AU2015331353B2 - Yeast capable of producing ethanol from xylose - Google Patents

Abstract

　The purpose of the present invention is to provide a yeast having an improved capability to produce ethanol from xylose. The present invention provides a transformed yeast in which a xylose utilization gene and enzyme genes of a pentose phosphate pathway and ethanol production pathway are expressibly introduced into a host yeast.

Description

YEAST CAPABLE OF PRODUCING ETHANOL FROM XYLOSE TECHNICAL FIELD

[0001] The present invention relates to a yeast capable of producing ethanol from xylose.

BACKGROUND ART

[0002] Recently, from the viewpoint of C02 emissions reduction, utilization of bioalcohol as an alternative fuel for petroleum has attracted attention. In particular, bioalcohol produced using plants as resources has been vigorously studied. Since the amount of xylose accounts for about 1/3 of the total amount of sugar obtained from cellulose-based biomass from plants and the like, xylose assimilating yeasts used in the production of such bioalcohol have been studied in various ways.

[0003] For the production of bioethanol, yeasts typified by Saccharomyces cerevisiae are mainly used. Saccharomyces cerevisiae has a high ability to produce ethanol from hexoses such as glucose and mannose and is highly resistant to ethanol. However, Saccharomyces cerevisiae cannot utilize pentoses such as xylose.

[0004] As a yeast having xylose assimilation ability, Scheffersomyces stipitis is known. The group of genes corresponding to the group of genes for assimilating xylose of Scheffersomyces stipitis exists inherent in Saccharomyces cerevisiae, but it is considered that many of these genes in Saccharomyces cerevisiae are not expressed, or that even if the genes are expressed, the expression level thereof is very low. Therefore, the improvement of Saccharomyces cerevisiae by introduction of genes derived from a xylose assimilating yeast has been promoted.

[0005] However, since these genes are exogenous genes for Saccharomyces cerevisiae, a yeast prepared according to the above-described method falls into a recombinant, and it is undesirable because various restrictions including need of means for preventing leakage of yeast cells to the outside are imposed on utilization thereof.

[0006] To date, preparation of a xylose assimilation ability-imparted yeast by activating xylose assimilation genes inherent in Saccharomyces cerevisiae has been studied (W02010/001906 (Patent Document 1), W02014/058034 (Patent Document 2). The yeasts obtained using these techniques have the advantage that they are not gene recombinants because xylose assimilation genes derived from the yeasts themselves are utilized.

[0007] However, the development of a yeast having a further improved ability to produce ethanol from xylose is still desired.

PRIOR ART DOCUMENTS PATENT DOCUMENTS

[0008] Patent Document 1: W02010/001906 Patent Document 2: W02014/058034

SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] The purpose of the present invention is to provide a yeast having an improved ability to produce ethanol from xylose. It is an object of the present invention to provide a yeast having a more improved ability to produce ethanol, than conventional xylose assimilating yeasts that utilize genes encoding xylose decomposing enzymes, and preferably, a transformed yeast having an excellent ethanol productivity by utilizing genes derived from the yeast itself; and/or to at least provide the public with a useful choice.

MEANS FOR SOLVING THE PROBLEMS

[0010] The present inventors have diligently made researches in order to solve the above-described problems. As a result, the inventors have found that when a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase are introduced into a conventional xylose assimilating yeast into which three genes, namely, a gene encoding xylose reductase, a gene encoding xylulose kinase and a gene encoding xylitol dehydrogenase have been introduced, a yeast having an enhanced ability to produce ethanol from xylose can be obtained. That is to say, the present inventors have found that a yeast, into which a combination of the aforementioned six genes has been introduced, can efficiently produce ethanol from xylose. The inventors have found that the thus obtained yeast has a higher ability to utilize xylose than conventional xylose assimilating yeasts and can improve the efficiency of producing ethanol from xylose, thereby completing the present invention.

SUMMARY OF THE INVENTION

[0010a] In first aspect the present invention provides a transformed yeast, into which a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are functionally introduced, wherein the yeast has an ability to produce ethanol from xylose, and wherein the gene encoding xylose reductase is GRE3.

[001Ob] In a second aspect the present invention provides a method for producing ethanol, which comprises culturing the transformed yeast according to the first aspect in a xylose-containing medium, and then collecting ethanol from the obtained culture.

[0011] In addition, the present disclosure describes the following embodiments:

[1] A transformed yeast, into which a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are functionally introduced.

[2] The yeast according to item [1], wherein the genes are endogenous genes of the yeast.

[3] The yeast according to item [1] or [2], wherein the genes are functionally inserted onto the chromosome of a host yeast.

[4] The yeast according to any one of items [1] to [3], wherein the gene encoding xylose reductase is GRE3.

[5] The yeast according to any one of items [1] to [4], wherein the gene encoding xylulose kinase is XKS1.

[6] The yeast according to any one of items [1] to [5], wherein the gene encoding xylitol dehydrogenase is SORI.

[7] The yeast according to any one of items [1] to [6], wherein the gene encoding transaldolase is TAL1.

[8] The yeast according to any one of items [1] to [7], wherein the gene encoding transketolase is TKL1.

[9] The yeast according to any one of items [1] to [8], wherein the gene encoding alcohol dehydrogenase is ADHI.

[10] The yeast according to any one of items [1] to [9], which has an ability to produce ethanol from xylose.

[11] The yeast according to any one of items [1] to [10], wherein the host yeast has a hexose assimilation ability, but does not have a pentose assimilation ability.

[12] The yeast according to any one of items [1] to [11], wherein the host yeast is a yeast belonging to the genus Saccharomyces.

[13] The yeast according to any one of items [1] to [12], wherein the host yeast is a yeast belonging to Saccharomyces cerevisiae.

[14] A method for producing ethanol, which comprises culturing the transformed yeast according to any one of items [1] to [13] in a xylose-containing medium, and then collecting ethanol from the obtained culture.

ADVANTAGEOUS EFFECT OF THE INVENTION

[0012] According to the present disclosure, a transformed yeast, into which a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase have been introduced, is described. In one embodiment described herein, the transformed yeast described herein is produced by being transformed by genes possessed by the yeast itself. Accordingly, the transformed yeast described herein does not fall into a gene recombinant, and thus, is preferable in terms of safety and easy handleability. In addition, the transformed yeast described herein can efficiently produce ethanol from xylose. Therefore, according to the present disclosure, a method for producing ethanol from xylose, and a transformed yeast capable of producing ethanol from xylose, which can be used in the aforementioned method, are described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

[Figure 1] Figure 1 is a view showing the ethanol yield of a transformed yeast (shochu yeast) in a medium containing xylose as a substrate. The black bar indicates the results of the xylose assimilation ability-imparted yeast obtained in Example 2, the gray bar indicates the results of the TAL1/TKL1 gene overexpressed strain obtained in Example 3, and the white bar indicates the results of the TAL/TKL1/ADH1 gene overexpressed strain obtained in Example 4.

[Figure 2] Figure 2 is a view showing an ethanol yield in a transformed yeast (shochu yeast) in a medium containing glucose and ethanol as substrates. The black bar indicates the results of the xylose assimilation ability-imparted yeast obtained in Example 2, the gray bar indicates the results of the TAL/TKL1 gene overexpressed strain obtained in Example 3, and the white bar indicates the results of the TAL1/TKL1/ADH1 gene overexpressed strain obtained in Example 4.

[Figure 3] Figure 3 is a view showing an ethanol yield in a transformed yeast (shochu yeast) in a medium containing glucose and ethanol as substrates. The gray bar indicates the results of the TAL1/TKL1 gene overexpressed strain obtained in Example 3, and the white bar indicates the results of the TAL/TKL1/ADH1 gene overexpressed strain obtained in Example 4.

[Figure 4] Figure 4 is a view showing the results obtained by analyzing the expression level of an ADHI gene. The gray bar indicates the results of the TAL1/TKL1 gene overexpressed strain obtained in Example 3, and the white bar indicates the results of the TAL1/TKL1/ADH1 gene overexpressed strain obtained in Example 4.

[Figure 5] Figure 5 is a view showing the results obtained by measuring the ADHI activity of a yeast. The gray bar indicates the results of the TAL/TKL1 gene overexpressed strain obtained in Example 3, and the white bar indicates the results of the TAL1/TKL1/ADH1 gene overexpressed strain obtained in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to the description. In addition to the following examples, the present invention can be suitably changed and then practiced by those skilled in the art within a range in which the effects of the present invention are not reduced.

[0015] 1. Outline of the present invention The present invention is based on the findings that a transformed yeast produced by functionally introducing genes regarding six types of enzymes, which are associated with assimilation of xylose and a pentose phosphate pathway and an ethanol production pathway, namely, a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase, into a host yeast, has an excellent ability to produce ethanol.

[0016] The transformed yeast described herein can be produced by functionally introducing xylose assimilation genes such as a gene encoding xylose reductase, a gene encoding xylulose kinase and a gene encoding xylitol dehydrogenase, and a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase, into a host yeast. Alternatively, the transformed yeast described herein can also be produced by increasing the expression levels of a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and/or a gene encoding alcohol dehydrogenase, which are possessed by a host yeast, by substitution of a promoter, etc.

[0017] In the present specification, the "xylose assimilation gene" is a gene encoding an enzyme associated with assimilation of xylose. The xylose assimilation genes to be introduced into a host yeast in the present disclosure are at least three genes, namely, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose kinase. In the present disclosure, it is preferable to use a gene encoding sorbitol dehydrogenase as such a gene encoding xylitol dehydrogenase.

[0018] In addition, in the present specification, at least three genes, namely, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are also introduced into a host yeast. These three genes are genes encoding enzymes regarding a pentose phosphate pathway and an ethanol production pathway.

[0019] In another embodiment described herein, these genes to be introduced into a host yeast are genes derived from the host yeast. That is to say, in another embodiment of the present disclosure, one feature of the transformed yeast of described herein is that the expression of (endogenous) enzyme genes originally possessed by the yeast is activated, and that the activity of the enzymes possessed by the yeast itself is enhanced.

[0020] In another embodiment of the present disclosure, one feature of the transformed yeast described herein is that these genes to be introduced into a host yeast are inserted onto the chromosome of the host yeast.

[0021] In addition, in another embodiment of the present disclosure, the transformed yeast, into which the above-described six types of enzyme genes are introduced, has an excellent ability to produce ethanol from xylose.

[0022] Among yeasts, there are yeasts which do not have the ability to assimilate a pentose such as xylose because the group of xylose assimilation enzymes do not substantially function, that is, are in a so-called dormant state. For example, yeasts belonging to the genus Saccharomyces have the group of genes encoding the group of xylose assimilation enzymes, but cannot produce ethanol by utilizing xylose.

[0023] Into such a yeast which is regarded as not having ethanol production ability, a xylose assimilating gene derived from the yeast itself is introduced, or a promoter thereof is substituted with another promoter, thereby increasing the activity of the xylose assimilation endogenous gene and imparting xylose assimilation ability thereto. Moreover, not only the xylose assimilation gene derived from the yeast itself, but also enzyme genes derived from the yeast itself, which are associated with a pentose phosphate pathway and an ethanol production pathway (a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase), are introduced, or the promoters of these genes are substituted with other promoters, thereby increasing the activity of these genes, and further improving the efficiency of producing ethanol from xylose. Therefore, the transformed yeast described herein may be a yeast produced by introducing a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase into a yeast, onto the chromosome of which xylose assimilation genes have been inserted. Furthermore, according to the present disclosure, a yeast regarded as not having ethanol production ability is allowed to efficiently produce ethanol by utilizing xylose. Also, the present disclosure can be effectively applied to a yeast regarded as not having ethanol production ability or a yeast whose ethanol production ability is to be improved.

[0024] Further, since the transformed yeast described herein has an excellent ability to produce ethanol from xylose, the present disclosure also describes a method for producing ethanol, which comprises culturing the above-described transformed yeast, and then collecting ethanol from the obtained culture.

[0025] 2. Transformed yeast of the present invention The transformed yeast described herein is a yeast produced by functionally introducing xylose assimilation genes such as a gene encoding xylose reductase, a gene encoding xylulose kinase and a gene encoding xylitol dehydrogenase, and a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase into a host yeast.

[0026] (1) Host yeast In the present disclosure, a yeast to be targeted for gene introduction or transformation is preferably a yeast which does not have the ability to assimilate a pentose such as xylose. Regarding the aforementioned yeast, it is sufficient when it does not have the ability to assimilate a pentose before gene introduction or transformation, and the yeast may have the ability to assimilate a hexose such as glucose. The "ability to assimilate a pentose" refers to the ability to grow using a pentose such as xylose as a carbon source. Yeasts having the ability to assimilate a pentose can grow in a medium to which only the pentose is added as a carbon source, and therefore, the ability to assimilate a pentose can be confirmed by measuring the turbidity at a wavelength of 600 nm, 660 nm or the like as the growth level of a yeast in a medium to which only a pentose is added as a carbon source.

[0027] Alternatively, in the present disclosure, the yeast to be targeted for gene introduction or transformation can also be selected for the purpose of improving ethanol productivity. Examples of such yeasts include a yeast to which xylose assimilation ability is imparted, and a yeast whose xylose assimilation ability is activated.

[0028] In the present disclosure, the yeast to be targeted for gene introduction or transformation is not particularly limited. An example of such yeast is a yeast belonging to the genus Saccharomyces. Examples of the yeast belonging to the genus Saccharomyces include Saccharomyces cerevisiae including laboratory yeast strains and the like. Further, in the present disclosure, as the yeast to be targeted for gene introduction or transformation, not only a haploid yeast, but also a diploid yeast can be used. The diploid yeast is excellent as a practical yeast, and examples thereof include baker's yeasts and brewery yeasts such as sake yeasts, shochu yeasts, and wine yeasts.

[0029] Further, in the present disclosure, the yeast to be targeted for gene introduction or transformation is preferably an ethanol-resistant brewery yeast, and examples of such yeasts include, but are not particularly limited to, yeasts belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae).

[0030] Accordingly, in the present disclosure, the yeast to be targeted for gene introduction or transformation is preferably a yeast belonging to the genus Saccharomyces, and more preferably Saccharomyces cerevisiae.

[0031] (2) Genes to be introduced In the present disclosure, the xylose assimilation genes are at least three genes, namely, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase and a gene encoding xylulose kinase, and among them, the xylitol dehydrogenase is preferably sorbitol dehydrogenase. More specifically, the gene encoding xylose reductase, the gene encoding xylitol dehydrogenase and the gene encoding xylulose kinase are preferably GRE3 (aldo-keto reductase gene 3), SORI (sorbitol dehydrogenase gene 1) and XKS1 (xylulose kinase gene 1), respectively.

[0032] In addition, in the present specification, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase are also introduced into the yeast. 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 ADH I(alcohol dehydrogenase gene 1), respectively, and these genes are introduced into the yeast.

[0033] In the present disclosure, the xylose assimilation genes, and also, the gene encoding transaldolase, the gene encoding transketolase and the gene encoding alcohol dehydrogenase, can be either exogenous genes or endogenous genes. Between them, endogenous genes are preferable. The "endogenous gene" refers to a gene possessed by a yeast targeted for gene insertion, a gene derived from a yeast targeted for gene insertion and a gene derived from a yeast of the same species as that of a yeast targeted for gene insertion. Accordingly, a yeast having the endogenous gene introduced therein does not fall into a recombinant.

[0034] (Genes encoding xylose reductase) As genes encoding xylose reductase possessed by the yeast, GRE3, YJR096w, YPR1, GCYl, ARAl and YDR124w have been known. Accordingly, in the present disclosure, as the gene encoding xylose reductase, GRE3, YJR096w, YPR1, GCYl, ARAl or YDR124w can be used. In the present specification, the gene encoding xylose reductase is described using GRE3 as an example thereof, but the description regarding GRE3 in the specification can be applied to YJR096w, YPR1, GCYl, ARAl and YDR124w, and these genes can be used in the same way in the present disclosure.

[0035] A person skilled in the art can obtain the nucleotide sequence information of GRE3, YJR096w, YPR1, GCYl, ARAl and YDR124w from a publicly-known database such as Genbank. Accession numbers related to the sequence information of the respective genes of Saccharomyces cerevisiaeare described below. GRE3: U00059, YJR096w: Z49596, YPR1: X80642, GCYl: X13228, ARA1: M95580, and YDR124w: Z48758.

[0036] In the present disclosure, GRE3 (aldo-keto reductase gene 3) is a gene comprising a nucleotide sequence encoding aldo-keto reductase, and for example, it is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 21 derived from Saccharomyces cerevisiae or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 22. It is known that the protein encoded by GRE3 also functions as xylose reductase in the yeast. In addition, the GRE3 protein is a protein having identity (homology) at the amino acid sequence level to XYL1 (xylose reductase (XR)) of Scheffersomyces stipitis.

[0037] In the present disclosure, GRE3 can be obtained, for example, from a yeast library or genome library by means of the gene amplification technique, using a primer designed based on the nucleotide sequence represented by SEQ ID NO: 21.

[0038] GRE3 to be used herein includes a gene encoding a mutant of GRE3 protein. The gene encoding a mutant of GRE3 protein includes, for example, a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 21 under stringent conditions and encodes a protein having xylose reductase activity. The xylose reductase activity will be described later.

[0039] The DNA encoding a mutant of GRE3 protein can be obtained from a cDNA library and genome library by means of a publicly-known hybridization method such as colony hybridization, plaque hybridization and Southern blotting, using the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 21 or a fragment thereof as a probe. Regarding the method for preparing a library, "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Press (2012)), etc. can be referred to. Further, a commercially-available cDNA library and genome library may also be used.

[0040] In this regard, as washing conditions after hybridization, examples of stringent conditions include "2xSSC, 0.1% SDS, 42°C" and "1xSSC, 0.1% SDS, 37°C", and examples of more stringent conditions include "1xSSC, 0.1% SDS, 65°C" and "0.5xSSC, 0.1% SDS, 50°C".

[0041] Hybridization can be performed according to a publicly-known method. Regarding the hybridization method, for example, "Molecular Cloning, A Laboratory Manual 4th ed." (Cold Spring Harbor Laboratory Press (2012)), "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997)), etc. can be referred to.

[0042] Further, in the present specification, the DNA which hybridizes under stringent conditions includes, for example, a DNA comprising a nucleotide sequence having at least 50%, preferably 70% or more, 80% or more, or 85% or more, more preferably at least 90%, 95%, 96%, 97% or 98%, even more preferably 99% or more, still more preferably 99.7% or more, and particularly preferably 99.9% identity (homology) to the nucleotide sequence represented by SEQ ID NO: 21. The value indicating the identity can be calculated by utilizing a publicly-known program such as BLAST.

[0043] Further, examples of the DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 21 under stringent conditions include a DNA comprising the nucleotide sequence represented by SEQ ID NO: 21 having mutation such as deletion, substitution or addition of one or several nucleic acids. Examples of such DNAs include: (i) a DNA in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2) bases in the nucleotide sequence represented by SEQ ID NO: 21 are deleted; (ii) a DNA in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2) bases in the nucleotide sequence represented by SEQ ID NO: 21 are substituted with other bases; (iii) a DNA in which one to several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2) bases are added to the nucleotide sequence represented by SEQ ID NO: 21; and (iv) a DNA in which the above-described mutations are combined, each of the DNAs encoding a protein having xylose reductase activity.

[0044] In the present disclosure, confirmation of a nucleotide sequence can be carried out by sequencing according to a commonly-used method. For example, it can be carried out according to the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) or the like. It is also possible to utilize an appropriate DNA sequencer to analyze a sequence.

[0045] In the present disclosure, for example, GRE3 (aldo-keto reductase gene 3) derived from Saccharomyces cerevisiae includes those encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 22. In the present disclosure, a gene encoding GRE3 protein derived from Saccharomyces cerevisiae or a mutant thereof is also included in GRE3 (aldo-keto reductase gene 3).

[0046] Examples of the mutant of GRE3 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 22 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 22 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 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 22; and (iv) a protein in which the above-described mutations are combined, each of the proteins having xylose reductase activity.

[0047] In this regard, the "xylose reductase activity" refers to the activity to convert xylose to xylitol in the presence of NAD+ (or NADP+). In the present disclosure, the degree of the xylose reductase activity of the mutant of GRE3 protein is not particularly limited as long as the mutant has xylose reductase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 22. The xylose reductase activity of the protein can be measured by a publicly-known method.

[0048] (Genes encoding xylitol dehydrogenase) As genes encoding xylitol dehydrogenase possessed by the yeast, SORI, SOR2 and YLR070c have been known. Accordingly, in the present disclosure, as the gene encoding xylitol dehydrogenase, SOR, SOR2 or YLR070c can be used, and among them, SORI is preferable. In the present specification, the gene encoding xylitol dehydrogenase is described using SORI as an example thereof, but the description regarding SORI in the specification can also be applied to SOR2 and YLR070c. It is to be noted that SORI and SOR2 have 99.9% identity at the gene sequence level to each other.

[0049] A person skilled in the art can obtain the nucleotide sequence information of SORI, SOR2 and YLR070c from a publicly-known database such as Genbank.

Accession numbers related to the sequence information of the respective genes of Saccharomyces cerevisiae are described below. SORI: L11039, SOR2: Z74294, and YLR070c: Z73242.

[0050] In the present disclosure, SORI (sorbitol dehydrogenase gene 1) is a gene comprising a nucleotide sequence encoding sorbitol dehydrogenase, and for example, it is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 23 derived from Saccharomyces cerevisiae or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 24. It is known that the protein encoded by SORI also functions as xylitol dehydrogenase in the yeast. Moreover, the SORI protein is a protein having identity (homology) (53%) at the amino acid sequence level to XYL2 (xylitol dehydrogenase (XDH)) of Scheffersomyces stipitis.

[0051] SORI to be used in the present disclosure includes a gene encoding a mutant of SORI protein. The gene encoding a mutant of SORI protein includes, for example, a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 23 derived from Saccharomyces cerevisiae under stringent conditions and encodes a protein having xylitol dehydrogenase activity.

[0052] Further, SORI to be used in the present disclosure may be a gene encoding a mutant of the following SORI proteins: (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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 24 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 24 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 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 24; and (iv) a protein in which the above-described mutations are combined, each of the proteins having xylitol dehydrogenase activity.

[0053] In this regard, the "xylitol dehydrogenase activity" refers to the activity to dehydrogenate xylitol to form xylulose. In the present disclosure, the degree of the xylitol dehydrogenase activity of the mutant of SORI protein is not particularly limited as long as the mutant has xylitol dehydrogenase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 24. The xylitol dehydrogenase activity of the protein can be measured by a publicly-known method.

[0054]

Regarding DNAs included in the above-described DNA which hybridizes, hybridization conditions, etc., the explanation above can be applied thereto. Further, in the present disclosure, SORI can be obtained or produced according to the same method as that for GRE3 described herein.

[0055] (Gene encoding xylulose kinase) In the present disclosure, as a gene encoding xylulose kinase, XKS1 (xylulose kinase gene 1) can be used. A person skilled in the art can obtain the nucleotide sequence information of XKS1 from a publicly-known database such as Genbank. For example, the accession number of XKS1 of Saccharomyces cerevisiae is Z72979.

[0056] In the present disclosure, XKS1 (xylulose kinase gene 1) is a gene comprising a nucleotide sequence encoding xylulose kinase, and for example, it is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 25 derived from Saccharomyces cerevisiae or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 26.

[0057] XKS1 to be used in the present disclosure includes a gene encoding a mutant of XKS1 protein. The gene encoding a mutant of XKS1 protein includes, for example, a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 25 derived from Saccharomyces cerevisiae under stringent conditions and encodes a protein having xylulose kinase activity.

[0058] Further, XKS1 to be used in the present disclosure may be a gene encoding a mutant of the following XKS1 proteins: (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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 26 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 26 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 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 26; and (iv) a protein in which the above-described mutations are combined, each of the proteins having xylulose kinase activity.

[0059] In this regard, the "xylulose kinase activity" refers to the activity to phosphorylate xylulose. In the present disclosure, the degree of the xylulose kinase activity of the mutant of XKS1 protein is not particularly limited as long as the mutant has xylulose kinase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 26. The xylulose kinase activity of the protein can be measured by a publicly-known method.

[0060] Regarding DNAs included in the above-described DNA which hybridizes, hybridization conditions, etc., the explanation above can be applied thereto. Further, in the present disclosure, XKS1 can be obtained or produced according to the same method as that for GRE3 described herein.

[0061] (Genes encoding transaldolase) In the present disclosure, as a gene encoding transaldolase, TAL1 (transaldolase gene 1) or TAL2 (transaldolase gene 2) can be used. A person skilled in the art can obtain information regarding the nucleotide sequences of TAL1 and TAL2 from a publicly-known database such as Genbank. For example, the accession numbers of Saccharomyces cerevisiae TAL1 and TAL2 are X15953 and X59720, respectively. In the present specification, TAL1 is explained as an example of the gene encoding transaldolase. However, the description regarding TALl in the present specification can also be applied to TAL2.

[0062] In the present disclosure, TALl (transaldolase gene 1) is a gene comprising a nucleotide sequence encoding transaldolase, and it is, for example, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 27 derived from Saccharomyces cerevisiae, or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 28.

[0063] TALl to be used in the present disclosure includes a gene encoding a mutant of TALl protein. An example of the gene encoding a TALl protein mutant is a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 27 derived from Saccharomyces cerevisiae under stringent conditions, and encodes a protein having transaldolase activity.

[0064] Moreover, TALlused in the present disclosure may be a gene encoding a mutant of the following TALl proteins: (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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 28 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 28 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 or 2) amino acids are added to the amino acid sequence represented by

SEQ ID NO: 28; and (iv) a protein in which the above-described mutations are combined, each of the proteins having transaldolase activity.

[0065] In this regard, the "transaldolase activity" refers to the activity to catalyze the reaction of sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate <> erythrose-4 phosphate + fructose-6-phosphate. In the present disclosure, the degree of the transaldolase activity of the mutant of TAL1 protein is not particularly limited as long as the mutant has transaldolase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 28. The transaldolase activity of the protein can be measured by a publicly-known method.

[0066] Regarding DNAs included in the above-described DNA which hybridizes, hybridization conditions, etc., the explanation above can be applied thereto. Furthermore, in the present disclosure, TAL1 can be obtained or produced according to the same method as that for GRE3 described herein.

[0067] (Genes encoding transketolase) In the present disclosure, as a gene encoding transketolase, TKL1 (transketolase gene 1) or TKL2 (transketolase gene 2) can be used. A person skilled in the art can obtain information regarding the nucleotide sequences of TKL1 and TKL2 from a publicly-known database such as Genbank. For example, the accession numbers of Saccharomyces cerevisiae TKL1 and TKL2 are X73224 and X73532, respectively. In the present specification, TKL1 is explained as an example of the gene encoding transketolase. However, the description regarding TKL1 in the present specification can also be applied to TKL2.

[0068] In the present disclosure, TKL1 (transketolase gene 1) is a gene comprising a nucleotide sequence encoding transketolase, and it is, for example, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 29 derived from Saccharomyces cerevisiae, or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 30.

[0069] TKL1 to be used in the present disclosure includes genes encoding a mutant of TKL1 protein. An example of the gene encoding a TKL1 protein mutant is a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 29 derived from Saccharomyces cerevisiae under stringent conditions, and encodes a protein having transketolase activity.

[0070]

Moreover, TKL1 to be used in the present disclosure may be a gene encoding a mutant of the following TKL1 proteins: (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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 30 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 or 2) amino acids in the amino acid sequence represented by SEQ ID NO: 30 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 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 30; and (iv) a protein in which the above-described mutations are combined, each of the proteins having transketolase activity.

[0071] In this regard, the "transketolase activity" refers to the activity to catalyze the reaction of sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate <> xylulose-5 phosphate + ribose-5-phosphate. In the present disclosure, the degree of the transketolase activity of the mutant of TKL1 protein is not particularly limited as long as the mutant has transketolase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 30. The transketolase activity of the protein can be measured by a publicly-known method.

[0072] Regarding DNAs included in the above-described DNA which hybridizes, hybridization conditions, etc., the explanation above can be applied thereto. Furthermore, in the present disclosure, TKL1 can be obtained or produced according to the same method as that for GRE3 described herein.

[0073] (Gene encoding alcohol dehydrogenase) In the present disclosure, as a gene encoding alcohol dehydrogenase, ADHI (alcohol dehydrogenase 1) can be used. A person skilled in the art can obtain information regarding the nucleotide sequence of ADHI from a publicly-known database such as Genbank. For example, the accession number of Saccharomyces cerevisiae ADHI is X83121.

[0074] In the present disclosure, ADHI (alcohol dehydrogenase 1) is a gene comprising a nucleotide sequence encoding alcohol dehydrogenase, and it is, for example, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 31 derived from Saccharomyces cerevisiae, or a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 32.

[0075] ADHI to be used in the present disclosure includes genes encoding a mutant of ADHI protein. An example of the gene encoding an ADHI protein mutant is a DNA which hybridizes to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 31 derived from Saccharomyces cerevisiae under stringent conditions, and encodes a protein having alcohol dehydrogenase activity.

[0076] Moreover, ADHI to be used in the present disclosure may be a gene encoding a mutant of the following ADHI proteins: (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 or 2) amino acids in the amino acid sequence represented by 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 or 2) amino acids in the amino acid sequence represented by 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 or 2) amino acids are added to the amino acid sequence represented by SEQ ID NO: 32; and (iv) a protein in which the above-described mutations are combined, each of the proteins having alcohol dehydrogenase activity.

[0077] In this regard, the "alcohol dehydrogenase activity" refers to the activity to oxidize alcohol to aldehyde. In the present disclosure, the degree of the alcohol dehydrogenase activity of the mutant of ADH Iprotein is not particularly limited as long as the mutant has alcohol dehydrogenase activity, but for example, it is sufficient when the mutant has about 10% or more of the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 32. The alcohol dehydrogenase activity of the protein can be measured by a publicly-known method.

[0078] Regarding DNAs included in the above-described DNA which hybridizes, hybridization conditions, etc., the explanation above can be applied thereto. Furthermore, in the present disclosure, ADHI can be obtained or produced according to the same method as that for GRE3 described herein.

[0079] In the present disclosure, it is anticipated that, by introducing the above described six types of genes into a host yeast of the same species as the origins of the genes, the enzyme activity of the yeast itself can be improved in comparison to the enzyme activity thereof before the introduction of the genes. For example, as shown in Examples below, the alcohol dehydrogenase activity of the yeast described herein is increased. In addition, when a non-recombinant yeast is produced, the above-described six types of genes to be introduced into a host yeast need to be endogenous genes.

[0080] (3) Introduction of genes into yeast In the present disclosure, xylose assimilation genes (e.g., three genes, namely, a gene encoding xylose reductase, a gene encoding xylulose kinase, and a gene encoding xylitol dehydrogenase), and a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase are functionally introduced into a host yeast, thereby producing the transformed yeast described herein. Alternatively, the promoters of these genes are substituted with promoters for increasing the expression levels of the genes, thereby producing the transformed yeast described herein.

[0081] The transformed yeast described herein is preferably a yeast, into which GRE3, SORI, XKS1, TAL1, TKL1 and ADH are functionally introduced. In the present specification, the term "functionally (introduced or inserted)" means that the genes are introduced or inserted into a yeast, such that they can be expressed in the transformed yeast under predetermined conditions.

[0082] In the present disclosure, the gene encoding xylose reductase, the gene encoding xylulose kinase, and the gene encoding xylitol dehydrogenase are preferably functionally inserted onto the chromosome of a yeast. In addition, in the present disclosure, the gene encoding transaldolase, the gene encoding transketolase and the gene encoding alcohol dehydrogenase are preferably functionally inserted onto the chromosome of a yeast. That is, in the present disclosure, introduction of the genes into a yeast includes insertion of the genes onto the chromosome of the yeast. The transformed yeast described herein is preferably a yeast, onto the chromosome of which the gene encoding xylose reductase, the gene encoding xylulose kinase, xylitol dehydrogenase, the gene encoding transaldolase, the gene encoding transketolase and the gene encoding alcohol dehydrogenase are functionally inserted, and is more preferably a yeast, on the chromosome of which GRE3, SORI, XKS1, TAL1, TKL1 and ADH Iare functionally inserted.

[0083] In the present disclosure, the number of each of the genes to be inserted onto the chromosome is not limited, and it is one or more than one. In addition, the order of inserting each of the genes to be inserted onto the chromosome is not particularly limited. Moreover, the position of the chromosome onto which the genes are to be inserted is not particularly limited, but the site that does not function in the yeast is preferable. Examples of such sites include an XYL2 site (Genbank Accession No. Z73242), an HXT13 site, an HXT17 site, and an AURI site. It is also possible to insert the genes in a site on the chromosome that does not encode genes. An example of such a site on the chromosome that does not encode genes is a 6 sequence as one of Ty factors. It has been known that a plurality of6 sequences (about 100 copies) are present on the chromosome of a yeast. Information regarding the positions and sequences of the 6 sequences on the yeast chromosome is publicly-known (e.g., Science 265, 2077 (1994)). For example, by introducing into a yeast, a plasmid in which a xylose assimilation gene has been inserted into a 6 sequence, one or more than one copies of the gene can be inserted into a position of interest on the chromosome. Moreover, in addition to the 6 sequences, the genes can also be inserted into a 3 sequence and a t sequence, which are also Ty factors. Furthermore, the genes can also be inserted into a ribosome gene site such as NTS2.

[0084] When the genes are inserted onto the chromosome, the genes may be individually inserted onto the chromosome, or an expression cassette may be produced by connecting two or more genes with one another in tandem under the control of a promoter and the expression cassette may be then inserted onto the chromosome. When the genes are connected with one another in tandem, the order of positioning the genes and the number of the genes to be connected are not particularly limited, and all of possible combinations may be applied.

[0085] A plasmid can be utilized for introduction of the genes into a yeast. Such a plasmid can comprise one or more enzyme genes used in the present disclosure. For example, three genes, namely, a gene encoding xylose reductase, a gene encoding xylulose kinase and a gene encoding xylitol dehydrogenase can be comprised in a single plasmid. Moreover, for example, three genes, namely, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase can be comprised in a single plasmid. When a plurality of genes are comprised in a single plasmid, the order of individual genes on the plasmid and the number of the genes are not particularly limited. The present disclosure includes a plasmid or an expression cassette comprising these genes. Examples of such a plasmid or an expression cassette include: a plasmid or an expression cassette, in which GRE3, SORI and XLS1 are connected with one another under the control of a promoter; and a plasmid or an expression cassette, in which TAL1 and TKL are connected with each other under the control of a promoter. A plurality of genes are connected with one another, such that each of the genes can be expressed therein when they are introduced into a yeast, and in particular, onto the chromosome of a yeast. When two genes are connected with each other to produce a fusion gene, a linker sequence, a restriction enzyme site and the like may be appropriately added, if necessary. Such operations can be carried out using a genetic engineering technique well known and commonly used in the art. By using such a plasmid or an expression cassette, 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 chromosome of a yeast.

[0086] The plasmid to be used in the present disclosure can be prepared by functionally inserting the above-described genes into a vector for yeast expression. For inserting the genes into the vector, a ligase reaction, a topoisomerase reaction or the like can be utilized. For example, it is possible to employ a method, wherein: a purified DNA is cleaved by an appropriate restriction enzyme; and the obtained DNA fragment is inserted into an appropriate restriction enzyme site, a multicloning site or the like in a vector, so that the DNA fragment can be connected with the vector.

[0087] The plasmid to be used in the present disclosure is not particularly limited by the origin of the vector as the base of the plasmid. For example, a plasmid derived from Escherichiacoli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, etc., can be used. For example, a commercially available vector such as pGADT7 and pAUR135 can also be used. When the transformed yeast described herein is prepared using a plasmid derived from a host yeast, the transformed yeast described herein does not fall in a gene recombinant.

[0088] The plasmid described herein may include a multicloning site, a promoter, an enhancer, a terminator, a selection marker cassette, etc., as long as the target gene can be expressed. Moreover, a linker or a restriction enzyme site may be suitably added thereto if required at the time of inserting a DNA. Such operations can be carried out using a genetic engineering technique well known and commonly used in the art.

[0089] The promoter can be incorporated upstream of the target gene. The promoter is not particularly limited as long as a target protein can be appropriately expressed in a transformant. For example, a PGK promoter, an ADH promoter, a TDH promoter, an ENO promoter, a CIT promoter, a TEF promoter, a CDC promoter, a GPM promoter, a PDC promoter, etc., can be used.

[0090] The terminator can be incorporated downstream of the target gene, and for example, a PGK terminator, a CIT terminator, a TEF terminator, a CDC terminator, a GPM terminator, a PDC terminator, etc., can be used. In the present disclosure, for efficient expression of the target gene in the yeast, it is preferred to use a PGK promoter and/or a PGK terminator.

[0091] Examples of the selection marker include drug resistance genes such as an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene and a hygromycin resistance gene, a dihydrofolate reductase gene, a leucine synthetase gene, and a uracil synthetase gene. In the case where a synthesis gene cassette of an amino acid such as leucine, histidine and tryptophan or a uracil synthesis gene cassette is contained in the vector, the transformed yeast can be selected by culturing the yeast in a medium which does not contain the amino acid or uracil.

[0092] The transformed yeast can be prepared by introducing the plasmid or expression cassette described herein into the host yeast described herein as the target for gene introduction.

[0093]

The method for introducing the plasmid described herein into the host yeast is not particularly limited. Examples thereof include publicly-known methods such as the lithium acetate method, the electroporation method, the calcium phosphate method, the lipofection method and the DEAE-dextran method. According to these methods, the transformed yeast described herein is described.

[0094] Moreover, the transformed yeast described herein can also be produced by incorporating the target gene onto the chromosome of a host yeast according to homologous recombination. A person skilled in the art can produce the transformed yeast described herein according to homologous recombination by a publicly-known method.

[0095] The xylose utilization ability is not imparted to the yeast not having pentose assimilation ability, unless all of the three genes, i.e., the gene encoding xylose reductase, the gene encoding xylitol dehydrogenase and the gene encoding xylulose kinase are expressed. Accordingly, by culturing the yeast subjected to gene introduction as described above in a xylose-containing (glucose-free) medium, a transformed yeast, into which at least xylose assimilation genes are functionally introduced, can be selected.

[0096] Thus, by functionally introducing the six genes into a yeast, and preferably, by introducing the six genes onto the chromosome of a yeast, the transformed yeast described herein can be produced.

[0097] Furthermore, the transformed yeast described herein may also be a yeast, in which the expression of a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase, which are possessed by the host cells is activated. That is to say, the present disclosure also includes a yeast, in which the expression levels of the above-described genes originally present on the chromosome of a host yeast are increased. By introducing a promoter from the outside, or by substituting the promoters possessed by the genes themselves with stronger promoters, or the like, the genes originally possessed by the yeast are functionally activated, and the target protein can be appropriately expressed. For example, the method for activating the expression of such an endogenous gene is not limited, and examples thereof include a method in which a promoter by which a target protein can be appropriately expressed is incorporated onto the chromosome by means of gene substitution using a publicly-known genetic recombination technique. As such a method of gene substitution, the method of Akada et al. Yeast 23: 399-405 (2006) (non-patent document) can be used. As such a promoter for substitution, publicly known promoters 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, a PDC promoter, etc., can be used.

[0098] 3. Method for producing ethanol Since the transformed yeast described herein is a yeast, into which xylose assimilation genes, and a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase, for example, GRE3, SORI, XKS1, TAL1, TKL1 and ADHI are introduced, the transformed yeast described herein has an ability to assimilate xylose and can produce ethanol at a high yield. Accordingly, the transformed yeast described herein can be used in the method for producing ethanol from xylose.

[0099] The transformed yeast described herein can be cultured according to the usual method used for yeast cultivation. A person skilled in the art can select an appropriate medium from publicly-known media such as SD medium, SCX medium, YPD medium, YPX medium, and CBS medium to culture a yeast under preferred culture conditions. In the case where a yeast is cultured in a liquid medium, shaking culture is preferred.

[0100] Ethanol can be produced by culturing the transformed yeast described herein and collecting ethanol from the obtained culture. When producing ethanol, the transformed yeast described herein is cultured in the presence of 2 to 66 g/L, preferably 4 to 55 g/L of xylose used as a carbon source. Otherwise, when the medium comprises both glucose and xylose as carbon sources, the transformed yeast described herein is cultured in the presence of 40 to 330 g/L, preferably 80 to 275 g/L of glucose and xylose as carbon sources. To the transformed yeast described herein, known culture methods such as batch culture that does not involve the additional supply of sugar as a carbon source, fed-batch culture that involves continuous/intermittent additional supply of sugar, or continuous culture, can be used. When sugar is additionally supplied as a carbon source, the concentration of the sugar is monitored such that it is maintained in the above-described concentration range, and the supply of sugar is desirably controlled.

[0101] Prior to the main culture, the transformed yeast may be precultured. For preculture, for example, the transformed yeast described herein is inoculated into a small amount of medium and cultured for 12 to 24 hours. A preculture solution in an amount of 0.1% to 10%, preferably 1% of the culture amount of the main culture is added to the medium of the main culture, and then the main culture is started. The main culture is carried out by means of shaking culture using a xylose-containing medium for 0.5 to 200 hours, preferably 10 to 150 hours, and more preferably 24 to 137 hours, at 20°C to 40°C, and preferably 30°C.

[0102]

The produced ethanol can be collected from a culture obtained by culturing the yeast described herein in the above-described way. The culture refers to a culture solution (culture supernatant), a cultured yeast, a fractured product of the cultured yeast, or the like. Ethanol can be purified from the culture according to a publicly-known purification method, and then collected. In the present disclosure, ethanol is secreted from the transformed yeast mainly into the culture supernatant, and therefore, it is preferred to collect ethanol from the culture supernatant.

[0103] The production amount of ethanol can be measured by analyzing ethanol contained in the medium by means of liquid chromatography, gas chromatography or a commercially-available ethanol measurement kit. Moreover, by measuring the production amount of ethanol, the ethanol production ability of the transformed yeast described herein can be confirmed.

[0104] When ethanol is produced using the transformed yeast described herein, the ethanol can be produced at a higher yield, in comparison to conventional xylose assimilation yeasts (e.g., GRE3, SORI and XKS1-introduced yeasts) or yeasts obtained by allowing the above-described xylose assimilation yeasts to overexpress TAL1 and TKL1.

[0105] Hereinafter, the nucleotide sequences or amino acid sequences represented by sequence numbers in the present specification will be described. SEQ ID NOS: 1 to 20 represent the nucleotide sequences of primers used in Examples. SEQ ID NO: 21 represents the nucleotide sequence of GRE3 of Saccharomyces cerevisiae. SEQ ID NO: 22 represents the amino acid sequence of GRE3 protein of Saccharomyces cerevisiae. SEQ ID NO: 23 represents the nucleotide sequence of SORI of Saccharomyces cerevisiae. SEQ ID NO: 24 represents the amino acid sequence of SORI protein of Saccharomyces cerevisiae. SEQ ID NO: 25 represents the nucleotide sequence of XKS1 of Saccharomyces cerevisiae. SEQ ID NO: 26 represents the amino acid sequence of XKS1 protein of Saccharomyces cerevisiae. SEQ ID NO: 27 represents the nucleotide sequence of TAL1 of Saccharomyces cerevisiae. SEQ ID NO: 28 represents the amino acid sequence of TAL1 protein of Saccharomyces cerevisiae.

SEQ ID NO: 29 represents the nucleotide sequence of TKL1 of Saccharomyces cerevisiae. SEQ ID NO: 30 represents the amino acid sequence of TKL1 protein of Saccharomycescerevisiae. SEQ ID NO: 31 represents the nucleotide sequence of ADHI of Saccharomycescerevisiae. SEQ ID NO: 32 represents the amino acid sequence of ADHI protein of Saccharomyces cerevisiae.

[0106] Hereinafter, the present invention will be more specifically described by way of illustrative examples, but the present invention is not limited thereto.

Example 1

[0107] Synthesis of GRE3, SOR, XKS1, TAL1, TKL1, ADHI, a PGK1 promoter, and a PGK1 terminator by PCR Oligonucleotides 1 to 16 shown in Table 1 were synthesized, and they were then used as primers for PCR reaction, to obtain DNA fragments, namely, individual DNA fragments of GRE3, SORI, XKS1, TAL1, TKL1, ADHI, a PGK1 promoter, and a PGK1 terminator. In addition, Oligonucleotides 17 to 20 shown in Table 1 were synthesized to obtain DNA fragments of HXT17 (a first half and a second half). As a template used in the PCR reaction, a chromosomal DNA extracted from the strain Saccharomyces cerevisiae CEN.PK2-1C was used. The PCR reaction was carried out using PrimeSTAR HS DNA polymerase (TAKARA BIO INC.), by employing TaKaRa PCR Thermal Cycler Dice Gradient TP600 (TAKARA BIO INC.). Amplification conditions applied in the PCR are shown in Table 2.

[0108] GRE3, SORI, XKS1, TAL, TKL1, ADHI, the PGK1 promoter, and the PGK1 terminator were derived from Saccharomyces cerevisiae. The GRE3 protein has identity (homology) at the amino acid level to the XYL1 (xylose reductase (XR)) of Scheffersomyces stipitis, and the SORI protein has identity (homology) at the amino acid sequence level to the XYL2 (xylitol dehydrogenase (XDH)) of Scheffersomyces stipitis. The XKS1 protein is xylulose kinase, the TAL1 protein is transaldolase, the TKL1 protein is transketolase, and the ADHI protein is alcohol dehydrogenase. It has been known that the PGK1 promoter and the PGK1 terminator function in Saccharomycescerevisiae.

[0109]

[Table 1]

Table 1. Nucleotide sequences of Oligonucleotides 1-20

Nucleotide sequence SEQ ID NO (GRE3) Oligonudeotide 1: GGACGGCGCGTCGACATGTCTTCACTGGTTACTCT 1 Oligonucleotide 2 GGACGGCGCGTCGACTCAGGCAAAAGTGGGGAATT 2 (SORI) Ollgonudeotide 3: GGACGGCGCGTCGACATGACTGACTTAACTACACAAG 3 Oligonudeotide4: GGACGGCGCGTCGACTCATTCCGGGCCCTCAATG 4 (XKS1) Oligonudeotde5: GGACGGCGCGTCGACATGTTGTGTTCAGTAATTCAG 5 Oligonudeotide 6. GGACGGCGCGTCGACTTAGATGAGAGTCTTTTCCAG 6 (TAL1) Oligonudeotide7: GGACGGCGCGTCGACATGTCTGAACCAGCTCAAAAGA 7 Oligonudeotide8: GGACGGCGCGTCGACTTAAGCGGTAACTTTCTTTTC 8 (TKL1) Oligonudeotide 9: GGACGGCGCGTCOACATGACTCAATTCACTGACATTGA 9 Oligonudeotide10: GGACGGCGCGTCGACTTAGAAAGCTTTTTTCAAAGGAGA 10 (ADHI) Oligonudeotide11: GGACGGCGCGTCGACATGTCTATCCCAGAAACTCAAA 11 Oligonudeotide12: GGACGGCGCGTCGACTTATTTAGAAGTGTCAACAACG 12 (PGK1 promoter) Oligonudeotide13: GGCGGGATCCGCTTCACCCTCATACTATTA 13 Oligonudeotide14: GGCGCGTCGACTGTTTTTATATTTGTTGTAAA 14 (PGK1 terminator) Oligonudeotide15: GGCGCGTCGACATTGAATTGAATTGAAATCG 15 Oligonudeotide16: GGCGCCTGCAGGAATTTTCGAGTTATTAAAC 16 (First half of HXT17) Oligonudeotide17: ATGCAGCTGGCTCGAGATGGACAACTTTAAAATGAACTTC 17 Oligonudeotide18: TACCGAGCTCGAATTCTACCAACGAAGTATTGGTACCA 18 (Second half of HXT17) Oligonudeotide19: GAGTCGACCTGCAGGCATGCAGATTATCTATGGTCTCGGTGC 19 Oligonudeotide20: CGCCAGCTGGAGATCTTCAATCAGATATCTTGGGGACT 20

[0110]

[Table 2]

Table 2. PCR amplification conditions

denaurion Annealing Elongation

980C 550C 72 0C GRE3 10 sec 5 sec 60 see 980C 550C 72°C SORI 10 sec 5 sec 60 see XKS1 980C 55 0C 72 0C 10 sec 5 sec 90 see TAL1 98C 55°C 72°C 10 see 5 sec 60 see 980C 550C 72°C TKL1 10 sec 5 see 120 see 980C 550C 72°C ADHI 10 sec 5 see 60sec PGK1 98°C 550C 720C promoter 10 sec 5 sec 60 sec PGK1 98C 550C 720C terminator 10 sec 5 sec 30 sec First half of 980C 550C 720C HXT17 10 see 5 see 30 sec Second half of 980C 550C 720C HXT17 10 see 5 see 30 see

Example 2

[0111] Production of xylose assimilation ability-imparted yeast The gene fragments, GRE3, SORI and XKS1, amplified in Example 1 were connected with one another in the order of GRE3, SOR1 and XKS1 under the control of the PGK1 promoter and the PGK1 terminator to produce an expression cassette I. In addition, XhoI and BglII cleavage sites were introduced into a commercially available vector pUC18 according to site-directed mutagenesis. For the site-directed mutagenesis, PrimeSTAR Mutagenesis Basal Kit (TAKARA BIO INC.) was used. Into the obtained XhoI and EcoRI cleavage sites in pUC18, the gene fragment of first-half HXT17 amplified in Example 1 was inserted, whereas into the SphI and BglII cleavage sites, the gene fragment of second-half HXT17 amplified in Example 1 was introduced. The obtained expression cassette I was cleaved with EcoRI and SphI, and the cleaved portion was then inserted between the gene fragments of the first-half and second half of HXT17, so as to produce an expression cassette II.

[0112]

Using the produced expression cassette II, the above-described gene was introduced into the HXT17 site on the chromosome according to a lithium acetate method, to obtain a xylose assimilation ability-imparted yeast. As a host to which xylose assimilation ability was to be imparted, a shochu yeast was used.

Example 3

[0113] Production of TAL1/TKL1 gene overexpressed strain Into the SmaI site of a commercially available expression vector pAUR135 (TAKARA BIO INC.), the gene fragment of TAL1 that was under the control of the PGK1 promoter and the PGK1 terminator was introduced. Thereafter, the gene fragment of TKL1 that was under the control of the PGK1 promoter and the PGK1 terminator was introduced into the SphI site of the pAUR13 vector, into which the gene fragment of TAL1 had been thus introduced. The obtained TAL1/TKL1 expression vector was cleaved with Stul, and the obtained gene fragment was then introduced into the AURI site on the chromosome of the xylose assimilation ability-imparted yeast produced in Example 2 according to the lithium acetate method. The obtained strain was defined as a TAL1/TKL1 gene overexpressed strain.

Example 4

[0114] Production of TAL1/TKL1/ADH1 gene overexpressed strain Into the EcoRI site of the TAL1/TKL1 expression vector produced in Example 3, the gene fragment of ADHI that was under the control of the PGK1 promoter and the PGK1 terminator was introduced. The obtained expression vector was cleaved with Stu, and the obtained gene fragment was then introduced into the AURI site on the chromosome of the xylose assimilation ability-imparted yeast produced in Example 2 according to the lithium acetate method. The obtained strain was defined as a TAL1/TKL1/ADH1 gene overexpressed strain.

Example 5

[0115] Evaluation of fermentability The strains obtained in Examples 2, 3 and 4 were precultured in a YPD medium (20 g/L glucose), and were then placed in a 50-mL flask comprising 15 mL of a medium containing only xylose as a substrate, or 15 mL of a modified CBS medium containing glucose and xylose as substrates ((H. B. Klinke et al., Biotechnol. Bioeng., 2003, Vol. 81, pp. 738-747: pH 5.0), Table 3). Thereafter, the obtained mixtures were subjected to a shaking culture in an initial inoculation amount of 2 x 108 cells/mL at 140 rpm and at 30°C. Sampling was carried out over time to evaluate fermentability. At the same time, a strain, into which only a vector containing no genes to be introduced was incorporated, was also produced in the same manner as described above, and the thus produced strain was used as a control strain in the subsequent experiments.

[0116]

[Table 3] Table 3. Composition of modified CBS medium (g/L)

Carbon source Xylose Glucose and Xylose Glucose - 80 Xylose 50 50 KH 2PO 4 3.5 3.5 MgSO4 -7H 2 0 0.75 0.75 (NH 4 ) 2 SO 4 7.5 7.5 C8 H4 (COOH)(COOK) 10.21 10.21

[0117] The sampled culture solution was centrifuged to remove a cell mass, a supernatant was then filtrated through a 0.2-pm polypropylene filter, and the resultant was then used as a measurement sample. The amounts of glucose, xylose and ethanol in the measurement sample were quantified by HPLC. Analysis conditions applied in the HPLC are shown in Table 4.

[0118]

[Table 4]

Table 4. HPLC analysis conditions

Apparatus Shimadzu RID-O10A Column Shodex SUGAR SP0810 Mobile phase Ultrapure water Flow rate 0.8mL/min Detector Refractive index detector Column temperature 800C

[0119]

The fermentability evaluation results of individual strains are shown in Figure 1 and Figure 2. Figure 1 shows the results of the medium containing only xylose as a substrate, and Figure 2 shows the results of the medium containing glucose and xylose as substrates. Fermentability was evaluated in the form of an ethanol yield (%). In a case where the amount of ethanol generated was identical to the amount of ethanol calculated by multiplying the amount of added substrate(s) by a theoretical yield (which was 0.51 in both cases of glucose and xylose), it was set at 100%.

[0120] It was demonstrated that when TAL1 and TKL1 were introduced onto the chromosome of a xylose assimilation ability-imparted yeast, onto the chromosome of which GRE3, SORI and XKS1 had been introduced (a TAL1/TKL1 gene overexpressed strain, gray), it became possible to utilize xylose rather than the original xylose assimilation ability-imparted yeast (black), and the amount of ethanol produced was thereby increased (Figure 1 and Figure 2). Moreover, it was also demonstrated that when TAL, TKL1 and ADHI were introduced onto the chromosome of a xylose assimilation ability-imparted yeast, onto the chromosome of which GRE3, SORI and XKS1 had been introduced (a TAL1/TKL1/ADH1 gene overexpressed strain, white), it became possible to utilize xylose much more than the original xylose assimilation ability-imparted yeast (black) and the TAL1/TKL1 gene overexpressed strain (gray), and the amount of ethanol produced was thereby increased (Figure 1 and Figure 2). Furthermore, a xylose assimilation ability-imparted yeast (which was a shochu yeast), onto the chromosome of which TALl, TKL1 and ADHI had been introduced (TAL1/TKL1/ADH1 gene overexpressed strain, white), achieved a higher ethanol yield in a medium containing glucose and xylose as substrates (Figure 2), than in a medium containing only xylose as a substrate (Figure 1), and the ethanol yield in this yeast was also improved more significantly than the xylose assimilation ability-imparted yeast (black) and the TAL1/TKL1 gene overexpressed strain (gray).

[0121] From these results, it was demonstrated that a yeast comprising a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase can produce ethanol from xylose, more efficiently than a conventional xylose assimilation ability-imparted yeast.

Example 6

[0122] Verification of ADHI gene-enhancing effect The strains obtained in Examples 3 and 4 were precultured in a YPD (20 g/L glucose) medium, and were then cultured in a 1-L jar comprising 600 mL of a YPDX (80 g/L glucose and 40 g/L xylose) medium containing glucose and xylose as substrates, in an initial inoculation amount of 1 X 107 cells/mL at 380 rpm and at 30°C. Sampling was carried out over time, and a supernatant was analyzed by the method described in Example 5. Also, for the measurement of ADH1 activity and the analysis of the expression of an ADHi gene, a cell mass of yeast was recovered.

[0123] The results obtained by analyzing ethanol in the culture supernatant are shown in Table 5 and Figure 3. The ethanol yield was calculated by the method described in Example 5.

[0124] In all of the sampling times, the ethanol yield in a yeast into which TAL1, TKL1 and ADHI had been introduced (Example 4, TAL1/TKL1/ADH1 gene overexpressed strain, white) was higher than that in the TAL1/TKL1 gene overexpressed strain (Example 3, gray), and thus, it was demonstrated that the amount of ethanol produced is increased by the enhancement of an ADHi gene.

[0125]

[Table 5]

Ethanol yield, %

Time, h TAL1I/TKL1 gene TAL1/TKL1/ADH1 gene overexpressed strain overexpressed strain

0 0 0 24 65.4 65.8 30 68.1 69.0 48 73.9 75.2

[0126] Analysis of expression of ADH1 gene Yeast cells were recovered from a cell mass that had been cultured in ajar, the medium was then removed, and the cells were then washed with 2 ml of sterilized distilled water twice. Thereafter, the resulting cells were suspended in 400 pl of an acetate buffer (50mM sodium acetate pH 5.3, 10 mM EDTA). After that, 40 pl of SDS (sodium dodecyl sulfate) was added to the suspension, and 440 pl of phenol that had been incubated at 65°C was then added thereto. The thus obtained mixture was fully stirred, and was then incubated at 65°C for 4 minutes. Thereafter, the reaction mixture was quenched in ice. The reaction mixture was centrifuged at 12000 x g for 5 minutes, and a supernatant was then transferred into a 1.5-ml tube. After that, a mixed solution of phenol and chloroform was added to the supernatant in an equal amount of the supernatant, and the obtained mixture was then fully stirred. The reaction mixture was centrifuged at 12000 x g for 5 minutes, and a supernatant was then transferred into a 1.5-ml tube. After that, 3 M sodium acetate (pH 5.3) was added to the supernatant in an amount of 1/10 of the supernatant, and ethanol precipitation was then carried out. Centrifugation was carried out at 12000 x g for 10 minutes, and the obtained pellets were washed with 80% ethanol twice and were then dried and hardened. Thereafter, the obtained product was dissolved in RNase free water, and the obtained solution was defined as an RNA solution. cDNA was synthesized from the RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). Expression analysis was carried out by real time PCR using SYBR Green (a relative quantification method using a standard curve). A fluorescent reagent was prepared using LightCycler FastStart DNA Master PLUS SYBR Green I (Roche). As an analysis apparatus, LightCycler 1.5 (Roche) was used. The results obtained by analyzing the expression of ADHI in the TAL1/TKL1 gene overexpressed strain (Example 3) and the TAL/TKL/ADH1 gene overexpressed strain (Example 4) are shown in Table 6 and Figure 4.

[0127]

[Table 6]

ADH1 gene expression level

Time, h TAL1/TKL1 gene TALI/TKLI/ADH1 gene overexpressed strain overexpressed strain

24 L00 1.80 30 1.00 1.55 48 1.00 2.00

[0128] The expression of the ADHI gene in the TAL1/TKL gene overexpressed strain and the TAL1/TKL1/ADH1 gene overexpressed strain was analyzed. As a result, in all of the sampling times, the expression level of the ADHI gene in the TAL1/TKL1/ADH1 gene overexpressed strain (white) was higher than that in the TAL1/TKL1 gene overexpressed strain (gray). It was demonstrated that, for example, after completion of the culture for 48 hours, the expression level of the ADH1 gene was increased to two times as a result of the enhancement of the ADH1 gene.

[0129] Measurement of ADH1 activity Yeast cells were recovered from a cell mass that had been cultured in ajar, the medium was then removed, and the cells were then washed with 2 ml of distilled water twice. Thereafter, 1 ml of Y-PER Yeast Protein Extraction Reagent (Pierce, Rockford, IL) was added to the resulting cells, so that they were suspended therein. Thereafter, the suspension was shaken at room temperature for 20 minutes. The suspension was centrifuged at 13,000 rpm for 10 minutes to obtain a cell extract. ADHI activity was measured as follows. That is, 10 l of a crude yeast extract was added to 960 tl of a solution containing 50 mM Tris-HCl (pH 8.5) and 1 mM NAD', and 30 tl of ethanol was then added to the solution, so as to initiate the reaction. Using ethanol as a substrate, the amount of NADH increased was calculated by measuring the absorbance at 340 nm. The activity necessary for reducing 1I mol NAD+ per minute was defined as 1 U, and the ADH1 activity (U/mg) per mg of protein in the cell extract was obtained. The ADHI activity in each of the TAL/TKL1 gene overexpressed strain (Example 3) and the TAL1/TKL1/ADH1 gene overexpressed strain (Example 4) is shown in Table 7 and Figure 5. The trial was carried out in two series.

[0130]

[Table 7]

ADH activity, U/mg

Time, h TAL1/TKL1 gene TAL1/TKL1/ADH1 gene overexpressed strain overexpressed strain

24 0. 81t0. 06 4. 78 0. 18 30 1.53±0t.26 3. 32±0. 09 48 0.53±0.07 0. 92±0. 18

[0131] The ADH1 activity in each of the TAL1/TKL1 gene overexpressed strain and the TAL1/TKL1/ADH1 gene overexpressed strain was measured. As a result, in all of the sampling times, the ADHI activity in the TAL/TKL/ADH1 gene overexpressed strain (white) was higher than that in the TAL1/TKL1 gene overexpressed strain (gray). It was demonstrated that, for example, after completion of the culture for 24 hours, the ADH1 activity was increased to about six times as a result of the enhancement of the ADHI gene.

[0132] From these results, it was demonstrated that the expression level of an ADH1 gene and the ADHI activity are increased by enhancing the ADHI gene, and that the ethanol productivity of the TAL1/TKL1/ADH1 gene overexpressed strain is higher than that of the TAL1/TKL1 gene overexpressed strain.

[0133] Moreover, it was also demonstrated that the present invention can be applied, not only to the culture of cells in a flask at a laboratory level, but also to the culture of cells in a culture tank at an industrial level or under culture conditions similar to such an industrial level.

INDUSTRIAL APPLICABILITY

[0134] According to the present disclosure, a transformed yeast produced by introducing a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and a gene encoding alcohol dehydrogenase into a host yeast is described. In one embodiment described herein, the transformed yeast described herein is produced by being transformed by genes possessed by the yeast itself. Accordingly, the transformed yeast described herein does not fall in a gene recombinant, and thus, it is preferable in terms of safety and easy handleability. Moreover, the transformed yeast described herein can efficiently produce ethanol from xylose. Therefore, according to the present disclosure, a method for producing ethanol from xylose, and a transformed yeast capable of producing ethanol from xylose, which can be used in the aforementioned method, are described.

Sequence Listing Free Text

[0135] SEQ ID NOS: 1 to 20: primers

[0136] The term "comprising" as used in this specification and claims means "consisting at least in part of'. When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

[0137] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0138] In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

Claims (12)

1. A transformed yeast, into which a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are functionally introduced, wherein the yeast has an ability to produce ethanol from xylose, and wherein the gene encoding xylose reductase is GRE3.

2. The yeast according to claim 1, wherein the genes are endogenous genes of the yeast.

3. The yeast according to claim 1 or 2, wherein the genes are functionally inserted onto the chromosome of a host yeast.

4. The yeast according to any one of claims 1 to 3, wherein the gene encoding xylulose kinase is XKS1.

5. The yeast according to any one of claims 1 to 4, wherein the gene encoding xylitol dehydrogenase is SORI.

6. The yeast according to any one of claims 1 to 5, wherein the gene encoding transaldolase is TAL1.

7. The yeast according to any one of claims 1 to 6, wherein the gene encoding transketolase is TKL1.

8. The yeast according to any one of claims 1 to 7, wherein the gene encoding alcohol dehydrogenase is ADHI.

9. The yeast according to any one of claims 1 to 8, wherein the host yeast has a hexose assimilation ability, but does not have a pentose assimilation ability.

10. The yeast according to any one of claims 1 to 9, wherein the host yeast is a yeast belonging to the genus Saccharomyces.

11. The yeast according to any one of claims 1 to 10, wherein the host yeast is a yeast belonging to Saccharomyces cerevisiae.

12. A method for producing ethanol, which comprises culturing the transformed yeast according to any one of claims 1 to 11 in a xylose-containing medium, and then collecting ethanol from the obtained culture.