JPWO2014058034A1 - Yeast producing ethanol from xylose - Google Patents

Abstract

An object of the present invention is to provide a yeast that produces ethanol from xylose. In the present invention, GDH2 (glutamate dehydrogenase gene 2) and SOR1 (GRE3 (glutamate dehydrogenase gene 2) and SOR1 (xylulose phosphorylase gene 3) were introduced into a yeast into which three genes, GRE3 (Aldo keto enzyme gene 3), SOR1 and XKS1 (xylulose phosphorylase gene) were introduced. The present invention relates to a transformed yeast into which at least one gene selected from sorbitol dehydrogenase gene 1) has been introduced, or to a transformed yeast into which three genes GRE3, SOR1 and XKS1 have been introduced on the chromosome.

Description

  The present invention relates to a yeast that produces ethanol from xylose.

In recent years, the use of bioethanol as a transportation fuel has attracted attention from the viewpoint of reducing CO ₂ emissions. Above all, production from cellulosic biomass is being studied as a bioethanol production method. Since about one-third of the sugar obtained from cellulosic biomass is occupied by xylose, it is necessary to develop a microorganism that efficiently produces ethanol from xylose in order to effectively use biomass resources.

  Currently, yeasts represented by Saccharomyces cerevisiae are mainly used as brewing yeasts for ethanol production. Saccharomyces cerevisiae has a high ability to produce ethanol from hexoses such as glucose and mannose, and has high resistance to ethanol. However, Saccharomyces cerevisia cannot use pentoses such as xylose.

  Schiffosomyces stipitis is known as a yeast having the ability to assimilate xylose. Saccharomyces cerevisiae has a gene group corresponding to the gene group for assimilating xylose possessed by Cifazomyces stippitis, but many of these genes in Saccharomyces cerevisiae are not expressed or expressed. Even if so, the amount is considered to be very small. Therefore, improvement of Saccharomyces cerevisiae by gene introduction derived from xylose-assimilating yeast is being promoted.

  However, since these genes are foreign genes for Saccharomyces cerevisiae, the yeast prepared by the above method falls under the category of recombinants, and there is a need for means to prevent the leakage of bacterial cells to the outside world. Since various restrictions arise, it is not preferable.

  On the other hand, production of a yeast imparted with xylose utilization ability by activating a xylose utilization gene endogenous to Saccharomyces cerevisiae has been studied (WO 2010/001906, Patent Document 1). Yeast obtained using this technique has the advantage that it is not a gene recombinant because it utilizes a xylose-utilizing gene derived from the yeast itself.

  However, there is still a demand for the development of a yeast that further improves the ability to produce ethanol from xylose.

WO2010 / 001906

  There is a demand for the development of a yeast that has been further improved in the ability to produce ethanol from xylose. Accordingly, an object of the present invention is to improve a yeast into which a xylose-assimilating gene has been introduced, and to provide a yeast that is excellent in the ability to produce xylose from ethanol and a yeast that produces a small amount of by-products.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that GRE3 (Aldo-keto reductase gene 3), SOR1 (sorbitol dehydrogenase gene 1) and XKS1 (xylulose) on the yeast chromosome. When three genes of phosphorylase gene 1) are introduced and at least one of GDH2 (glutamate dehydrogenase gene 2) and SOR1 (sorbitol dehydrogenase gene 1) is overexpressed, ethanol productivity is increased. It was found that high yeast was obtained, and that the amount of by-products such as xylitol or glycerol decreased in the GDH2 overexpressing strain. Furthermore, as a result of intensive studies to solve the above-mentioned problems, the present inventors have also introduced ethanol fusion genes of GRE3 and SOR1 and SOR1 and XKS1 onto yeast chromosomes. It has been found that a highly productive yeast can be obtained. And it discovered that the yeast obtained in this way has high xylose utilization ability, and can be used for producing ethanol from xylose, and completed this invention.

That is, the present invention relates to the following.
(1) A transformed yeast in which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted on the chromosome,
The three genes link a gene encoding xylose reductase and a gene encoding xylitol dehydrogenase, and a gene encoding xylitol dehydrogenase and a gene encoding xylulose phosphorylase. The yeast, which has been inserted into the chromosome as a modified gene.
(2) The yeast according to (1), wherein the gene is an endogenous gene of the yeast.
(3) The yeast according to (1) or (2), wherein the gene encoding xylose reductase is a gene selected from the group consisting of GRE3, YJR096w, YPR1, GCY1, ARA1, and YDR124w.
(4) The yeast according to any one of (1) to (3), wherein the gene encoding xylitol dehydrogenase is a gene selected from the group consisting of SOR1, SOR2, and YLR070c.
(5) Any of (1) to (4), wherein the gene encoding xylose reductase, the gene encoding xylitol dehydrogenase, and the gene encoding xylulose phosphorylase are GRE3, SOR1, and XKS1, respectively. The yeast according to item 1.
(6) The yeast according to any one of (1) to (5), wherein the yeast belongs to the genus Saccharomyces.
(7) The yeast according to any one of (1) to (6), wherein the yeast is Saccharomyces cerevisiae.
(8) The yeast according to any one of (1) to (7), which has an ability to produce ethanol from xylose.
(9) A method for producing ethanol, comprising culturing the transformed yeast according to any one of (1) to (8) in a xylose-containing medium and collecting ethanol from the obtained culture.
The present invention also relates to the following.
[1] Encode glutamate dehydrogenase into host yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase, are inserted on the chromosome. A transformed yeast into which at least one gene selected from a gene and a gene encoding xylitol dehydrogenase has been introduced.
[2] The yeast according to claim 1, wherein the gene is derived from a host yeast.
[3] At least one gene selected from the gene encoding glutamate dehydrogenase and the gene encoding xylitol dehydrogenase is inserted so as to be expressible on the chromosome of the host yeast, [1] or The yeast according to [2].
[4] Any one of [1] to [3], wherein at least one gene selected from GDH2 and SOR1 is introduced into a host yeast into which three genes GRE3, SOR1 and XKS1 are inserted on a chromosome so as to allow expression. 2. The transformed yeast according to item 1.
[5] Yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted, belongs to the genus Saccharomyces [1 ] The yeast of any one of [4].
[6] The yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted, is Saccharomyces cerevisiae, [1]- The yeast according to any one of [5].
[7] The yeast according to any one of [1] to [6], which has an ability to produce ethanol from xylose.
[8] A method for producing ethanol, comprising culturing the transformed yeast according to any one of [1] to [7] in a xylose-containing medium and collecting ethanol from the obtained culture.
[9] whether at least one gene selected from a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase introduced into the transformed yeast is a gene encoding glutamate dehydrogenase, Alternatively, the method according to [8], which is both a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase.
[10] At least one gene selected from a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase introduced into the transformed yeast is a gene encoding xylitol dehydrogenase, The method according to [8], wherein the culturing step is performed in the presence of NAD +.

According to the present invention, a transformed yeast capable of producing ethanol from xylose is provided.
In one embodiment of the present invention, the transformed yeast of the present invention is not a genetic recombinant because it is transformed using the gene of the yeast itself, and is preferable in terms of safety and ease of handling. .
Moreover, since the transformed yeast of the present invention has an excellent ability to produce ethanol from xylose, ethanol can be efficiently produced from xylose by using the transformed yeast of the present invention.
In addition, since the transformed yeast of the present invention showed a reduction in the production of by-products such as xylitol and glycerol, the by-products accumulated when ethanol was produced using the transformed yeast of the present invention. The problem of doing can be avoided.

Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and those skilled in the art can appropriately modify and implement other than the following examples without departing from the spirit of the present invention.
In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.

1. SUMMARY OF THE INVENTION The present invention relates to a host yeast having enhanced activity of a xylose-utilizing endogenous gene derived from yeast itself and imparted xylose-assimilating ability to sorbitol dehydrogenase gene 1 (SOR1) and / or glutamate dehydrogenase. This is based on the knowledge that transformed yeast overexpressing gene 2 (GDH2) has an excellent ethanol production ability.
In the present invention, it was also found that the production amount of by-products such as xylitol and glycerol decreases in the GDH2 overexpression strain in which GDH2 is introduced into the host yeast. Since GDH2 is a glutamate dehydrogenase gene that converts NADH, which is a reduced form of NAD +, into NAD +, it was shown that NAD + is involved in the suppression of byproduct production. Therefore, by culturing the SOR1 overexpressing strain in which SOR1 is introduced into the host yeast in the presence of NAD +, or by co-expressing SOR1 and GDH2 in the host yeast, the by-product as in the GDH2 overexpressing strain can be obtained. It can be said that production volume decreases.

One of the features of the transformed yeast of the present invention is that it is produced using a host yeast in which a xylose-assimilating gene, preferably the yeast's own xylose-assimilating gene is introduced on the chromosome. The “xylose utilization (sex) gene” is a gene encoding a protein involved in utilization of xylose. The xylose utilization gene introduced into the host yeast in the present invention is at least three genes: a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose kinase.
In one embodiment of the present invention, the transformed yeast of the present invention comprises a gene encoding xylitol dehydrogenase and / or a gene encoding glutamate dehydrogenase, preferably sorbitol dehydrogenase gene 1 (SOR1) and One of the features is that it is produced by introducing glutamic acid dehydrogenase gene 2 (GDH2) into host yeast. In another embodiment of the present invention, the gene to be introduced into the host yeast is a gene derived from the host yeast.
Moreover, in another aspect of the present invention, the transformed yeast of the present invention is characterized in that the amount of by-products produced is low.

  Among yeasts, there are yeasts that do not have the ability to assimilate pentoses such as xylose because they are in a so-called dormant state in which the xylose-utilizing enzyme group does not substantially function. For example, yeast belonging to the genus Saccharomyces cannot produce ethanol using xylose, despite having a gene group encoding a xylose-utilizing enzyme group.

  Introducing a self-derived xylose-assimilating gene into such yeast that does not have ethanol-producing ability can enhance the activity of xylose-utilizing endogenous gene and impart xylose-assimilating ability. it can. Therefore, in the transformed yeast of the present invention, the gene (XR) encoding xylose reductase, the gene (XDH) encoding xylitol dehydrogenase and the gene (XK) encoding xylulose phosphorylase are inserted on the chromosome. Yeast may be used. A transformed yeast obtained by overexpressing sorbitol dehydrogenase gene 1 (SOR1) and / or glutamate dehydrogenase gene 2 (GDH2) in a yeast introduced with a xylose-assimilating gene derived from itself is surprising. In particular, the ability to produce ethanol from xylose is improved compared to the control strain. In addition, the amount of by-products produced decreases under certain culture conditions. That is, according to the present invention, ethanol can be efficiently produced using xylose in yeast that does not have ethanol-producing ability. Furthermore, the transformed yeast of the present invention in which the above three genes are introduced into the chromosome as a fusion gene of XR and XDH and a fusion gene of XDH and XK has an improved ability to produce ethanol from xylose. By setting it as the form of the said fusion gene, the introduction | transduction number to the yeast chromosome of the gene (XDH) which codes a xylitol dehydrogenase among said three genes can be increased.

  The present invention also provides a method for producing ethanol by culturing the transformed yeast and collecting ethanol from the resulting culture. In the present invention, it is possible to significantly reduce the amount of by-products to be produced as compared with the conventional method.

2. Transformed yeast of the present invention The transformed yeast of the present invention is a host yeast into which a xylose-assimilating gene has been introduced onto a chromosome, and is selected from at least a gene encoding xylitol dehydrogenase and a gene encoding glutamate dehydrogenase. Yeast into which one gene, preferably at least one gene selected from sorbitol dehydrogenase gene 1 (SOR1) and glutamate dehydrogenase gene 2 (GDH2), has been further introduced.
The transformed yeast of the present invention may be a yeast in which a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted on the chromosome.

(1) Host yeast in which a xylose-assimilating gene is introduced onto a chromosome In the present invention, the yeast to be subjected to gene introduction or transformation is a yeast that does not have the ability to assimilate pentoses such as xylose. It is preferable. The yeast may have any ability to assimilate pentose before gene introduction or transformation, and may have ability to assimilate hexose such as glucose. “5 carbon sugar assimilation ability” refers to the ability to grow using 5 carbon sugars such as xylose as a carbon source. Since yeast having pentose assimilation ability can grow in a medium to which only pentose is added as a carbon source, the pentose utilization ability is in a medium to which only pentose is added as a carbon source. The degree of yeast growth in can be confirmed by measuring turbidity at a wavelength such as 600 nm or 660 nm.

  In the present invention, yeast that does not have pentose assimilation ability is not particularly limited, and examples thereof include yeast belonging to the genus Saccharomyces. Examples of yeast belonging to the genus Saccharomyces include Saccharomyces cerevisiae such as CEN.PK2-1C strain. In the present invention, the yeast to be the target of gene transfer or transformation can be not only haploid but also diploid yeast. Diploid yeast is excellent as a practical yeast. For example, association yeast such as Association No. 7, shochu yeast such as shochu yeast S-2 strain, wine yeast, Red Star strain, Taiken No. 396 strain, etc. Can do.

  In the present invention, the yeast to be subjected to gene transfer or transformation is preferably a brewing yeast having resistance to ethanol, and such yeast is not particularly limited, Examples include yeast belonging to the genus Saccharomyces (for example, Saccharomyces cerevisiae).

  Therefore, the yeast that is the target of gene transfer or transformation in the present invention is preferably a yeast belonging to the genus Saccharomyces, more preferably Saccharomyces cerevisiae.

  In the present invention, the xylose utilization gene inserted on the chromosome of the host yeast is a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase, Are three genes, GRE3 (Aldo keto reductase gene 3), SOR1 (sorbitol dehydrogenase gene 1) and XKS1 (xylulose phosphorylase gene). In the present invention, as the xylose utilization gene, either a foreign gene or an endogenous gene can be used, but an endogenous gene is preferred. “Endogenous gene” means a gene of a yeast to be gene-inserted, a gene derived from a yeast to be gene-inserted, or a gene derived from a yeast of the same kind as the yeast to be gene-inserted. Therefore, yeast introduced with an endogenous gene is not a recombinant.

  Known genes encoding yeast xylose reductase are GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w. Therefore, in the present invention, 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 apply the description in this specification regarding GRE3, and similarly in the present invention. Can be used.

The base sequence information of GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w can be obtained from a known database such as Genbank by those skilled in the art. The accession numbers for the sequence information of each gene in Saccharomyces cerevisia are shown below.
GRE3: U00059, YJR096w: Z49596, YPR1: X80642, GCY1: X13228, ARA1: M95580, YDR124w: Z48758.

In the present invention, GRE3 (Aldo keto reductase gene 3) is a gene containing a base sequence encoding aldo keto reductase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 1 derived from Saccharomyces cerevisiae, Or it is DNA which codes the protein which consists of an amino acid sequence shown by sequence number 2. It is known that the protein encoded by GRE3 also functions as a xylose reductase in yeast.
In the present invention, GRE3 can be obtained from a yeast library or a genomic library by a gene amplification technique by designing a primer based on the base sequence represented by SEQ ID NO: 1, for example.

  GRE3 used in the present invention includes a gene encoding a mutant of GRE3 protein. A gene encoding a mutant of the GRE3 protein hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1, and exhibits xylose reductase activity. A DNA encoding a protein having the same; The xylose reductase activity will be described later.

  DNA encoding a mutant of GRE3 protein is cDNA live by a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc. using the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof as a probe. Libraries and genomic libraries. For the method for preparing the library, “Molecular Cloning, A Laboratory Manual 4th ed.” (Cold Spring Harbor Press (2012)) and the like can be referred to. Commercially available cDNA libraries and genomic libraries may also be used.

Here, the stringent conditions are, for example, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent conditions. Examples of the conditions include “1 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 50 ° C.”, and the like.
Hybridization can be performed by a known method. Hybridization methods include, 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. You can refer to it.

  In the present specification, the DNA hybridizing under stringent conditions includes, for example, at least 50% or more, preferably 70% or more, 80% or more, or 85% or more with the base sequence represented by SEQ ID NO: 1, More preferably 90% or more, 95% or more, 96% or more, 97% or more or 98% or more, more preferably 99% or more, still more preferably 99.7% or more, particularly preferably 99.9% identity (homology) DNA containing a base sequence having A value indicating identity can be calculated by using a known program such as BLAST.

  The DNA that hybridizes under stringent conditions with the DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 is, for example, one or several nucleic acids in the base sequence represented by SEQ ID NO: 1. DNA containing a nucleotide sequence in which mutation such as deletion, substitution or addition occurs. Examples of such DNA include (i) 1 to several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1) in the base sequence represented by SEQ ID NO: 1. Is a DNA in which 1 to 2 bases are deleted, (ii) 1 to several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 1) of the base sequence represented by SEQ ID NO: 1. DNA in which 3 bases, more preferably 1 to 2 bases are substituted with other bases, (iii) 1 to several (for example 1 to 10, preferably 1 to 1) in the base sequence represented by SEQ ID NO: 1 A DNA having 5 bases, more preferably 1 to 3 bases, more preferably 1 to 2 bases) and (iv) a DNA having a combination of these mutations, and having a protein having xylose reductase activity. Examples include DNA to be encoded.

  In the present invention, the base sequence can be confirmed by sequencing by a conventional method. For example, the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer.

  In the present invention, for example, GRE3 (Aldo keto reductase gene 3) derived from Saccharomyces cerevisiae includes those encoding a protein having the amino acid sequence represented by SEQ ID NO: 2. In the present invention, a gene encoding a GRE3 protein derived from Saccharomyces cerevisiae or a variant thereof is also included in GRE3 (Aldo keto reductase gene 3).

  The variant of GRE3 protein is (i) 1 to several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 2. (-2) a protein in which amino acids are deleted, (ii) 1 to several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 2 , More preferably 1 to 2 amino acids substituted with other amino acids, (iii) 1 to several (for example 1 to 10, preferably 1 to 5) in the amino acid sequence represented by SEQ ID NO: 2 , More preferably 1 to 3, and even more preferably 1 to 2 amino acids) and (iv) a protein in which these mutations are combined and a protein having xylose reductase activity. Is .

  Here, “xylose reductase activity” means an activity of converting xylose into xylitol in the presence of NAD + (or NADP +). In the present invention, the mutant of GRE3 protein is not particularly limited to the extent of its activity as long as it has xylose reductase activity. For example, it has an activity of about 10% or more of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. If you do. The xylose reductase activity of the protein can be measured by a known method.

  Yeast is known to have SOR1, SOR2, and YLR070c as genes encoding xylitol dehydrogenase. Therefore, in the present invention, SOR1, SOR2, or YLR070c can be used as a gene encoding xylitol dehydrogenase. In the present specification, SOR1 is described as an example of a gene encoding xylitol dehydrogenase. However, SOR2 and YLR070c can be used in the present invention in the same manner as described herein with respect to SOR1.

Those skilled in the art can obtain the nucleotide sequence information of SOR1, SOR2, and YLR070c from a known database such as Genbank. The accession numbers for the sequence information of each gene in Saccharomyces cerevisia are shown below.
SOR1: L11039, SOR2: Z74294, YLR070c: Z73242.

  In the present invention, SOR1 (sorbitol dehydrogenase gene 1) is a gene containing a base sequence encoding sorbitol dehydrogenase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 3 derived from Saccharomyces cerevisiae, or a sequence DNA encoding a protein consisting of the amino acid sequence shown by No. 4. It is known that the protein encoded by SOR1 also functions as xylitol dehydrogenase in yeast.

SOR1 used in the present invention includes a gene encoding a mutant of SOR1 protein. A gene encoding a mutant of SOR1 protein hybridizes under stringent conditions with, for example, a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 3 derived from Saccharomyces cerevisiae, and DNA encoding a protein having xylitol dehydrogenase activity is included.
The SOR1 used in the present invention may be a gene encoding a mutant of the following SOR1 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 4 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 4, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) A protein in which an amino acid (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) is substituted with another amino acid, (iii) represented by SEQ ID NO: 4 Proteins having 1 to several amino acids (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) amino acids added to the amino acid sequence, and (iv) their Tampa combined with mutation A quality and a protein having xylitol dehydrogenase activity.

  Here, “xylitol dehydrogenase activity” means the activity of dehydrogenating xylitol to xylulose. In the present invention, the mutant of SOR1 protein is not particularly limited as long as it has xylitol dehydrogenase activity. For example, the mutant of SOR1 protein has an activity of about 10% or more of the protein consisting of the amino acid sequence shown by SEQ ID NO: 4. It only has to have. The xylitol dehydrogenase activity of the protein can be measured by a known method.

  The above description can be similarly applied to the DNA contained in the DNA to be hybridized, the hybridization conditions, and the like. In the present invention, SOR1 can be obtained or manufactured by the same method as described for GRE3.

  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 a known database such as Genbank. For example, the accession number of XKS1 of Saccharomyces cerevisia is Z72979.

  In the present invention, XKS1 (xylulose phosphorylase gene 1) is a gene containing a base sequence encoding xylulose phosphorylase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 5 derived from Saccharomyces cerevisiae, Or it is DNA which codes the protein which consists of an amino acid sequence shown by sequence number 6.

XKS1 used in the present invention includes a gene encoding a mutant of XKS1 protein. A gene encoding a mutant of the XKS1 protein hybridizes under stringent conditions with, for example, a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 5 derived from Saccharomyces cerevisiae, and DNA encoding a protein having xylulose kinase activity is included.
XKS1 used in the present invention may be a gene encoding a variant of the following XKS1 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 6 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 6, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2). A protein in which an amino acid (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) is substituted with another amino acid, (iii) represented by SEQ ID NO: 6 Proteins having 1 to several amino acids (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) amino acids added to the amino acid sequence, and (iv) their Tampa combined with mutation A quality, and protein having xylulose kinase activity.

  Here, “xylulose kinase activity” means an activity of phosphorylating xylulose. In the present invention, the mutant of the XKS1 protein is not particularly limited as long as it has xylulose phosphatase activity. For example, the activity of about 10% or more of the protein consisting of the amino acid sequence represented by SEQ ID NO: 6 As long as it has. The xylulose kinase activity of the protein can be measured by a known method.

  The above description can be similarly applied to the DNA contained in the DNA to be hybridized, the hybridization conditions, and the like. In the present invention, XKS1 can also be obtained or produced by a method similar to that described for GRE3.

  In the present invention, the host yeast of the present invention can be obtained by inserting the three genes xREose utilization genes GRE3, SOR1 and XKS1 onto the yeast chromosome. By inserting the above xylose-utilizing endogenous gene into the yeast chromosome, the yeast's own xylose reductase, xylitol dehydrogenase and xylulose phosphorylase activities are improved as compared with those before gene introduction, It will be possible to grant assimilation ability.

  In the present invention, non-recombinant yeast is preferably employed as the host yeast. In producing non-recombinant yeast, it is necessary that the xylose-assimilating gene introduced on the yeast chromosome is a gene derived from the same species as the yeast. Moreover, the yeast into which the gene is introduced needs to be the same kind of yeast as the origin of the gene.

In the present invention, three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding silulose phosphatase, preferably three genes of GRE3, SOR1 and XKS1, are preferably of the same type. Insert into the yeast chromosome. Each of these genes may be inserted into the chromosome individually, or an expression cassette linked in tandem under the control of a promoter may be prepared and inserted into the chromosome. When connecting in tandem, the order of arrangement of the three genes is not particularly limited, and any conceivable combination may be used. Alternatively, a gene may be introduced onto the yeast chromosome using a plasmid containing three genes, GRE3, SOR1, and XKS1. The above three genes may be contained in one plasmid or in different plasmids. Moreover, the number of each gene inserted is not limited, and is one or more. The order of gene introduction into the chromosome is not particularly limited.
Three genes of xylose utilization genes are a gene in which a gene (XR) encoding xylose reductase and a gene (XDH) encoding xylitol dehydrogenase are linked in tandem, and a gene encoding xylitol dehydrogenase (XDH) and a gene encoding xylulose kinase (XK) may be inserted into a yeast chromosome as a gene linked in tandem. That is, the present invention relates to the above three types of xylose-assimilating genes, a gene in which a gene encoding xylose reductase and a gene encoding xylitol dehydrogenase are linked, and a gene encoding xylitol dehydrogenase and xylol It includes transformed yeast inserted on a chromosome as a gene linked to a gene encoding rosin kinase. For example, a xylose-assimilating gene can be introduced onto a yeast stain using a plasmid containing a GRE3-SOR1 fusion gene and a plasmid containing a SOR1-XKS1 fusion gene. The order of the arrangement of the genes in the fusion gene is not particularly limited. For example, the gene in which XR and XDH are linked may be either XR-XDH or XDH-XR. Similarly, the gene obtained by linking XDH and XK may be XDH-XK or XK-XDH. When the linked gene cassette is used, the expression level of the gene encoding xylitol dehydrogenase (SOR1) is particularly increased. Such a transformed yeast has particularly excellent xylose utilization ability and can efficiently produce ethanol from xylol.

  Further, the position of the chromosome into which the gene is inserted is preferably a site that does not function in yeast, and examples include, but are not limited to, the XYL2 site (Genbank accession number Z73242), the HXT13 site, and the HXT17 site. It is also possible to insert at a site on a chromosome that does not encode a gene. An example of a site on a chromosome that does not encode a gene is a δ sequence that is one of Ty factors. It is known that a plurality of (approximately 100 copies) δ sequences are present on the yeast chromosome. The position and sequence information of the δ sequence in the yeast chromosome are known (for example, Science 265, 2077 (1994)). For example, by introducing a plasmid having a xylose-assimilating gene inserted in the middle of the δ sequence into yeast, one or more copies of the gene can be inserted at a desired position on the chromosome. In addition to the δ sequence, it can also be inserted into the σ and τ sequences, which are also Ty factors. It can also be inserted into a ribosomal gene site such as NTS2.

A person skilled in the art can appropriately prepare a cassette or a plasmid for inserting a gene on a chromosome, select an insertion position on a chromosome, or insert into a chromosome (for example, lithium acetate method) based on a known method. Can be implemented. The present invention includes an expression cassette or plasmid in which three genes, GRE3, SOR1 and XKS1, are linked in tandem under the control of a promoter. Examples of such a plasmid include a plasmid in which GRE3 and SOR1 are linked under the control of a promoter (for example, PGK promoter), or a plasmid in which SOR1 and XKS1 are linked under the control of a promoter. Two genes are linked so that each gene can be expressed when inserted into a chromosome. If necessary when linking two genes and preparing a fusion gene, a linker sequence 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. By using these plasmids, three types of xylose utilization genes may be introduced onto the yeast chromosome.
Moreover, the plasmid used in the present invention is not particularly limited as long as it can introduce a gene into the yeast chromosome, and for example, a commercially available vector such as pUC18 can be used.
If yeast that does not have pentose utilization ability does not express all three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose kinase, Xylose availability is not provided. Therefore, a transformed yeast can be selected by culturing the yeast introduced with the gene as described above in a xylose-containing (ethanol-free) medium.

Thus, the host yeast in the present invention can be prepared by introducing three genes, GRE3, SOR1 and XKS1, onto the yeast chromosome.
Since the host yeast of the present invention preferably contains endogenous xylose utilization genes, that is, endogenous GRE3, SOR1 and XKS1 genes on the chromosome, the expression of GRE3, SOR1 and XKS1 can be activated. . Here, “expression of a xylose-utilizing gene is activated” means that the gene present in the host yeast is activated in an expressible form and can appropriately express the target protein. Means that. Moreover, in the host yeast of this invention, since the expression of a xylose utilization gene can be activated, xylose utilization ability can be acquired. Therefore, the host yeast of the present invention can be a yeast imparted with xylose utilization capability, preferably a brewery yeast imparted with xylose utilization capability.

(2) Transformed yeast of the present invention The transformed yeast of the present invention comprises at least one gene selected from a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase, preferably It is a yeast in which at least one gene selected from GDH2 (glutamate dehydrogenase gene 2) and the aforementioned SOR1 (sorbitol dehydrogenase gene 1) is introduced so as to allow expression.

  In the present invention, GDH2 (glutamate dehydrogenase gene 2) can be used as a gene encoding glutamate dehydrogenase. GDH2 is an enzyme that uses NAD as a coenzyme.

  Those skilled in the art can obtain GDH2 nucleotide sequence information from a known database such as Genbank. The accession number of GDH2 in Saccharomyces cerevisia is S66436.

  In the present invention, GDH2 (glutamate dehydrogenase gene 2) is a gene containing a base sequence encoding glutamate dehydrogenase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 7 derived from Saccharomyces cerevisiae, Or it is DNA which codes the protein which consists of an amino acid sequence shown by sequence number 8.

GDH2 used in the present invention includes a gene encoding a mutant of GDH2 protein. A gene encoding a mutant of the GDH2 protein hybridizes under stringent conditions with, for example, a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 7 derived from Saccharomyces cerevisiae, and DNA encoding a protein having glutamate dehydrogenase activity is included.
In addition, GDH2 used in the present invention may be a gene encoding a variant of the following GDH2 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 8 (Ii) 1 to several in the amino acid sequence represented by SEQ ID NO: 8, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2). A protein in which (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) amino acids are substituted with other amino acids, (iii) represented by SEQ ID NO: 8 Proteins having 1 to several amino acids (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, more preferably 1 to 2) amino acids added to the amino acid sequence, and (iv) their Tampa combined with mutation A quality and a protein having a glutamate dehydrogenase activity.

  Here, “glutamic acid dehydrogenase activity” means the activity of interconverting glutamic acid and α-ketoglutaric acid. In the present invention, the mutant of the GDH2 protein is not particularly limited as long as it has glutamate dehydrogenase activity. For example, the GDH2 protein mutant has an activity of about 10% or more of the protein consisting of the amino acid sequence represented by SEQ ID NO: 8. It only has to have. The glutamate dehydrogenase activity of the protein can be measured by a known method.

  The above description can be similarly applied to the DNA contained in the DNA to be hybridized, the hybridization conditions, and the like. In the present invention, GDH2 can be obtained or produced by a method similar to the above method.

  In the present invention, the aforementioned SOR2 or YLR070c having xylitol dehydrogenase activity can be used instead of SOR1 introduced into the host yeast. SOR2 and YLR070c apply the description herein regarding SOR1 and can be used in the present invention as well.

  The gene introduced into the host cell of the present invention is preferably a gene sequence endogenous to the host yeast, but may be an exogenous gene.

  In the present invention, SOR1 is introduced into a host yeast so that it can be expressed. Moreover, in this invention, GDH2 is introduce | transduced into host yeast so that expression is possible. Moreover, in this invention, both SOR1 and GDH2 are introduce | transduced into a host yeast so that expression is possible. The gene transfer can be performed using a plasmid containing the gene of interest in a form that can be expressed. When both SOR1 and GDH2 are introduced into host yeast, SOR1 and GDH2 may be contained in one plasmid or in different plasmids. The present invention includes plasmids containing these genes.

  The plasmid used in the present invention can be prepared by inserting the above gene into a yeast expression vector so that the gene can be expressed. The gene can be inserted into the vector using a ligase reaction, a topoisomerase reaction, or the like. For example, a method in which purified DNA is cleaved with an appropriate restriction enzyme, and the resulting DNA fragment is inserted into an appropriate restriction enzyme site or a multicloning site in the vector to be ligated to the vector, etc. it can.

  The plasmid used in the present invention is not particularly limited to the origin of the basic vector. For example, E. coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, and the like can be used. For example, commercially available vectors such as pGADT7 and pAUR135 can also be used.

  The plasmid of the present invention may contain a multicloning site, a promoter, an enhancer, a terminator, a selection marker cassette and the like as long as the target gene can be expressed. 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.

The promoter can be incorporated upstream of the gene of interest. The promoter is not particularly limited as long as it can appropriately express the target protein in the transformant, and PGK promoter, ADH promoter, TDH promoter, ENO promoter and the like can be used.
The terminator can be incorporated downstream of the target gene.
In the present invention, it is preferable to use a PGK promoter and / or PGK terminator in order to efficiently express a target gene in yeast.

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

  A transformed yeast can be produced by introducing the plasmid of the present invention into the host yeast of the present invention to be introduced.

  The method for introducing the plasmid of the present invention into the host yeast is not particularly limited, and examples thereof include known methods such as lithium acetate method, electroporation method, calcium phosphate method, lipofection method, DEAE dextran method and the like. . By these methods, the transformed yeast of the present invention is provided.

  The present invention also includes a transformed yeast in which GDH2 (glutamate dehydrogenase gene 2) and SOR1 (sorbitol dehydrogenase gene 1) are integrated on the host yeast chromosome by homologous recombination. When a gene is inserted into the host yeast chromosome, the insertion site is not particularly limited. Further, the method for inserting GDH2 and SOR1 into the host yeast chromosome so as to allow expression is not limited, and a method using a known gene recombination technique can be used.

  Furthermore, the transformed yeast of the present invention may be one in which the expression of GDH2 or SOR1 possessed by the host cell is activated. That is, the present invention also relates to a yeast having an increased expression level of GDH2 originally present on the host yeast chromosome, or a yeast having an increased expression level of SOR1 originally present on the host yeast chromosome or SOR1 introduced into the host yeast. Is included. GDH2 or SOR1 can be activated in an expressible form by introducing a promoter from the outside, or by replacing the promoter of the gene itself with a stronger promoter, and can appropriately express the target protein.

  The method for activating the expression of the endogenous gene in this way is not limited, but a method for incorporating a promoter capable of appropriately expressing the target protein into the chromosome by gene replacement using a known gene recombination technique, etc. Is mentioned. As a gene replacement method, 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 PGK promoter, ADH promoter, TDH promoter, ENO promoter, etc. can be used.

3. Ethanol Production Method The transformed yeast of the present invention is a host yeast to which xylose assimilation ability is imparted, at least one gene selected from a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase, such as SOR1 And / or GDH2 is introduced, the transformed yeast of the present invention has the ability to assimilate xylose. In addition, the transformed yeast of the present invention can produce ethanol from xylose.
Further, since the host yeast of the present invention is a yeast imparted with xylose utilization ability, it is possible to produce ethanol from xylose. In particular, a gene linking a gene encoding xylose reductase and a gene encoding xylitol dehydrogenase, and a gene linking a gene encoding xylitol dehydrogenase and a gene encoding xylulose phosphorylase. A transformed yeast in which both are introduced into the yeast chromosome is particularly suitable for producing ethanol from xylose. Hereinafter, the method for producing ethanol will be described by taking the transformed yeast of the present invention as an example, but the transformed yeast of the present invention having the ability to assimilate xylose can also be used in the ethanol production method.

  The transformed yeast of the present invention can be cultured according to a usual method used for yeast culture. A person skilled in the art can select an appropriate medium from known mediums such as SD medium, SCX medium, YPD medium, YPX medium, and cultivate yeast under preferable culture conditions. When culturing yeast in a liquid medium, shaking culture is preferred.

Ethanol can be produced by culturing the transformed yeast of the present invention and collecting ethanol from the resulting culture.
When ethanol is produced, the transformed yeast of the present invention is cultured in the presence of xylose at 10 to 150 g / L, preferably 70 g / L. Prior to the main culture, the transformed yeast may be precultured. The preculture may be performed, for example, by inoculating a small amount of the transformed yeast of the present invention and culturing for 12 to 24 hours. A preculture solution of 0.1 to 10%, preferably 1%, of the culture amount of the main culture is added to the main culture medium, and the main culture is started. The main culture is carried out in a xylose-containing medium for 0.5 to 200 hours, preferably 10 to 150 hours, more preferably 24 to 137 hours, and shaking culture at 20 to 40 ° C, preferably 30 ° C.

  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 solution (culture supernatant), cultured yeast, a disrupted culture yeast, or the like. Ethanol can be purified and collected from the culture by a known purification method. In the present invention, since ethanol is mainly secreted from the transformed yeast into the culture supernatant, it is preferably collected from the culture supernatant.

The amount of ethanol produced can be measured by analyzing ethanol contained in the medium with liquid chromatography, gas chromatography, or a commercially available ethanol measurement kit.
Moreover, the ethanol production ability of the transformed yeast of the present invention can be confirmed by measuring the production amount of ethanol.

  As the transformed yeast of the present invention, a transformed yeast obtained by introducing a gene encoding glutamate dehydrogenase into the host yeast of the present invention (for example, a GDH2 overexpression strain into which GDH2 has been introduced), or a glutamic acid dehydrated strain into the host yeast of the present invention. When ethanol is produced using a transformed yeast (for example, GDH2 and SOR1 overexpressing strains into which both GDH2 and SOR1 are introduced) into which both a gene encoding elementary enzyme and a gene encoding xylitol dehydrogenase are introduced, The production of by-products xylitol and glycerol is reduced compared to the control strain.

In addition, when a transformed yeast obtained by introducing a gene encoding xylitol dehydrogenase into the host yeast of the present invention (for example, a SOR1 overexpression strain into which SOR1 has been introduced) is used as the transformed yeast of the present invention, the presence of NAD + By culturing in a xylose-containing medium below, the production of by-products xylitol and glycerol can be reduced compared to the control strain. NAD + may be added to the xylose-containing medium so that the final concentration is 0.01 to 10,000 μM, preferably 0.1 to 1000 μM, more preferably 1 to 100 μM.
NAD + may be present in the culture system during the entire culture period, or may be present only during a certain period of culture. Therefore, for example, NAD + may be added to the culture system before the start of the culture, or may be added to the culture system after a predetermined time has elapsed since the start of the culture.

The amount of xylitol and glycerol produced can be measured by analyzing xylitol or glycerol contained in the medium by liquid chromatography, gas chromatography, or a commercially available measurement kit.
Moreover, by measuring the production amounts of xylitol and glycerol, the ability to suppress by-product production and the ethanol production efficiency of the transformed yeast of the present invention can be confirmed.

SEQ ID NO: 1 shows the base sequence of Saccharomyces cerevisiae GRE3.
SEQ ID NO: 2 This shows the amino acid sequence of Saccharomyces cerevisiae GRE3 protein.
SEQ ID NO: 3 This shows the base sequence of Saccharomyces cerevisiae SOR1.
SEQ ID NO: 4 This shows the amino acid sequence of Saccharomyces cerevisiae SOR1 protein.
SEQ ID NO: 5: This shows the base sequence of Saccharomyces cerevisiae XKS1.
SEQ ID NO: 6 This shows the amino acid sequence of Saccharomyces cerevisiae XKS1 protein.
SEQ ID NO: 7: This shows the base sequence of Saccharomyces cerevisiae GDH2.
SEQ ID NO: 8: This shows the amino acid sequence of Saccharomyces cerevisiae GDH2 protein.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. The yeasts used in the examples are all Saccharomyces cerevisiae.

[Enzyme activity measurement]
GRE3 (aldoketoreductase 3) xylose reductase activity, SOR1 (sorbitol dehydrogenase 1) xylitol dehydrogenase activity, and XKS1 (xylulose phosphate 1) xylulose phosphate enzyme activity are as follows. Measured with
The crude extract was prepared from yeast culture using Y-PER Protein Extraction Reagent (Pierce).
After preparing a reaction solution having the composition shown below, the mixture was allowed to stand at 30 ° C. for 5 minutes, and the absorbance was measured. The activity was measured by measuring the decrease in absorbance at 340 nm based on oxidation of NADH for GRE3 and XKS1, and by measuring the increase in absorbance at 340 nm based on reduction of NAD + for SOR1.
The composition of the reaction solution is as follows.
Aldoketo reductase (GRE3):
50 mM sodium phosphate buffer, pH 6.8
0.2mM NADPH
100 mM D-xylose
Crude extract sorbitol dehydrogenase (SOR1):
50 mM Tris-HCl pH 8.5
1mM NAD ⁺
5 mM MgCl ₂
100 mM xylitol
Crude extract xylulose kinase (XKS1):
20 mM sodium phosphate buffer, pH 6.8
100 mM KCl
5 mM MgCl ₂
5mM ATP
0.2mM NADH
1 mM phosphoenolpyruvate
5U / ml pyruvate kinase
7U / ml lactate dehydrogenase
5mM xylulose
Crude extract

1. Production of xylose-assimilating yeast Xylose-utilizing endogenous gene GRE3 (aldo-keto reductase gene: used as an alternative to xylose reductase gene), SOR1 (sorbitol dehydrogenase gene: xylitol dehydrogenase gene) And an expression cassette in which XKS1 (xylulose phosphorylase gene) was linked in tandem under the control of the PGK1 promoter. Using the prepared expression cassette, the above gene was introduced into the XYL2 site on the chromosome to obtain a yeast for brewing with xylose utilization ability.

2. Production of overexpression strain SOR1 or GDH2 (glutamate dehydrogenase gene) placed under the control of the PGK1 promoter was incorporated into a commercially available expression vector pAUR135 (Takara Bio Inc.). Using the obtained recombinant vector, the above 1. SOR1 or GDH2 was introduced into the AUR1 site on the chromosome of the yeast for brewing with xylose-assimilating ability prepared in step 1. The obtained strains were designated as an SOR1 overexpression strain and a GDH2 overexpression strain, respectively. In addition, a strain in which only the vector was incorporated was similarly prepared and used as a control strain in the following experiment.

3. Evaluation of fermentability 2. The strain prepared in (1) was pre-cultured with YPD (containing 2% glucose) and then modified CBS medium containing 5% xylose (Non-patent Document HB Klinke et al., Biotechnol. Bioeng., 81, 738 (2003): pH 5.0) In a 50 ml Erlenmeyer flask containing 15 ml, the initial inoculation amount OD ₆₀₀ = 20, 30 ° C., swirling culture at 140 rpm, sampling over time, and fermentability was evaluated. Fermentation metabolites were quantified by HPLC using a Shodex SUGAR SP0810 column. The bacterial cell concentration was measured with a spectrophotometer (λ = 600 nm).

4). Fermentability evaluation results Table 1 shows the results. The SOR1 overexpressing strain did not change the xylose consumption rate compared to the control strain, and the ethanol production was improved, while the production of by-products xylitol and glycerol was slightly decreased. The GDH2-overexpressing strain had reduced production of by-products xylitol and glycerol and improved ethanol production.

1. Preparation of Chromosome Transfer Vector A commercially available vector pUC18 was cleaved with EcoRI and KpnI, and the first half part (1-240 bp) amplified from the yeast (Association No. 7) chromosome was introduced. The obtained vector was cleaved with PstI and SphI, and the latter half of the δ sequence (241 to 334 bp) also amplified from yeast (Association No. 7) was introduced to obtain vector pUC-δ. pUC-δ was cleaved with SmaI, and GRE3-SOR1 and XKS1-SOR1 fragments in which two genes were linked in tandem under the control of the PGK1 promoter were introduced. A SOR1 vector was obtained. The GRE3, SOR1 and XKS1 genes are derived from Association No. 7.

2. Production of xylose-assimilating yeast The pUC-δ-GRE3-SOR1 and pUC-δ-XKS1-SOR1 vectors obtained in 1 above were digested with StuI to form a linear fragment, mixed in equal amounts, and then mixed with the lithium acetate method for the S-2 yeast strain S-2 Introduced. The yeast culture solution into which the gene fragment has been introduced is applied to an SD-xylose agar plate containing amino acids (xylose 5%), incubated at 30 ° C. for 7 to 10 days, and the chromosome integration strain (improved strains A to G) is obtained. Obtained. The transformed strain was cultured in SC-X (xylose 5%) liquid medium (2 ml / 15 ml culture tube, 140 rpm, 30 ° C.), and the culture of the strain with good growth was evaluated.
In addition, as a control strain, GRE3-SOR1-XKS1 linked in tandem under the control of the PGK1 promoter was introduced into the XYL2 site to prepare a xylose-assimilating yeast (XYL2 strain). This yeast (XYL2 strain) was also evaluated for culture.

3. Evaluation of fermentability 2. The strain prepared in (1) was precultured with YPD (containing 2% glucose), and then in a 50 mL Erlenmeyer flask containing 15 mL of the medium having the composition shown in Table 2, the initial inoculum OD ₆₀₀ = 20, 30 ° C, 140 Rotating culture was performed at rpm, sampling was performed over time, and fermentability was evaluated. Fermentation metabolites were quantified by HPLC using a Shodex SUGAR SP0810 column. The bacterial cell concentration was measured with a spectrophotometer.

4). The results of fermentability evaluation are shown in Table 3. Improved strains A to G prepared by the above method showed high ethanol yield. Improved strains A to G showed improved ethanol production compared to xylose-assimilating yeast (XYL2 strain) produced by introducing GRE3-SOR1-XKS1 into XYL2 site in tandem under the control of PGK1 promoter. .

The association No. 7 yeast was tested in the same manner as in Example 2.
Example 2 1. Using each gene of GRE3, SOR1 and XKS1 obtained from Association No. 7 yeast The pUC-δ-GRE3-SOR1 vector and the pUC-δ-XKS1-SOR1 vector were obtained by the same method as described above. Example 2 using the obtained pUC-δ-GRE3-SOR1 and pUC-δ-XKS1-SOR1 vectors and yeast (Association No. 7) In the same manner as above, xylose-assimilating yeast was prepared (improved strains HM).

  2. Fermentability evaluation was performed on the improved strains H to M in Example 2. The same method was used. SOR1 overexpressing strain (Association No. 7) prepared by the same method as in Example 1, and XYL2 strain prepared by introducing GRE3-SOR1-XKS1 tandemly linked under the control of PGK1 promoter into the XYL2 site as a control strain ( Association No. 7) was also evaluated for fermentability.

  The results are shown in Table 4. The SOR1 overexpressing strain and the improved strains HM showed higher ethanol yields than the control strain. In particular, the improved strains H to M showed a better improvement in ethanol production.

According to the present invention, a transformed yeast capable of producing ethanol from xylose is provided.
In one embodiment of the present invention, the transformed yeast of the present invention is not a genetic recombinant because it is transformed using the gene of the yeast itself, and is preferable in terms of safety and ease of handling. .
Moreover, since the transformed yeast of the present invention has an excellent ability to produce ethanol from xylose, ethanol can be efficiently produced from xylose by using the transformed yeast of the present invention.
In addition, since the transformed yeast of the present invention showed a reduction in the production of by-products such as xylitol and glycerol, the by-products accumulated when ethanol was produced using the transformed yeast of the present invention. The problem of doing can be avoided.

Claims (15)

A transformed yeast in which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose kinase are inserted on the chromosome,
The three genes link a gene encoding xylose reductase and a gene encoding xylitol dehydrogenase, and a gene encoding xylitol dehydrogenase and a gene encoding xylulose phosphorylase. The yeast, which has been inserted into the chromosome as a modified gene.   The yeast according to claim 1, wherein the gene is an endogenous gene of the yeast.   The yeast according to claim 1 or 2, wherein the gene encoding xylose reductase is a gene selected from the group consisting of GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w.   The yeast according to any one of claims 1 to 3, wherein the gene encoding xylitol dehydrogenase is a gene selected from the group consisting of SOR1, SOR2, and YLR070c.   The gene encoding xylose reductase, the gene encoding xylitol dehydrogenase, and the gene encoding xylulose phosphorylase are GRE3, SOR1, and XKS1, respectively. yeast.   A gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase into a host yeast into which a gene encoding glutamate dehydrogenase and xylitol are inserted into a chromosome. A transformed yeast into which at least one gene selected from a gene encoding a dehydrogenase is introduced so as to allow expression.   The yeast according to claim 6, wherein the gene is derived from a host yeast.   8. The gene according to claim 6 or 7, wherein at least one gene selected from the gene encoding glutamate dehydrogenase and the gene encoding xylitol dehydrogenase has been inserted so as to be expressed on the chromosome of the host yeast. Yeast.   The at least 1 gene chosen from GDH2 and SOR1 was introduced so that expression was possible in the host yeast which inserted three genes, GRE3, SOR1, and XKS1, on a chromosome, The statement of any one of Claims 6-8 Transformed yeast.   The yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted, belongs to the genus Saccharomyces. The yeast of any one of these.   The yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted, is Saccharomyces cerevisiae. The yeast according to claim 1.   The yeast according to any one of claims 1 to 11, which has an ability to produce ethanol from xylose.   A method for producing ethanol, comprising culturing the transformed yeast according to any one of claims 1 to 12 in a xylose-containing medium and collecting ethanol from the resulting culture.   At least one gene selected from a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase, which is introduced into the transformed yeast according to any one of claims 6 to 12, is glutamate dehydrogenase. 14. The method according to claim 13, which is a gene encoding, or both a gene encoding glutamate dehydrogenase and a gene encoding xylitol dehydrogenase.   The at least one gene selected from the gene encoding glutamate dehydrogenase and the gene encoding xylitol dehydrogenase introduced into the transformed yeast according to any one of claims 6 to 12, wherein xylitol dehydrogenase is The method according to claim 13, wherein the gene is an encoding gene, and the culturing step is performed in the presence of NAD +.