method, lipofection method, DEAE-dextran method, etc. The transformed yeast of the present invention can be provided by these methods.

[0149] The transformed yeast of the present invention can also be prepared by integrating a gene of interest into a chromosome of a parent yeast by homologous recombination. Those skilled in the art can prepare the transformed yeast of the present invention by homologous recombination using known methods.

[0150] A yeast that does not have the ability to utilize pentose cannot be endowed with the ability to utilize xylose unless it expresses all three genes, the gene encoding xylose reductase, the gene encoding xylitol dehydrogenase, and the gene encoding xylulose phosphorylase. Therefore, by culturing the yeast into which the gene has been introduced as described above in a xylose-containing medium, preferably a medium containing glucose and xylose, it is possible to select a transformed yeast into which at least the xylose utilization enzyme gene has been introduced in an expressible manner.

[0151] In this manner, for example, the transformed yeast of the present invention can be produced by expressibly introducing a gene encoding pyruvate decarboxylase and / or by expressibly introducing six xylose assimilation-related genes into yeast together with attenuating the gene encoding pyruvate dehydrogenase. Preferably, the transformed yeast of the present invention can be produced by expressibly introducing a gene encoding pyruvate decarboxylase and / or by expressibly introducing six xylose assimilation-related genes into yeast together with attenuating the gene encoding pyruvate dehydrogenase onto the chromosome of the yeast.

[0152] Furthermore, the transformed yeast of the present invention may be one in which the expression of a gene encoding pyruvate decarboxylase in the parent yeast is activated and / or the expression of a gene encoding pyruvate dehydrogenase is attenuated, and the expression of a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase in the parent yeast is activated. That is, the present invention also includes yeast in which the expression level of the above-mentioned genes originally present on the chromosome of the parent yeast is increased. By introducing a promoter from outside or replacing the promoter of the gene itself with a stronger promoter, the gene originally possessed by the yeast can be activated in an expressible form and the target protein can be appropriately expressed. For example, a method for activating the expression of an endogenous gene in this way is not limited, but includes a method of incorporating a promoter capable of appropriately expressing a target protein into a chromosome by gene replacement using a known gene recombination technique. As a method for gene replacement, the method of Akada et al. Yeast 23: 399-405 (2006) (Non-Patent Document) can be used. As a promoter to be replaced, a known promoter such as a PGK promoter, an ADH promoter, a TDH promoter, an ENO promoter, a CIT promoter, a TEF promoter, a CDC promoter, a GPM promoter, or a PDC promoter can be used.

[0153] <How ethanol is produced> The transformed yeast of the present invention has a gene encoding pyruvate decarboxylase introduced in an expressible manner and / or has a gene encoding pyruvate dehydrogenase attenuated, and has the ability to produce ethanol from xylose, so that the transformed yeast of the present invention can suppress xylitol accumulation during ethanol production from xylose and produce ethanol with high efficiency. Therefore, the transformed yeast of the present invention can be used in a method for producing ethanol from xylose. Here, "accumulation" of xylitol refers to suppression of xylitol production from xylose and / or promotion of xylulose production from xylitol.

[0154] The transformed yeast of the present invention can be cultured according to a conventional method used for culturing yeast. Those skilled in the art can select an appropriate medium from known media such as SD medium, SCX medium, YPD medium, YPX medium, CBS medium, etc., and culture the yeast under preferred culture conditions. When culturing yeast in a liquid medium, shaking culture is preferred.

[0155] Ethanol can be produced by culturing the transformed yeast of the present invention and collecting ethanol from the resulting culture. When producing ethanol, the transformed yeast of the present invention can be cultured in the presence of, for example, 10 to 50 g / L, preferably 10 to 40 g / L, of xylose as a carbon source in the above medium. In addition to xylose as a carbon source, it is preferable to further contain, for example, 40 to 300 g / L, preferably 60 to 200 g / L, of glucose. When both glucose and xylose are contained as carbon sources, the transformed yeast of the present invention can be cultured in the presence of, for example, 40 to 300 g / L, preferably 60 to 200 g / L in total. The transformed yeast of the present invention can be cultured by known culture methods such as batch culture without additional supply of sugar as a carbon source, fed-batch culture in which sugar is additionally supplied continuously / intermittently, or continuous culture. When additional supply of sugar as a carbon source is performed, it is desirable to monitor the concentration of sugar so as to maintain it within the above concentration range, and to control the supply of sugar.

[0156] The transformed yeast may be precultured before the main culture. For example, the preculture may be performed by inoculating the transformed yeast of the present invention into a small amount of medium and culturing it for 12 to 72 hours. For example, the main culture may be started by adding a preculture solution of 0.1 to 10%, preferably 1%, of the culture volume of the main culture to the medium. Alternatively, the main culture may be started by adding a yeast suspension obtained by suspending the yeast collected from the preculture solution in water or the like to the medium of the main culture. Here, the amount of the yeast suspension added to the medium of the main culture is not particularly limited as long as it does not interfere with the effects of the present invention, and it is preferable to add the yeast suspension so that the OD600 of the medium of the main culture becomes 10 to 20, and more preferably to add the yeast suspension so that the OD600 becomes 15. The main culture may be cultured with shaking at 20 to 40°C, preferably 30°C, for a culture time of 0.5 to 200 hours, preferably 10 to 150 hours, more preferably 24 to 137 hours in a xylose-containing medium, preferably a xylose and glucose-containing medium.

[0157] The produced ethanol can be collected from a culture obtained by culturing the yeast of the present invention as described above. The culture means a culture liquid (culture supernatant), cultured yeast, or a crushed product of cultured yeast, etc. Ethanol can be purified from the culture by a known purification method and collected. In the present invention, ethanol is mainly secreted into the culture supernatant from the transformed yeast, so it is preferable to collect it from the culture supernatant.

[0158] The amount of ethanol produced can be measured by analyzing the ethanol contained in the medium by liquid chromatography. Specifically, the amount of ethanol produced can be measured by the method used in Example 7. Furthermore, the ethanol producing ability of the transformed yeast of the present invention can be confirmed by measuring the amount of ethanol produced.

[0159] The amount of accumulated xylitol and the rate of consumption of xylose can be measured by analyzing the amounts of xylitol and xylose contained in the medium by liquid chromatography. Specifically, the amount of accumulated xylitol and the rate of consumption of xylose can be measured by the method used in Example 7.

[0160] The amount of glycerol produced can be measured by analyzing the amount of glycerol contained in the medium by liquid chromatography. Specifically, the amount of glycerol produced can be measured by the method used in Example 7.

[0161] The nucleotide sequences or amino acid sequences represented by SEQ ID NOs in the present invention are described below. SEQ ID NOs: 1 to 30: Show the base sequences of primers used in the examples. Sequence number 31: shows the base sequence of Saccharomyces cerevisiae PDC1. SEQ ID NO: 32: Shows the amino acid sequence of Saccharomyces cerevisiae PDC1 protein. Sequence number 33: Shows the base sequence of Saccharomyces cerevisiae PDA1. SEQ ID NO: 34: Shows the amino acid sequence of Saccharomyces cerevisiae PDA1 protein. Sequence number 35: shows the base sequence of Saccharomyces cerevisiae GRE3. SEQ ID NO: 36: Shows the amino acid sequence of Saccharomyces cerevisiae GRE3 protein. Sequence number 37: Shows the base sequence of Saccharomyces cerevisiae SOR1. SEQ ID NO: 38: Shows the amino acid sequence of Saccharomyces cerevisiae SOR1 protein. Sequence number 39: Shows the base sequence of Saccharomyces cerevisiae XKS1. SEQ ID NO: 40: Shows the amino acid sequence of Saccharomyces cerevisiae XKS1 protein. Sequence number 41: Shows the base sequence of Saccharomyces cerevisiae TAL1. SEQ ID NO: 42: Shows the amino acid sequence of Saccharomyces cerevisiae TAL1 protein. Sequence number 43: Shows the base sequence of Saccharomyces cerevisiae TKL1. SEQ ID NO: 44: Shows the amino acid sequence of Saccharomyces cerevisiae TKL1 protein. Sequence number 45: Shows the base sequence of Saccharomyces cerevisiae ADH1. SEQ ID NO: 46: Shows the amino acid sequence of Saccharomyces cerevisiae ADH1 protein. EXAMPLES

[0162] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, all percentages and ratios used in the present invention are by weight. Furthermore, unless otherwise specified, the units and measurement methods described in this specification are in accordance with the Japanese Industrial Standards (JIS).

[0163] Example 1: Synthesis of GRE3, SOR1, XKS1, TAL1, TKL1, ADH1, PGK1 promoters and PGK1 terminator by PCR Oligonucleotides 1 to 16 shown in Table 1 were synthesized and used as primers for PCR reaction to obtain DNA fragments of GRE3, SOR1, XKS1, TAL1, TKL1, ADH1, PGK1 promoter and PGK1 terminator (hereinafter, DNA fragments containing genes such as GRE3 are also referred to as gene fragments). Oligonucleotides 17 to 20 shown in Table 1 were also synthesized to obtain DNA fragments of HXT17 (first and second halves). Chromosomal DNA extracted from Saccharomyces cerevisiae CEN.PK2-1C strain was used as a template for PCR reaction. PCR reaction was performed using PrimeSTAR HS DNA polymerase (Takara Bio Inc.) with TaKaRa PCR Thermal Cycler Dice Gradient TP600 (Takara Bio Inc.). The amplification conditions for PCR are shown in Table 2. In PCR, after a heat denaturation step at 94°C for 2 minutes, 30 cycles of heat denaturation, annealing and extension steps were repeated.

[0164] GRE3, SOR1, XKS1, TAL1, TKL1, ADH1, PGK1 promoter and PGK1 terminator are derived from Saccharomyces cerevisiae. GRE3 protein has amino acid sequence identity (homology) with XYL1 (xylose reductase (XR)) of Scheffersomyces stipitis, and SOR1 protein has amino acid sequence identity (homology) with XYL2 (xylitol dehydrogenase (XDH)) of Scheffersomyces stipitis. XKS1 protein is xylulose phosphorylase, TAL1 protein is transaldolase, TKL1 protein is transketolase, and ADH1 protein is alcohol dehydrogenase. PGK1 promoter and PGK1 terminator are known to function in Saccharomyces cerevisiae.

[0165] [Table 1]

[0166] [Table 2]

[0167] Example 2: Preparation of yeast with xylose utilization ability The GRE3, SOR1, and XKS1 gene fragments amplified in Example 1 were ligated in this order under the control of the PGK1 promoter and PGK1 terminator to prepare an expression cassette I. In addition, Xho I and Bgl II cleavage sites were introduced into the commercially available vector pUC18 by site-specific mutagenesis. PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.) was used for the site-specific mutagenesis. The gene fragment of the first half of HXT17 amplified in Example 1 was inserted into the Xho I and EcoR I cleavage site of the obtained pUC18, and the gene fragment of the second half of HXT17 amplified in Example 1 was introduced into the Sph I and Bgl II cleavage site. The obtained expression cassette I was digested with EcoR I and Sph I and inserted between the first and second half of the HXT17 gene fragments to prepare expression cassette II.

[0168] The gene was introduced into the HXT17 site of the chromosome by the lithium acetate method using the constructed expression cassette II to obtain yeast strains with xylose assimilation ability. The yeast strain used was a brewing yeast strain of Saccharomyces cerevisiae.

[0169] Example 3: Construction of TAL1·TKL1 expression vector A TAL1 gene fragment under the control of the PGK1 promoter and PGK1 terminator was introduced into the SmaI site of a commercially available expression vector pAUR135 (Takara Bio Inc.). In addition, a TKL1 gene fragment under the control of the PGK1 promoter and PGK1 terminator was introduced into the SphI site of the pAUR135 vector into which the TAL1 gene fragment had been introduced.

[0170] Example 4: Preparation of parent yeast (TAL1·TKL1·ADH1 gene-transfected strain) An ADH1 gene fragment under the control of the PGK1 promoter and PGK1 terminator was introduced into the EcoRI site of the TAL1·TKL1 expression vector prepared in Example 3. The resulting expression vector was cleaved with StuI, and the resulting gene fragment was introduced, by the lithium acetate method, into the AUR1 site on the chromosome of the yeast with xylose assimilation ability prepared in Example 2. The resulting strain was used as the TAL1·TKL1·ADH1 gene-introduced strain (parent yeast) (hereinafter also referred to as the control strain). Furthermore, a URA3-deficient strain was prepared from the obtained control strain (hereinafter, also referred to as the control URA3-deficient strain). Specifically, the control URA3-deficient strain was obtained by subjecting the control strain to a mutation treatment using UV light, and then selecting a strain that grew on 5-FOA (5-fluoroorotic acid).

[0171] Example 5: Construction of transformed yeast PDA1 disruptant strain Oligonucleotides 21 to 22 shown in Table 3 were synthesized and used as primers for PCR reaction to obtain a DNA fragment of URA3. Oligonucleotides 23 to 26 shown in Table 3 were synthesized and used as primers for PCR reaction to obtain a DNA fragment of PDA1 (first half and second half). [Table 3] Chromosomal DNA extracted from Saccharomyces cerevisiae CEN.PK2-1C strain was used as a template for PCR. PCR was performed using LA Taq DNA Polymerase (Takara Bio) as the DNA amplification enzyme in a Thermal Cycler DICE TP600 (Takara Bio). The reaction conditions were as follows: (1) Thermal denaturation step 1: 94℃, 2 minutes (2) Thermal denaturation step 2: 94℃, 20 seconds (3) Annealing step: 50°C, 30 seconds (4) Extension step: 68°C, 2.5 minutes After the heat denaturation step 1 in (1), steps (2) to (4) were repeated 30 cycles.

[0172] PDA1 and URA3 are derived from Saccharomyces cerevisiae. The PDA1 protein is a pyruvate dehydrogenase, and the URA3 protein is a uracil synthase.

[0173] The URA3, PDA1 first half and PDA1 second half gene fragments amplified above were ligated in this order: PDA1 first half, URA3 and PDA1 second half to prepare a fusion gene fragment. The specific method for producing the above fusion gene fragment was as follows: the first half fragment of PDA1 (1-600 bp), URA3 (1037 bp), and the second half fragment of PDA1 (601-1263 bp) were each amplified by PCR as described above, and the amplified fragments were ligated by fusion PCR. The primers used in the fusion PCR reaction were oligonucleotide 23 and oligonucleotide 26. The DNA amplification enzyme used was Takara LA Taq polymerase (Takara Bio). The PCR reaction conditions were as follows: (1) Thermal denaturation step 1: 94℃, 2 minutes (2) Thermal denaturation step 2: 94℃, 20 seconds (3) Annealing step: 50°C, 30 seconds (4) Extension step: 68°C, 2.5 minutes After the heat denaturation step 1 in (1), steps (2) to (4) were repeated 30 cycles.

[0174] The obtained fusion gene fragment was introduced by the lithium acetate method into the PDA1 site on the chromosome of the URA3-deficient control strain obtained in Example 4. The obtained yeast was spread on a uracil-deficient SD medium plate (glucose 2%) and incubated at 30°C for 3 days.

[0175] Example 6: Preparation of transformed yeast PDC1-introduced strain Oligonucleotides 27 and 28 shown in Table 4 were synthesized and used as primers for PCR reaction to obtain a DNA fragment of PDC1. [Table 4] The template for the PCR reaction of PDC1 was yeast No. 7 chromosomal DNA. The PCR reaction was carried out using PrimeSTAR HS DNA polymerase (Takara Bio) as the DNA amplification enzyme, in a Thermal Cycler DICE TP600 (Takara Bio). The reaction conditions were as follows: (1) Heat denaturation step: 98℃, 10 seconds (2) Annealing step: 55°C, 5 seconds (3) Extension step: 72℃, 3 minutes Steps (1) to (3) were repeated 30 cycles.

[0176] The PGK1 promoter DNA fragment amplified in Example 1 was integrated into the BamHI and SalI cleavage sites of pUC18, and the PGK1 terminator DNA fragment amplified in Example 1 was integrated into the SalI and PstI cleavage sites to prepare pUC-PGKPT. Then, pUC-PGKPT was cleaved with SalI, and the PDC1 gene fragment amplified above was integrated into it to prepare pUC-PDC1.

[0177] The pUC-PDC1 obtained above was used as a template for PCR reaction, and oligonucleotides 29 and 30 shown in Table 5 were used as primers for PCR reaction to amplify the PGK1 promoter, PDC1, and PGK1 terminator. [Table 5] The PCR reaction was carried out using PrimeSTAR HS DNA polymerase (Takara Bio) as the DNA amplification enzyme in a Thermal Cycler DICE TP600 (Takara Bio) under the following reaction conditions. (1) Heat denaturation step: 98℃, 10 seconds (2) Annealing step: 55°C, 5 seconds (3) Extension step: 72℃, 3 minutes Steps (1) to (3) were repeated 30 cycles.

[0178] PDC1, PGK1 promoter and PGK1 terminator are from Saccharomyces cerevisiae. The PDC1 protein is pyruvate decarboxylase. The PGK1 promoter and PGK1 terminator are known to function in Saccharomyces cerevisiae.

[0179] The amplified fragment was digested with the restriction enzyme SmaI. The resulting fragment was subjected to agarose gel electrophoresis, excised from the gel and purified. This fragment was ligated with pAUR135 plasmid digested with SmaI to obtain the yeast introduction plasmid pAUR-PDC1. pAUR-PDC1 was cleaved with StuI, and the resulting PDC1 gene-containing fragment was introduced into the control strain obtained in Example 4 by the lithium acetate method. As a control, pAUR135 cleaved with StuI was introduced in the same manner. The resulting yeast was spread on an aureobasidin-containing (0.5 mg / l) YPD medium plate and incubated at 30° C. for 3 days.

[0180] Example 7: Fermentation performance evaluation 200 ml of YPD medium (peptone 2%, yeast extract 1%, glucose 1%) was placed in a 500 ml flask, and the strain obtained in Example 5 or 6 was inoculated and pre-cultured at 140 rpm and 30° C. for 20 hours. After collecting the bacteria from the preculture, the bacteria were washed twice with sterile distilled water, and the OD600 was adjusted with sterile distilled water to prepare a yeast suspension. OD600 was measured using UV1800 (Shimadzu Corporation). The OD600 was adjusted to about OD600 = 300 for the yeast of Example 5 and about OD600 = 200 for the yeast of Example 6. Phthalic acid-containing modified CBS medium (HB Klinke et al., Biotechnol. Bioeng., 2003, Vol. 81, pp. 738-747: pH 5.0) (carbon source: glucose 8%, xylose 1%, Table 6) and the yeast suspension were added to a 50 ml Erlenmeyer flask to make the total volume 15 ml (initial OD600 = 15), and the mixture was shaken and cultured at 30 ° C. and 140 rpm for 48 hours, and samples were taken over time to evaluate the fermentation performance. Furthermore, the control strain of Example 4 was used as a control for the PDA1-disrupted strain of Example 5 (hereinafter also referred to as the PDA1-disrupted control strain) to evaluate the above-mentioned fermentation performance. As a control for the PDC1-introduced strain of Example 6 (hereinafter also referred to as the PDC1-introduced control strain), a yeast in which pAUR135 cleaved with StuI was introduced into the control strain of Example 4 was used to evaluate the above-mentioned fermentation performance.

[0181] [Table 6]

[0182] The sampled culture solution was centrifuged to remove the bacterial cells, and the supernatant was filtered through a 0.2 μm polypropylene filter to prepare a measurement sample. The amounts of xylitol, glycerol, xylose, and ethanol in the measurement sample were quantified by HPLC. The analytical conditions for HPLC are shown in Table 7.

[0183] [Table 7]

[0184] The results of the fermentation performance evaluation for each strain are shown in Figures 1 to 3. Specifically, Figure 1 shows the results for the amount of xylitol accumulated after 48 hours of culture. Figure 2 shows the results for the xylose consumption rate after 24 hours of culture. Figure 3 shows the results for the amount of glycerol produced after 48 hours of culture. The xylose consumption rate can be calculated by the amount of xylose consumed / culture time. Since all xylose has been consumed after 48 hours of culture, the amount consumed after 24 hours of culture was used to calculate the xylose consumption rate. Figures 1 to 3 show the average ± standard deviation (n = 4).

[0185] As shown in Figure 1, the amount of xylitol accumulated in the PDA1-disrupted strain and the PDC1-introduced strain was significantly suppressed compared to the respective control strains (t-test, *: p<0.01 vs. the PDA1-disrupted control strain, *: p<0.01 vs. the PDC1-introduced control strain). In addition, as shown in Figures 2 and 3, the xylose consumption rate and the amount of glycerol produced were equivalent in the PDA1-disrupted strain, the PDA1-disrupted control strain, the PDC1-introduced strain, and the PDC1-introduced control strain. Furthermore, the amount of ethanol produced was measured to evaluate the ethanol yield (%). The ethanol yield was defined as 100% when the amount of ethanol produced matched the amount of ethanol calculated by multiplying the amount of substrate administered by the theoretical yield (0.51 for both glucose and xylose). As a result, the ethanol yield was equivalent in the PDA1-disrupted strain, the PDA1-disrupted control strain, the PDC1-introduced strain, and the PDC1-introduced control strain.

Claims

1. A transformed yeast having an ability to produce ethanol from xylose, the transformed yeast having an attenuated gene encoding pyruvate dehydrogenase, The parent yeast of the transformed yeast is not attenuated, and xylose assimilation-related genes including a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, and a gene encoding xylitol dehydrogenase have been introduced in advance in an expressible manner. The transformed yeast is a yeast belonging to the genus Saccharomyces.

2. The transformed yeast of claim 1 , wherein the parent yeast has at least the ability to produce pyruvic acid from xylose.

3. The transformed yeast according to claim 1 or 2, wherein the parent yeast has at least one xylose assimilation-related gene selected from the group consisting of a gene encoding a transaldolase, a gene encoding a transketolase, and a gene encoding an alcohol dehydrogenase further introduced in advance in an expressible manner.

4. The transformed yeast according to claim 3, wherein the xylose utilization-related genes are a gene encoding a xylose reductase, a gene encoding a xylulose phosphorylase, a gene encoding a xylitol dehydrogenase, a gene encoding a transaldolase, a gene encoding a transketolase, and a gene encoding an alcohol dehydrogenase.

5. The transformed yeast according to any one of claims 1 to 4, wherein at least one gene selected from the group consisting of a gene encoding pyruvate dehydrogenase and a gene related to xylose assimilation is an endogenous gene of the parent yeast.

6. The transformed yeast according to any one of claims 1 to 5, wherein the xylose utilization-related gene is inserted onto a chromosome in an expressible manner.

7. The transformed yeast according to any one of claims 1 to 6, wherein the gene encoding pyruvate dehydrogenase is PDA1.

8. The transformed yeast according to any one of claims 1 to 7, wherein the gene encoding xylose reductase is GRE3.

9. The transformed yeast according to any one of claims 1 to 8, wherein the gene encoding xylulose phosphorylase is XKS1.

10. The transformed yeast according to any one of claims 1 to 9, wherein the gene encoding xylitol dehydrogenase is SOR1.

11. The transformed yeast according to claim 3 or 4, wherein the gene encoding transaldolase is TAL1.

12. 12. The transformed yeast according to claim 3, wherein the gene encoding transketolase is TKL1.

13. 13. The transformed yeast according to any one of claims 3, 4, 11 and 12, wherein the gene encoding alcohol dehydrogenase is ADH1.

14. The transformed yeast according to any one of claims 1 to 13, wherein the parent yeast is a yeast belonging to the species Saccharomyces cerevisiae.

15. A method for producing ethanol, comprising culturing the transformed yeast according to any one of claims 1 to 14 in a medium containing glucose and xylose, and collecting ethanol from the resulting culture.

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