Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a yeast that produces ethanol from xylose.
• Saccharomyces cerevisiae Currently, yeasts represented by Saccharomyces cerevisiae are mainly used for brewing yeast in the production of ethanol. 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.
• Saccharomyces cerevisiae 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.
• 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.
• 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.
• GRE3 Aldo-keto reductase gene 3
• SOR1 sorbitol dehydrogenase gene 1
• XKS1 xylulose
• 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.
• the present invention relates to the following.
• 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.
• the yeast according to (1) wherein the gene is an endogenous gene of the yeast.
• the gene encoding xylose reductase is a gene selected from the group consisting of GRE3, YJR096w, YPR1, GCY1, ARA1, and YDR124w.
• the gene encoding xylitol dehydrogenase is a gene selected from the group consisting of SOR1, SOR2, and YLR070c.
• 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.
• 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.
• 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].
• Saccharomyces cerevisiae is a yeast into which three genes, a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding xylulose phosphorylase are inserted, [1] to 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.
• 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,
• a transformed yeast capable of producing ethanol from xylose is provided.
• 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. .
• 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.
• 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.
• 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.
• SOR1 sorbitol dehydrogenase gene 1
• GDH2 transformed yeast overexpressing gene 2
• 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.
• 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.
• 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
• GDH2 glutamic acid dehydrogenase gene 2
• the gene to be introduced into the host yeast is a gene derived from the host yeast.
• the transformed yeast of the present invention is characterized in that the amount of by-products produced is low.
• 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.
• yeast belonging to the genus Saccharomyces cannot produce ethanol using xylose, despite having a gene group encoding a xylose-utilizing enzyme group.
• 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.
• the ability to produce ethanol from xylose is improved compared to the control strain.
• 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.
• 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.
• 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 above transformed yeast and collecting ethanol from the obtained culture.
• it is possible to significantly reduce the amount of by-products to be produced as compared with the conventional method.
• 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.
• 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.
• 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.
• 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.
• the yeast having no pentose assimilation ability is not particularly limited, and examples thereof include yeast belonging to the genus Saccharomyces.
• yeast belonging to the genus Saccharomyces include Saccharomyces cerevisiae such as CEN.PK2-1C strain.
• 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.
• 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.
• 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).
• 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.
• 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,
• GRE3 Aldo keto reductase gene 3
• SOR1 sorbitol dehydrogenase gene 1
• XKS1 xylulose phosphorylase gene.
• 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.
• GRE3, YJR096w, YPR1, GCY1, ARA1 and YDR124w are known as genes encoding xylose reductase of yeast. Therefore, in the present invention, GRE3, YJR096w, YPR1, GCY1, ARA1 or YDR124w can be used as a gene encoding xylose reductase.
• 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.
• Genbank 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.
• 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 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.
• 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 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.
• 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.
• 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.
• the nucleotide sequence can be confirmed by sequencing by a conventional method.
• 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.
• GRE3 Aldo keto reductase gene 3
• Saccharomyces cerevisia includes that which encodes a protein having the amino acid sequence represented by SEQ ID NO: 2.
• 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 GRE3 protein variant 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: 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 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: 2 More preferably 1 to 3, more preferably 1 to 2 amino acids) and (iv) a protein in which these mutations are combined and a protein having xylose reductase activity. It is.
• xylose reductase activity means the activity of converting xylose to xylitol in the presence of NAD + (or NADP +).
• 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.
• SOR1 is described as an example of a gene encoding xylitol dehydrogenase.
• SOR2 and YLR070c can be used in the present invention in the same manner as described herein with respect to SOR1.
• SOR1 L11039
• SOR2 Z74294
• YLR070c Z73242.
• 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 (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: 4 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) their Tampa combined with mutation A quality and a protein having xylitol dehydrogenase activity.
• xylitol dehydrogenase activity means the activity of dehydrogenating xylitol to xylulose.
• the mutant of SOR1 protein is not particularly limited as long as it has xylitol dehydrogenase activity.
• 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.
• 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.
• Genbank the accession number of XKS1 of Saccharomyces cerevisia is Z72979.
• XKS1 xylulose phosphorylase gene 1
• XKS1 xylulose phosphorylase gene 1
• SEQ ID NO: 5 a DNA comprising the base sequence represented by 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 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 (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: 6 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) their Tampa combined with mutation A quality, and protein having xylulose kinase activity.
• xylulose kinase activity means an activity of phosphorylating xylulose.
• the mutant of the XKS1 protein is not particularly limited as long as it has xylulose phosphatase activity.
• 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.
• XKS1 can also be obtained or produced by a method similar to that described for GRE3.
• the host yeast of the present invention can be obtained by inserting three genes, GRE3, SOR1 and XKS1, which are xylose utilization genes, into the yeast chromosome.
• GRE3, SOR1 and XKS1 which are xylose utilization genes
• XKS1 xylose utilization genes
• non-recombinant yeast is preferably employed as the host yeast.
• the xylose-assimilating gene introduced on the yeast chromosome is a gene derived from the same species as the yeast.
• the yeast into which the gene is introduced needs to be the same kind of yeast as the origin of the gene.
• three genes a gene encoding xylose reductase, a gene encoding xylitol dehydrogenase, and a gene encoding silulose phosphorylase, 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 individually inserted on a chromosome, or an expression cassette linked in tandem under the control of a promoter may be prepared and inserted on a chromosome. When connecting in tandem, the order of arrangement of the three genes is not particularly limited, and any conceivable combination may be used.
• 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.
• 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.
• the 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.
• XR gene
• XDH xylitol dehydrogenase
• XK xylulose kinase
• the present invention relates to the above three kinds of xylose utilization genes, a gene obtained by linking a gene encoding xylose reductase and a gene encoding xylitol dehydrogenase, and a gene encoding xylitol dehydrogenase and xyl It includes transformed yeast inserted on a chromosome as a gene linked to a gene encoding rosin kinase.
• a xylose-assimilating gene can be introduced onto yeast staining using a plasmid containing a GRE3-SOR1 fusion gene and a plasmid containing a SOR1-XKS1 fusion gene.
• the order of gene arrangement within the fusion gene is not particularly limited.
• the gene in which XR and XDH are linked may be either XR-XDH or XDH-XR.
• the gene linking XDH and XK may be XDH-XK or XK-XDH.
• the linked gene cassette is used, the expression level of the gene encoding xylitol dehydrogenase (SOR1) is particularly increased.
• SOR1 xylitol dehydrogenase
• Such a transformed yeast has particularly excellent xylose utilization ability and can efficiently produce ethanol from xylol.
• 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.
• a site on a chromosome that does not encode a gene is a ⁇ sequence that is one of Ty factors. It is known that a plurality of (approximately 100 copies) ⁇ sequences are present on the yeast chromosome.
• the position and sequence information of the ⁇ 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 ⁇ sequence into yeast, one or more copies of the gene can be inserted at a desired position on the chromosome. In addition to the ⁇ sequence, it can also be inserted into the ⁇ and ⁇ 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.
• 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.
• telomeres 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.
• Yeast that does not have the ability to assimilate pentose must 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 gene-transferred as described above in a xylose-containing (ethanol-free) medium.
• 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. .
• “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.
• 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.
• 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.
• GDH2 glutamate dehydrogenase gene 2
• SOR1 sorbitol dehydrogenase gene 1
• GDH2 glutamate dehydrogenase gene 2
• GDH2 is an enzyme that uses NAD as a coenzyme.
• the base sequence information of GDH2 can be obtained from a known database such as Genbank by those skilled in the art.
• Genbank The accession number of GDH2 in Saccharomyces cerevisia is S66436.
• GDH2 glutamate dehydrogenase gene 2
• GDH2 glutamate dehydrogenase gene 2
• SEQ ID NO: 7 derived from Saccharomyces cerevisiae
• SEQ ID NO: 8 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.
• the GDH2 used in the present invention may be a gene encoding the following mutant of 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 amino acids 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 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: 8 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) their Tampa combined with mutation A quality and a protein having a glutamate dehydrogenase activity.
• Glutamic acid dehydrogenase activity means the activity of interconverting glutamic acid and ⁇ -ketoglutaric acid.
• the mutant of the GDH2 protein is not particularly limited as long as it has glutamate dehydrogenase activity.
• 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.
• GDH2 can be obtained or produced by a method similar to the above method.
• 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.
• SOR1 is introduced into the host yeast so that it can be expressed.
• GDH2 is introduce
• both SOR1 and GDH2 are introduce
• the gene transfer can be performed using a plasmid containing the gene of interest in a form that can be expressed.
• 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.
• 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.
• a plasmid derived from E. coli a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like can be used.
• 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.
• the selection marker examples 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.
• 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.
• an amino acid synthesis gene cassette such as leucine, histidine, tryptophan, or a uracil synthesis gene cassette
• 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 into the host yeast chromosome by homologous recombination.
• GDH2 glutamate dehydrogenase gene 2
• SOR1 sorbitol dehydrogenase gene 1
• the insertion site is not particularly limited.
• 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.
• 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.
• a gene replacement method the method of Akada et al. Yeast 23: 399-405 (2006) (non-patent document) can be used.
• a promoter to be replaced a known promoter such as PGK promoter, ADH promoter, TDH promoter, ENO promoter, etc. can be used.
• 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.
• the transformed yeast of the present invention can produce ethanol from xylose.
• the host yeast of the present invention is a yeast imparted with xylose utilization ability, it is possible to produce ethanol from xylose.
• 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.
• 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.
• shaking culture is preferred.
• Ethanol can be produced by culturing the transformed yeast of the present invention and collecting ethanol from the resulting culture.
• the transformed yeast of the present invention is cultured in the presence of 10 to 150 g / L, preferably 70 g / L, of xylose.
• the transformed yeast Prior to the main culture, the transformed yeast may be precultured.
• 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, and shaking culture 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.
• ethanol 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.
• 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.
• a transformed yeast for example, GDH2 and SOR1 overexpressing strains into which both GDH2 and SOR1 are introduced
• the production of by-products xylitol and glycerol is reduced compared to the control strain.
• 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
• 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 ⁇ M, preferably 0.1 to 1000 ⁇ M, more preferably 1 to 100 ⁇ M.
• 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.
• yeasts used in the examples are all Saccharomyces cerevisiae.
• GRE3 (aldoketoreductase 3) xylose reductase activity
• SOR1 sorbitol dehydrogenase 1 xylitol dehydrogenase activity
• XKS1 xylulose phosphate 1 xylulose phosphate enzyme activity
• 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 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 5 U / ml pyruvate kinase 7 U / ml lactate dehydrogenase 5 mM xylulose crude extract
• 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.
• 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.
• SC-X xylose 5% liquid medium
• 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.
• the results of fermentability evaluation are shown in Table 3.
• the improved strains A to G prepared by the above method showed a 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. .
• Example 2 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- ⁇ -GRE3-SOR1 vector and the pUC- ⁇ -XKS1-SOR1 vector were obtained by the same method as described above.
• Example 2 using the obtained pUC- ⁇ -GRE3-SOR1 and pUC- ⁇ -XKS1-SOR1 vectors and yeast (Association No. 7) In the same manner as described above, xylose-assimilating yeast was prepared (improved strains HM).
• Example 2 For fermented strains HM, fermentability evaluation was conducted in Example 2. 3. 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.
• a transformed yeast capable of producing ethanol from xylose is provided.
• 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. .
• 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.
• 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.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (2)

Applications Claiming Priority (2)

Publications (1)

Family

ID=50477497

Family Applications (1)

Country Status (3)

Cited By (4)

Citations (2)

Family Cites Families (1)

• 2013
  • 2013-10-10 JP JP2014540897A patent/JP5796138B2/ja active Active
  • 2013-10-10 WO PCT/JP2013/077674 patent/WO2014058034A1/fr not_active Ceased
  • 2013-10-10 BR BR112015007234-8A patent/BR112015007234B1/pt active IP Right Grant

Patent Citations (2)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events