WO2016060171A1

WO2016060171A1 - Yeast capable of producing ethanol from xylose

Info

Publication number: WO2016060171A1
Application number: PCT/JP2015/079070
Authority: WO
Inventors: 仁小西; 梢栄牟田口; 福田　明; 上村　毅
Original assignee: Ｊｘ日鉱日石エネルギー株式会社
Priority date: 2014-10-15
Filing date: 2015-10-14
Publication date: 2016-04-21
Also published as: JPWO2016060171A1; AU2015331353A1; JP6616311B2; AU2015331353B2; BR112017005959A2

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 producing ethanol from xylose

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

In recent years, the use of bioalcohol has attracted attention as an alternative fuel for petroleum from the viewpoint of reducing CO ₂ emissions. In particular, bioalcohol using vegetation as a resource has been actively studied. Since about one-third of the sugar obtained from cellulosic biomass such as plants is occupied by xylose, various researches have been conducted on xylose-assimilating yeast used in the production of bioalcohol.

For the production of bioethanol, yeasts represented by Saccharomyces cerevisiae are mainly used. 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.

As a yeast having xylose utilization ability, Schiffosomyces stipitis is known. Saccharomyces cerevisiae has a gene group corresponding to the gene group for assimilating xylose possessed by Syphazomyces stipitis, 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.

So far, production of yeast imparted with xylose utilization ability by activating a xylose utilization gene endogenous to Saccharomyces cerevisiae has been studied (WO2010 / 001906 (Patent Document 1), WO2014 / 058034). No. (Patent Document 2)). Yeasts produced using these techniques have the advantage that they are not genetic recombinants because they utilize xylose-utilizing genes derived from the yeasts themselves.

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

WO2010 / 001906 WO2014 / 058034

An object of the present invention is to provide a yeast having an improved ability to produce ethanol from xylose. The present invention is a yeast that has further improved ethanol-producing ability over conventional xylose-utilizing yeast using a gene encoding a xylose-degrading enzyme, preferably by using a gene derived from the yeast itself. It aims at providing the transformed yeast which has ethanol productivity.

As a result of intensive studies to solve the above problems, the present inventors have found that there are three genes: a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, and a gene encoding xylitol dehydrogenase. Introducing a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase into conventional xylose-assimilating yeast introduced with the gene enhances the ability to produce ethanol from xylose. It was found that the obtained yeast was obtained. That is, the present inventor has found that yeast introduced with the combination of the above six genes can efficiently produce ethanol from xylose. And it discovered that the yeast obtained in this way had high xylose utilization ability compared with the conventional xylose utilization yeast, and it could improve the production efficiency of ethanol from xylose, and completed this invention.

That is, the present invention relates to the following.
[1] A gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and alcohol dehydrogenase A transformed yeast into which a gene to be encoded has been introduced so that it can be expressed.
[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 is inserted into a chromosome of a host yeast so that the gene can be expressed.
[4] The yeast according to any one of [1] to [3], wherein the gene encoding xylose reductase is GRE3.
[5] The yeast according to any one of [1] to [4], wherein the gene encoding xylulose kinase is XKS1.
[6] The yeast according to any one of [1] to [5], wherein the gene encoding xylitol dehydrogenase is SOR1.
[7] The yeast according to any one of [1] to [6], wherein the gene encoding transaldolase is TAL1.
[8] The yeast according to any one of [1] to [7], wherein the gene encoding transketolase is TKL1.
[9] The yeast according to any one of [1] to [8], wherein the gene encoding alcohol dehydrogenase is ADH1.
[10] The yeast according to any one of [1] to [9], which has an ability to produce ethanol from xylose.
[11] The yeast according to any one of [1] to [10], wherein the host yeast has hexose assimilability but not pentose assimilability.
[12] The yeast according to any one of [1] to [11], wherein the host yeast is a yeast belonging to the genus Saccharomyces.
[13] The yeast according to any one of [1] to [12], wherein the host yeast is a yeast belonging to the species Saccharomyces cerevisiae.
[14] A method for producing ethanol, comprising culturing the transformed yeast according to any one of [1] to [13] in a xylose-containing medium and collecting ethanol from the resulting culture.

According to the present invention, a gene encoding xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and alcohol dehydrogenase A transformed yeast into which a gene encoding is introduced is provided.
In one embodiment of the present invention, the transformed yeast of the present invention is produced by transforming with a gene possessed by the yeast itself. For this reason, the transformed yeast of the present invention does not correspond to a gene recombinant, and is preferable in terms of safety and ease of handling.
In addition, the transformed yeast of the present invention can efficiently produce ethanol from xylose. Therefore, the present invention provides a method for producing ethanol from xylose and a transformed yeast capable of producing ethanol from xylose that can be used in the method.

It is the figure which showed the ethanol yield of the transformed yeast (shochu yeast) in the culture medium which contains xylose as a substrate. The black color represents the result of the xylose-assimilating yeast obtained in Example 2, the gray color represents the result of the TAL1 · TKL1 gene overexpression strain obtained in Example 3, and the white color was obtained in Example 4. The result of the TAL1 / TKL1 / ADH1 gene overexpression strain is shown. It is the figure which showed the ethanol yield of the transformed yeast (shochu yeast) in the culture medium which contains glucose and ethanol as a substrate. The black color represents the result of the xylose-assimilating yeast obtained in Example 2, the gray color represents the result of the TAL1 · TKL1 gene overexpression strain obtained in Example 3, and the white color obtained in Example 4. The results of TAL1, TKL1, and ADH1 gene overexpression strains are shown. It is the figure which showed the ethanol yield of the transformed yeast (shochu yeast) in the culture medium which contains glucose and ethanol as a substrate. Gray represents the result of the TAL1 · TKL1 gene overexpression strain obtained in Example 3, and white represents the result of the TAL1 · TKL1 · ADH1 gene overexpression strain obtained in Example 4. It is a figure which shows the result of having analyzed the expression level of ADH1 gene. Gray represents the result of the TAL1 · TKL1 gene overexpression strain obtained in Example 3, and white represents the result of the TAL1 · TKL1 · ADH1 gene overexpression strain obtained in Example 4. It is a figure which shows the result of having measured the ADH1 activity of yeast. Gray represents the result of the TAL1 · TKL1 gene overexpression strain obtained in Example 3, and white represents the result of the TAL1 · TKL1 · ADH1 gene overexpression strain obtained in Example 4.

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.

1. SUMMARY OF THE INVENTION The present invention relates to genes relating to six enzymes relating to xylose utilization and the pentose phosphate pathway and ethanol production pathway, that is, genes encoding xylose reductase, genes encoding xylulose kinase, xylitol dehydration A transformed yeast into which a gene encoding an elementary enzyme, a gene encoding a transaldolase, a gene encoding a transketolase, and a gene encoding an alcohol dehydrogenase have been introduced so that they can be expressed has excellent ethanol production ability Based on knowledge.

The transformed yeast of the present invention comprises a xylose utilization gene such as a gene encoding xylose reductase, a gene encoding xylulose phosphorylase and a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, It can be prepared by introducing a gene encoding a ketolase and a gene encoding an alcohol dehydrogenase in an expressible manner. Alternatively, the transformed yeast of the present invention may comprise a gene encoding a xylose reductase, a gene encoding xylulose kinase, a gene encoding xylitol dehydrogenase, a trans It can also be produced by increasing the expression level of a gene encoding an aldolase, a gene encoding a transketolase and / or a gene encoding an alcohol dehydrogenase.

In the present specification, the “xylose utilization gene” is a gene encoding an enzyme 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 the present invention, the gene encoding xylitol dehydrogenase is preferably a gene encoding sorbitol dehydrogenase.

In this specification, at least three genes, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase, are also introduced into the host yeast. These three genes are genes encoding enzymes related to the pentose phosphate pathway and the ethanol production pathway.

In another embodiment of the present invention, these genes introduced into the host yeast are genes derived from the host yeast. That is, in another embodiment of the present invention, one of the features is to activate the expression of an enzyme gene inherent in yeast (endogenous) and enhance the activity of the enzyme possessed by the yeast itself.

In another aspect of the present invention, one of the characteristics of these genes introduced into the host yeast is that they are introduced into the host yeast chromosome.

In another embodiment of the present invention, the transformed yeast introduced with the genes for the above six enzymes has an excellent productivity from xylose to ethanol.

Some yeasts do not have pentose assimilation ability such as xylose because they are in a so-called dormant state in which the xylose assimilating 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.

By introducing the xylose-utilizing gene derived from itself or replacing the promoter into yeast that does not have such ethanol-producing ability, the activity of the xylose-utilizing endogenous gene is increased and the xylose-utilizing ability is increased. Can be granted. In addition to yeast-derived xylose utilization genes, yeast-derived enzyme genes related to the pentose phosphate pathway and ethanol production pathway (transaldolase-encoding gene, transketolase-encoding gene, and alcohol dehydrogenase) When the activity of these genes is also increased by introducing the encoding genes) or replacing the promoters of these genes, the efficiency of ethanol production from xylose is further improved. Therefore, in the transformed yeast of the present invention, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase were introduced into yeast in which a xylose utilization gene was inserted on the chromosome. It may be yeast. In addition, according to the present invention, ethanol can be efficiently produced using xylose in a yeast that does not have ethanol-producing ability. In addition, the present invention can be effectively applied to yeasts that do not have ethanol-producing ability and yeasts that want to improve ethanol-producing ability.

Moreover, since the transformed yeast of the present invention has an excellent ability to produce ethanol from xylose, the present invention cultivates the transformed yeast and produces ethanol by collecting ethanol from the resulting culture. A method is also provided.

2. Transformed yeast of the present invention The transformed yeast of the present invention comprises a xylose utilization gene such as a gene encoding xylose reductase, a gene encoding xylulose phosphorylase and a gene encoding xylitol dehydrogenase, and a transaldolase. , A gene encoding a transketolase, and a gene encoding an alcohol dehydrogenase have been introduced so as to allow expression.

(1) Host Yeast In the present invention, it is preferable that the yeast to be subjected to gene transfer or transformation is a yeast that does not have the ability to assimilate pentoses such as xylose. 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.

Alternatively, in the present invention, the yeast to be subjected to gene transfer or transformation can be selected for the purpose of improving ethanol productivity. Examples of such yeasts include yeasts imparted with xylose utilization ability and yeasts with activated xylose utilization ability.

In the present invention, the target yeast for gene transfer or transformation is not particularly limited, and examples thereof include yeast belonging to the genus Saccharomyces. Examples of yeast belonging to the genus Saccharomyces include Saccharomyces cerevisia species such as laboratory yeast strains. In the present invention, the yeast to be the target of gene transfer or transformation can be not only haploid but also diploid yeast. The diploid yeast is excellent as a practical yeast, and examples thereof include brewing yeast such as baker's yeast, sake yeast, shochu yeast, and wine yeast.

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.

(2) Transgene In the present invention, the xylose utilization gene is at least three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose kinase. The xylitol dehydrogenase is preferably sorbitol dehydrogenase. More specifically, the gene encoding xylose reductase, the gene encoding xylitol dehydrogenase, and the gene encoding xylulose phosphorylase are respectively GRE3 (Aldo keto reductase gene 3) and SOR1 (sorbitol). Dehydrogenase gene 1) and XKS1 (xylulose kinase gene 1) are preferred.

In this specification, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are also introduced into yeast. More specifically, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase are TAL1 (transaldolase gene 1), TKL1 (transketolase gene 1) and ADH1 (alcohol dehydrogenase gene 1) is preferably introduced into the yeast.

In the present invention, the xylose utilization gene, the gene encoding transaldolase, the gene encoding transketolase, and the gene encoding alcohol dehydrogenase can be any foreign gene or endogenous gene. It is preferably a sex gene. “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.

(Gene encoding xylose reductase)
GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w are known as genes encoding xylose reductase in yeast. Therefore, in the present invention, 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: 21 derived from Saccharomyces cerevisiae, Or it is DNA which codes the protein which consists of an amino acid sequence shown by sequence number 22. It is known that the protein encoded by GRE3 also functions as a xylose reductase in yeast. The GRE3 protein is a protein having amino acid sequence identity (homology) with XYL1 (xylose reductase (XR)) of Shephasomyces staphytis.

In the present invention, GRE3 can be obtained from a yeast library or a genomic library by gene amplification technology by designing a primer based on the base sequence represented by SEQ ID NO: 21, 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: 21, 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 obtained by 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: 21 or a fragment thereof as a probe. Libraries and genomic libraries. Regarding the method for preparing the library, “Molecular Cloning, A Laboratory Manual 4th ed.” (Cold Spring Spring 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: 21, 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 shown in SEQ ID NO: 21 is, for example, one or several nucleic acids in the base sequence shown in SEQ ID NO: 21 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 nucleotide sequence represented by SEQ ID NO: 21 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: 21 DNA in which 3 bases, more preferably 1 to 2 bases are substituted with other bases, (iii) 1 to several (eg 1 to 10, preferably 1 to 3) in the base sequence represented by SEQ ID NO: 21 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 xylose reductase activity. Examples include DNA to be encoded.

In the present invention, the nucleotide 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 cerevisia includes a protein encoding a protein having the amino acid sequence represented by SEQ ID NO: 22. In the present invention, a gene encoding a GRE3 protein derived from Saccharomyces cerevisiae or a mutant thereof is also included in GRE3 (Aldo keto reductase gene 3).

The GRE3 protein mutant has (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: 22. (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: 22 Further, a protein in which 1 to 2 amino acids are 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: 22 More preferably 1 to 3, more preferably 1 to 2 amino acids) and (iv) a protein having a combination of these mutations and having xylose reductase activity And the like.

Here, “xylose reductase activity” means the activity of converting xylose to xylitol in the presence of NAD + (or NADP +). In the present invention, a mutant of GRE3 protein is not particularly limited in its activity as long as it has xylose reductase activity. For example, the GRE3 protein variant has an activity of about 10% or more of a protein consisting of the amino acid sequence represented by SEQ ID NO: 22. If you do. The xylose reductase activity of the protein can be measured by a known method.

(Gene encoding xylitol dehydrogenase)
SOR1, SOR2, and YLR070c are known as genes encoding xylitol dehydrogenase of yeast. Therefore, in the present invention, SOR1, SOR2, or YLR070c can be used as a gene encoding xylitol dehydrogenase, and SOR1 is preferred. In the present specification, SOR1 is described as an example of a gene encoding xylitol dehydrogenase, but SOR2 and YLR070c can apply the description in this specification regarding SOR1. SOR1 and SOR2 have 99.9% identity in gene sequence.

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 including a base sequence encoding sorbitol dehydrogenase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 23 derived from Saccharomyces cerevisiae, or a sequence DNA encoding a protein consisting of the amino acid sequence shown by No. 24. It is known that the protein encoded by SOR1 also functions as xylitol dehydrogenase in yeast. The SOR1 protein is a protein having amino acid sequence identity (homology) (53%) with XYL2 (Xylitol dehydrogenase (XDH)) of Shephasomyces staphytis.

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: 23 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: 24 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 24, 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) is represented by SEQ ID NO: 24 A protein to which 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 are added in the amino acid sequence, and (iv) Mutation combined A protein, 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 a protein consisting of the amino acid sequence represented by SEQ ID NO: 24. 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 hybridizing DNA, the hybridization conditions, and the like. In the present invention, SOR1 can be obtained or manufactured by the same method as described for GRE3.

(Gene encoding xylulose kinase)
In the present invention, 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 comprising a base sequence encoding xylulose phosphorylase, for example, a DNA comprising the base sequence represented by SEQ ID NO: 25 derived from Saccharomyces cerevisiae, Or it is DNA which codes the protein which consists of an amino acid sequence shown by sequence number 26.

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: 25 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: 26 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 26, 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) is represented by SEQ ID NO: 26 A protein to which 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 are added in the amino acid sequence, and (iv) Mutation combined A protein, and a 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: 26 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 hybridizing DNA, 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.

(Gene encoding transaldolase)
In the present invention, TAL1 (transaldolase gene 1) or TAL2 (transaldolase gene 2) can be used as a gene encoding transaldolase. Those skilled in the art can obtain the base sequence information of TAL1 and TAL2 from a known database such as Genbank. For example, Saccharomyces cerevisiae TAL1 and TAL2 accession numbers are X15953 and X59720, respectively. In the present specification, TAL1 is described as an example of a gene encoding transaldolase, but the description in the present specification regarding TAL1 can be similarly applied to TAL2.

In the present invention, TAL1 (transaldolase gene 1) is a gene containing a base sequence encoding transaldolase. For example, DNA comprising the base sequence represented by SEQ ID NO: 27 derived from Saccharomyces cerevisiae, or represented by SEQ ID NO: 28 DNA encoding a protein consisting of an amino acid sequence.

TAL1 used in the present invention includes a gene encoding a mutant of the TAL1 protein. A gene encoding a mutant of TAL1 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: 27 derived from Saccharomyces cerevisiae, and DNA encoding a protein having transaldolase activity is included.

TAL1 used in the present invention may be a gene encoding the following mutant of TAL1 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 28 (Ii) 1 to several amino acids in the amino acid sequence shown in SEQ ID NO: 28, 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) is represented by SEQ ID NO: 28 A protein to which 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 are added in the amino acid sequence, and (iv) Mutation combined A protein, and a protein having transaldolase activity.

Here, “transaldolase activity” means an activity that catalyzes the reaction of cedoheptulose-7-phosphate + glyceraldehyde-3-phosphate-erythrose-4-phosphate + fructose-6-phosphate. In the present invention, a mutant of the TAL1 protein is not particularly limited to the extent of its activity as long as it has transaldolase activity. It only has to be. The transaldolase activity of the protein can be measured by a known method.

The above description can be similarly applied to the DNA contained in the hybridizing DNA, the hybridization conditions, and the like. In the present invention, TAL1 can be obtained or produced by a method similar to that described for GRE3.

(Gene encoding transketolase)
In the present invention, TKL1 (transketolase gene 1) or TKL2 (transketolase gene 2) can be used as a gene encoding transketolase. The base sequence information of TKL1 and TKL2 can be obtained from a known database such as Genbank by those skilled in the art. For example, the accession numbers of Saccharomyces cerevisiae TKL1 and TKL2 are X73224 and X73532, respectively. In the present specification, TKL1 is described as an example of a gene encoding a transketolase, but the description in this specification regarding TKL1 can be similarly applied to TKL2.

In the present invention, TKL1 (transketolase gene 1) is a gene including a base sequence encoding transketolase. For example, DNA comprising the base sequence represented by SEQ ID NO: 29 derived from Saccharomyces cerevisiae, or SEQ ID NO: 30 DNA encoding a protein consisting of the amino acid sequence represented by

TKL1 used in the present invention includes a gene encoding a mutant of TKL1 protein. The gene encoding the mutant of TKL1 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: 29 derived from Saccharomyces cerevisiae, and DNA encoding a protein having transketolase activity is included.

Further, TKL1 used in the present invention may be a gene encoding a variant of the following TKL1 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 30 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 30, 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) shown in SEQ ID NO: 30 A protein to which 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 are added in the amino acid sequence, and (iv) Mutation combined A protein, and a protein having transketolase activity.

Here, “transketolase activity” means an activity that catalyzes the reaction of sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate xylulose-5-phosphate + ribose-5-phosphate. In the present invention, the mutant of TKL1 protein is not particularly limited as long as it has transketolase activity, but has, for example, an activity of about 10% or more of the protein consisting of the amino acid sequence shown by SEQ ID NO: 30. If you do. The transketolase activity possessed by the protein can be measured by a known method.

The above description can be similarly applied to the DNA contained in the hybridizing DNA, the hybridization conditions, and the like. In the present invention, TKL1 can also be obtained or produced by a method similar to that described for GRE3.

(Gene encoding alcohol dehydrogenase)
In the present invention, ADH1 (alcohol dehydrogenase gene 1) can be used as a gene encoding alcohol dehydrogenase. A person skilled in the art can obtain the base sequence information of ADH1 from a known database such as Genbank. For example, the accession number of Saccharomyces cerevisia ADH1 is X83121.

In the present invention, ADH1 (alcohol dehydrogenase gene 1) is a gene containing a base sequence encoding alcohol dehydrogenase. For example, DNA comprising the base sequence represented by SEQ ID NO: 31 derived from Saccharomyces cerevisiae, or a sequence This is a DNA encoding a protein consisting of the amino acid sequence shown by No. 32.

ADH1 used in the present invention includes a gene encoding a mutant of ADH1 protein. A gene encoding a mutant of ADH1 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: 31 derived from Saccharomyces cerevisiae, and DNA encoding a protein having alcohol dehydrogenase activity is included.

In addition, ADH1 used in the present invention may be a gene encoding a variant of the following ADH1 protein: (i) 1 to several (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 32 (Ii) 1 to several amino acids in the amino acid sequence represented by SEQ ID NO: 32, 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) SEQ ID NO: 32 A protein to which 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 are added in the amino acid sequence, and (iv) Mutation combined A protein, and a protein having an alcohol dehydrogenase activity.

Here, “alcohol dehydrogenase activity” means the activity of oxidizing alcohol to aldehyde. In the present invention, the ADH1 protein mutant is not particularly limited in its activity as long as it has alcohol dehydrogenase activity. It only has to have. The alcohol 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 hybridizing DNA, the hybridization conditions, and the like. In the present invention, ADH1 can be obtained or produced by a method similar to the method described for GRE3.

In the present invention, by introducing the above six genes into the same host yeast as the origin of the gene, it is expected that the enzyme activity of the yeast itself is improved more than before the gene introduction. For example, as shown in the Examples, the alcohol dehydrogenase activity is increased in the yeast of the present invention. Moreover, when producing non-recombinant yeast, it is necessary that the six genes introduced into the host yeast are endogenous genes.

(3) Introduction of gene into yeast In the present invention, three genes, a xylose utilization gene (for example, 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 so that they can be expressed in a host yeast, the transformed yeast of the present invention can be produced. . Moreover, the transformed yeast of the present invention can also be produced by replacing the promoters of these genes with promoters that increase the expression level of the genes.

The transformed yeast of the present invention is preferably a yeast into which GRE3, SOR1, XKS1, TAL1, TKL1, and ADH1 have been introduced so that they can be expressed. In the present specification, the expression “being expressible (introduction or insertion)” means that the introduced or inserted gene is introduced or inserted in such a form that it can be expressed in a transformant under a predetermined condition.

In the present invention, a gene encoding xylose reductase, a gene encoding xylulose kinase, and a gene encoding xylitol dehydrogenase are preferably inserted so as to be expressible on the yeast chromosome. In the present invention, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase are preferably inserted in a yeast chromosome so that they can be expressed. That is, in the present invention, introduction of a gene into yeast includes insertion of the gene onto the yeast chromosome. The transformed yeast of the present invention is preferably a gene encoding xylose reductase, a gene encoding xylulose kinase, xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and alcohol A yeast into which a gene encoding a dehydrogenase is inserted so as to be expressed on a chromosome is more preferable, and a yeast into which GRE3, SOR1, XKS1, TAL1, TKL1, and ADH1 are inserted so as to be expressed on a chromosome is more preferable.

In the present invention, the number of each gene inserted into a chromosome is not limited and is one or more. Moreover, the order of each gene inserted in a chromosome is not specifically limited. Further, the position of the chromosome into which the gene is inserted is not particularly limited, but a site that does not function in yeast is preferable. For example, an XYL2 site (Genbank accession number Z73242), HXT13 site, HXT17 site, AUR1 site, etc. Can be mentioned. 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.

When inserting genes into chromosomes, each gene may be inserted into the chromosome individually, or an expression cassette in which two or more genes are linked in tandem under the control of a promoter is created and inserted into the chromosome. May be. In the case of tandem linkage, the order of gene arrangement and the number of genes to be linked are not particularly limited, and any conceivable combination may be used.

A plasmid can be used for introduction of a gene into yeast. The plasmid may contain one or more of the enzyme genes used in the present invention. For example, one plasmid can contain three genes: a gene encoding xylose reductase, a gene encoding xylulose kinase, and a gene encoding xylitol dehydrogenase. Further, for example, three genes, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase, can be contained in one plasmid. When a plurality of genes are included in one plasmid, the order and number of each gene on the plasmid are not particularly limited. The present invention includes plasmids or expression cassettes containing these genes. Examples of such a plasmid or expression cassette include a plasmid or expression cassette in which GRE3, SOR1 and XLS1 are linked under the control of a promoter, or a plasmid or expression cassette in which TAL1 and TKL are linked under the control of a promoter. A plurality of genes are linked so that each gene can be expressed when introduced into yeast, particularly 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 or expression cassettes, three types of xylose utilization genes and / or genes encoding transaldolase, genes encoding transketolase and / or genes encoding alcohol dehydrogenase can be transformed into yeast chromosomes. It may be introduced above.

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, and for example, a plasmid derived from E. coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like can be used. For example, commercially available vectors such as pGADT7 and pAUR135 can also be used. When the transformed yeast of the present invention is prepared using a plasmid derived from a host yeast, the transformed yeast of the present invention does not correspond to a genetic recombinant.

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.

Promoter can be incorporated upstream of target gene. The promoter is not particularly limited as long as the target protein can be appropriately expressed in the transformant. However, the PGK promoter, ADH promoter, TDH promoter, ENO promoter, CIT promoter, TEF promoter, CDC promoter, GPM promoter or PDC promoter Etc. can be used.

The terminator can be incorporated downstream of the target gene. For example, a PGK terminator, a CIT terminator, a TEF terminator, a CDC terminator, a GPM terminator, a PDC terminator, or the like can be used.
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 selection markers 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 or expression cassette 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 transformed yeast of the present invention can also be produced by integrating the target gene into the host yeast chromosome by homologous recombination. A person skilled in the art can produce the transformed yeast of the present invention by homologous recombination by a known method.

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, by culturing the gene-transferred yeast as described above in a xylose-containing (glucose-free) medium, a transformed yeast into which at least the xylose-utilizing enzyme gene has been introduced can be selected.

Thus, the transformed yeast of the present invention can be prepared by introducing six genes so that they can be expressed in yeast, and preferably by introducing six genes onto the yeast chromosome.

Furthermore, the transformed yeast of the present invention includes a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, The expression of the gene encoding ketolase and the gene encoding alcohol dehydrogenase may be activated. That is, the present invention also includes yeast in which the expression level of the gene originally present on the host yeast chromosome is increased. By introducing a promoter from the outside or replacing the promoter of the gene itself with a stronger promoter, the gene originally possessed by yeast can be activated in a form that can be expressed, and the target protein can be expressed appropriately. . For example, the method for activating the expression of the endogenous gene in this way is not limited, but a promoter capable of appropriately expressing the target protein is incorporated into the chromosome by gene replacement using a known gene recombination technique. Methods and the like. As a gene replacement method, the method of Akada et al. Yeast 23: 399-405 (2006) (non-patent document) can be used. As the promoter to be replaced, a known promoter such as PGK promoter, ADH promoter, TDH promoter, ENO promoter, CIT promoter, TEF promoter, CDC promoter, GPM promoter or PDC promoter can be used.

3. Method for producing ethanol Transformed yeast of the present invention comprises a xylose utilization gene, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase, such as GRE3, SOR1, XKS1, TAL1, Since TKL1 and ADH1 are introduced, the transformed yeast of the present invention has the ability to assimilate xylose and can produce ethanol with high efficiency. Therefore, the transformed yeast of the present invention can be used in a method for producing ethanol from xylose.

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 media such as SD media, SCX media, YPD media, YPX media, CBS media, etc., and cultivate yeast under favorable 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 2-66 g / L, preferably 4-55 g / L, of xylose as a carbon source. Alternatively, when both glucose and xylose are included as a carbon source, the transformed yeast of the present invention is cultured in the presence of 40 to 330 g / L, preferably 80 to 275 g / L. The transformed yeast of the present invention may use a known culture method such as batch culture without additional supply of sugar as a carbon source, fed-batch culture in which additional supply of sugar is continuously / intermittently, or continuous culture. it can. In the case of additionally supplying sugar as a carbon source, it is desirable that the sugar supply be controlled by monitoring the sugar concentration so as to be maintained in the above-mentioned concentration range.

Prior to the main culture, the transformed yeast may be precultured. For the preculture, for example, the transformed yeast of the present invention may be inoculated into a small amount of medium and cultured for 12 to 24 hours. The main culture is started by adding 0.1 to 10%, preferably 1%, of the preculture solution to the culture medium of the main culture. 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 at 20 to 40 ° C, preferably 30 ° C.

The produced ethanol can be collected from the 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.

When ethanol is produced using the transformed yeast of the present invention, a conventional xylose-assimilating yeast (eg, GRE3, SOR1, and XKS1-introduced yeast), a yeast in which TAL1 and TKL1 are overexpressed in the xylose-assimilating yeast, and In comparison, ethanol can be produced in high yield.

The base sequence or amino acid sequence shown by SEQ ID NO in this specification is described below.
SEQ ID NOs: 1 to 20: This shows the base sequences of primers used in the examples.
SEQ ID NO: 21: This shows the base sequence of Saccharomyces cerevisiae GRE3.
SEQ ID NO: 22: This shows the amino acid sequence of Saccharomyces cerevisiae GRE3 protein.
SEQ ID NO: 23: This shows the base sequence of Saccharomyces cerevisiae SOR1.
SEQ ID NO: 24: This shows the amino acid sequence of Saccharomyces cerevisiae SOR1 protein.
SEQ ID NO: 25: This shows the base sequence of Saccharomyces cerevisiae XKS1.
SEQ ID NO: 26: This shows the amino acid sequence of Saccharomyces cerevisiae XKS1 protein.
SEQ ID NO: 27: This shows the base sequence of Saccharomyces cerevisiae TAL1.
SEQ ID NO: 28: This shows the amino acid sequence of Saccharomyces cerevisiae TAL1 protein.
SEQ ID NO: 29: This shows the base sequence of Saccharomyces cerevisiae TKL1.
SEQ ID NO: 30 This shows the amino acid sequence of Saccharomyces cerevisiae TKL1 protein.
SEQ ID NO: 31: This shows the base sequence of Saccharomyces cerevisiae ADH1.
SEQ ID NO: 32: This shows the amino acid sequence of Saccharomyces cerevisiae ADH1 protein.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Synthesis of GRE3, SOR1, XKS1, TAL1, TKL1, ADH1, PGK1 promoter and PGK1 terminator by PCR. Oligonucleotides 1 to 16 shown in Table 1 were synthesized and used as primers for the PCR reaction. GRE3, SOR1, XKS1, DNA fragments of TAL1, TKL1, ADH1, PGK1 promoter and PGK1 terminator were obtained. In addition, oligonucleotides 17 to 20 shown in Table 1 were synthesized to obtain DNA fragments of HXT17 (first half and second half). Chromosomal DNA extracted from Saccharomyces cerevisiae CEN.PK2-1C strain was used as a template for PCR reaction. PCR reaction was performed with TaKaRa PCR Thermal Cycler Dice Gradient TP600 (Takara Bio Inc.) using PrimeSTAR HS DNA polymerase (Takara Bio Inc.). Table 2 shows the amplification conditions in PCR.

GRE3, SOR1, XKS1, TAL1, TKL1, ADH1, PGK1 promoter and PGK1 terminator are derived from Saccharomyces cerevisiae. The GRE3 protein has amino acid sequence identity (homology) with XYL1 (xylose reductase (XR)) from Shephasomyces stipitsis, and SOR1 protein is XYL2 (xylitol dehydrogenation) from schephamyces stipitsis Enzyme (XDH)) is a protein having amino acid sequence identity (homology). XKS1 protein is xylulose kinase, TAL1 protein is transaldolase, TKL1 protein is transketolase, and ADH1 protein is alcohol dehydrogenase. The PGK1 promoter and PGK1 terminator are known to function in Saccharomyces cerevisiae.

Production of yeast with xylose utilization ability GRE3, SOR1, and XKS1 gene fragments amplified in Example 1 were ligated in the order of GRE3, SOR1, and XKS1 under the control of PGK1 promoter and PGK1 terminator to produce expression cassette I. did.
In addition, Xho I and Bgl II cleavage sites were introduced into a commercially available vector pUC18 by site-directed mutagenesis. For the site-specific mutation, PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.) was used. The gene fragment of the first half of HXT17 amplified in Example 1 was inserted into the Xho I and EcoR I cleavage sites of the obtained pUC18, and the gene of the second half of HXT17 amplified in Example 1 was inserted into the Sph I and Bgl II cleavage sites. Fragments were introduced.
The obtained expression cassette I was cleaved with EcoR I and Sph I, and inserted between the gene fragments of the first half and the second half of HXT17 to prepare expression cassette II.

Using the prepared expression cassette II, the above gene was introduced into the HXT17 site on the chromosome by the lithium acetate method to obtain a xylose-assimilating yeast. Shochu yeast was used as a host for imparting xylose utilization ability.

Preparation of TAL1 / TKL1 gene overexpression strain A gene fragment of TAL1 placed under the control of the PGK1 promoter and PGK1 terminator was introduced into the SmaI site of a commercially available expression vector pAUR135 (Takara Bio Inc.). In addition, the TKL1 gene fragment placed under the control of the PGK1 promoter and PGK1 terminator was introduced into the SphI site of the pAUR13 vector into which the TAL1 gene fragment was introduced. The gene fragment obtained by cleaving the obtained TAL1 / TKL1 expression vector with StuI was introduced into the AUR1 site on the chromosome of the xylose-assimilating yeast prepared in Example 2 by the lithium acetate method. The obtained strain was designated as a TAL1 / TKL1 gene overexpression strain.

Production of TAL1, TKL1, ADH1 gene overexpression strain A gene fragment of ADH1 placed under the control of the PGK1 promoter and PGK1 terminator was introduced into the EcoRI site of the TAL1 · TKL1 expression vector produced in Example 3. The gene fragment obtained by cleaving the obtained expression vector with StuI was introduced into the AUR1 site on the chromosome of the xylose-assimilating yeast prepared in Example 2 by the lithium acetate method. The obtained strain was designated as a TAL1 / TKL1 / ADH1 gene overexpression strain.

Evaluation of Fermentation Performance After the strains obtained in Examples 2, 3 and 4 were pre-cultured in a YPD (glucose 20 g / L) medium, a medium containing only xylose as a substrate, and a modified CBS containing glucose and xylose as substrates Medium ((HB Klinke et al., Biotechnol. Bioeng., 2003, Vol. 81, pp. 738-747: pH 5.0), Table 3) Into a 50 mL flask containing 15 mL, the initial inoculum was 2 ×. The cells were cultured with shaking at 10 ⁸ cells / mL, 140 rpm, 30 ° C., and sampled over time to evaluate the fermentation performance. In addition, a strain in which only a vector containing no transgene was incorporated was similarly prepared and used as a control strain in the following experiment.

The sampled culture solution was centrifuged to remove the cells, and the supernatant was filtered through a 0.2 μm polypropylene filter to obtain a measurement sample. The amounts of glucose, xylose, and ethanol in the measurement sample were quantified by HPLC. Table 4 shows the analytical conditions in HPLC.

The fermentation performance evaluation results for each strain are shown in FIG. 1 and FIG. FIG. 1 shows the result of a medium containing only xylose as a substrate, and FIG. 2 shows the result of a medium containing glucose and xylose as substrates. Fermentation performance is evaluated as ethanol yield (%), and the amount of ethanol produced corresponds to the amount of ethanol calculated by multiplying the administered basic mass by the theoretical yield (0.51 for both glucose and xylose). 100%.

When TAL1 and TKL1 are introduced onto the chromosome (Gal3, SOR1 and XKS1), the original xylose-assimilating yeast It became possible to use xylose more than (black), and it was shown that ethanol production increased (FIGS. 1 and 2). In addition, TAL1, TKL1 and ADH1 overexpressed strains (white, TAL1, TKL1 and ADH1 genes, white) on the chromosome of xylose-assimilating yeast in which GRE3, SOR1 and XKS1 have been introduced on the chromosome, It was shown that xylose can be further used compared to the original xylose-assimilating yeast (black) and the TAL1 / TKL1 gene overexpression strain (gray), and ethanol production was increased (FIG. 1). And FIG. 2).
Yeast with TAL1, TKL1, and ADH1 introduced into the chromosome of xylose-assimilating yeast (using shochu yeast) (TAL1, TKL1, ADH1 gene overexpressing strain, white) contains glucose and xylose as substrates. In the medium (Fig. 2), a higher ethanol yield was achieved compared to the medium containing only xylose as a substrate (Fig. 1), and xylose-assimilating yeast (black) and TAL1 · TKL1 gene overexpression strain The improvement in ethanol yield compared to (gray) was also significant.

From this result, a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase and alcohol dehydrogenase It was shown that the yeast containing the gene encoding can more efficiently produce ethanol from xylose than the conventional xylose-assimilating yeast.

Verification of ADH1 gene enhancement effect The strains obtained in Examples 3 and 4 were pre-cultured in a YPD (glucose 20 g / L) medium, and then YPDX (glucose 80 g / L, xylose 40 g / L) containing glucose and xylose as substrates. The cells were cultured in a 1 L jar containing 600 mL of medium at an initial inoculum of 1 × 10 ⁷ cells / mL, 380 rpm, 30 ° C. Sampling was performed over time, and the supernatant was analyzed by the method described in Example 5. In addition, yeast cells were collected for measurement of ADH1 activity and expression analysis of the ADH1 gene.

The results of ethanol analysis of the culture supernatant are shown in Table 5 and FIG. The ethanol yield was calculated by the method described in Example 5.

At any sampling time, the ethanol yield of the yeast into which TAL1, TKL1 and ADH1 were introduced (Example 4 TAL1, TKL1, ADH1 gene overexpressing strain, white) was determined by the TAL1 · TKL1 gene overexpressing strain (Example 3, It was shown that ethanol production was increased by enhancing the ADH1 gene.

Expression analysis of ADH1 gene
The yeast cells were collected from the cells cultured in jar culture, the medium was removed, the cells were washed twice with 2 ml of sterile distilled water, and then 400 μl of acetate buffer (50 mM sodium acetate pH 5.3, 10 mM EDTA) It was suspended in. 40 μl of SDS (sodium dodecyl sulfate) was added, 440 μl of phenol kept at 65 ° C. was added, and the mixture was stirred well, then kept at 65 ° C. for 4 minutes and rapidly cooled in ice. The mixture was centrifuged at 12000 × g for 5 minutes, the supernatant was transferred to a 1.5 ml tube, an equal amount of a phenol / chloroform mixture was added, and the mixture was stirred well. After centrifugation at 12000 × g for 5 minutes, the supernatant was transferred to a 1.5 ml tube, and 1/10 amount of 3M sodium acetate (pH 5.3) was added, followed by ethanol precipitation. The pellet obtained by centrifugation at 12000 × g for 10 minutes was washed twice with 80% ethanol and dried, and then dissolved in RNase-free water to obtain an RNA solution. Synthesis of cDNA from RNA was performed by Transcriptor First Strand cDNA Synthesis Kit (Roche). Expression analysis was performed by real-time PCR using SYBR Green (relative quantification method using a standard curve). The fluorescent reagent was prepared using LightCycler FastStart DNA Master PLUS SYBR Green I (Roche). The analyzer used was LightCycler 1.5 (Roche). Table 6 and FIG. 4 show the ADH1 expression analysis results of the TAL1 · TKL1 gene overexpression strain (Example 3) and the TAL1 · TKL1 · ADH1 gene overexpression strain (Example 4).

Analysis of ADH1 gene expression in TAL1 / TKL1 gene overexpressing strains and TAL1, TKL1 and ADH1 gene overexpressing strains, ADH1 gene expression in TAL1, TKL1 and ADH1 overexpressing strains (white) at any sampling time The amount exceeded that of the TAL1 / TKL1 gene overexpression strain (gray). For example, in 48 hours of culture, it was shown that the ADH1 gene expression level doubled due to enhancement of the ADH1 gene.

Measurement of ADH1 activity Collect yeast cells from cells cultured in jar culture, remove the medium, wash the cells with 2 ml distilled water, and then add 1 Y-PER Yeast Protein Extraction Reagent (Pierce, Rockford, IL). After adding ml and suspending, the mixture was shaken at room temperature for 20 minutes. The cell extract was obtained by centrifugation at 13,000 rpm for 10 minutes. ADH1 activity was measured as follows. 10 μl of crude yeast extract was added to 960 μl of a solution containing 50 mM Tris-HCl (pH 8.5) and 1 mM NAD ^+, and 30 μl of ethanol was added to initiate the reaction. The increase in NADH was calculated by measuring the absorbance at 340 nm using ethanol as a substrate. The activity for reducing 1 μmol of NAD + per minute was defined as 1 U, and the activity per 1 mg of protein in the cell extract (U / mg) was determined. Table 7 and FIG. 5 show the ADH1 activity of the TAL1 · TKL1 gene overexpression strain (Example 3) and the TAL1 · TKL1 · ADH1 gene overexpression strain (Example 4). The test was performed in duplicate.

When the ADH1 activity of the TAL1 · TKL1 gene overexpressing strain and the TAL1 · TKL1 · ADH1 gene overexpressing strain was measured, the ADH1 activity of the TAL1 · TKL1 · ADH1 gene overexpressing strain (white) was TAL1 at any sampling time.・ It exceeded TKL1 gene overexpression strain (gray). For example, in 24 hours of culture, it was shown that ADH1 activity increased about 6-fold by enhancing the ADH1 gene.

Based on these results, by enhancing the ADH1 gene, the ADH1 gene expression level and ADH1 activity increase, and the ethanol productivity of the TAL1, TKL1, and ADH1 gene overexpressing strains is higher than that of the TAL1 and TKL1 gene overexpressing strains. It was shown that.

Further, it was shown that the present invention can be applied not only to culture at a laboratory level using a flask but also to culture at an industrial level using a culture tank or culture conditions close to the industrial level.

Sequence numbers 1 to 20: Primer

Claims

Gene encoding xylose reductase, gene encoding xylulose phosphorylase, gene encoding xylitol dehydrogenase, gene encoding transaldolase, gene encoding transketolase and gene encoding alcohol dehydrogenase Transformed yeast introduced so that can be expressed.
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 is inserted so as to be expressible on the chromosome of the host yeast.
The yeast according to any one of claims 1 to 3, wherein the gene encoding xylose reductase is GRE3.
The yeast according to any one of claims 1 to 4, wherein the gene encoding xylulose kinase is XKS1.
The yeast according to any one of claims 1 to 5, wherein the gene encoding xylitol dehydrogenase is SOR1.
The yeast according to any one of claims 1 to 6, wherein the gene encoding transaldolase is TAL1.
The yeast according to any one of claims 1 to 7, wherein the gene encoding transketolase is TKL1.
The yeast according to any one of claims 1 to 8, wherein the gene encoding alcohol dehydrogenase is ADH1.
The yeast according to any one of claims 1 to 9, which has an ability to produce ethanol from xylose.
The yeast according to any one of claims 1 to 10, wherein the host yeast has hexose sugar assimilation ability but does not have pentose sugar assimilation ability.
The yeast according to any one of claims 1 to 11, wherein the host yeast is a yeast belonging to the genus Saccharomyces.
The yeast according to any one of claims 1 to 12, wherein the host yeast is a yeast belonging to the species Saccharomyces cerevisiae.
A method for producing ethanol, comprising culturing the transformed yeast according to any one of claims 1 to 13 in a xylose-containing medium and collecting ethanol from the resulting culture.