WO2020032233A1 - Levure recombinante et procédé de production d'éthanol l'utilisant - Google Patents

Levure recombinante et procédé de production d'éthanol l'utilisant Download PDF

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WO2020032233A1
WO2020032233A1 PCT/JP2019/031560 JP2019031560W WO2020032233A1 WO 2020032233 A1 WO2020032233 A1 WO 2020032233A1 JP 2019031560 W JP2019031560 W JP 2019031560W WO 2020032233 A1 WO2020032233 A1 WO 2020032233A1
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gene
amino acid
recombinant yeast
protein
seq
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大西　徹
宣紀 多田
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トヨタ自動車株式会社
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    • 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
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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 recombinant yeast and a method for producing ethanol using the recombinant yeast.
  • Patent Document 1 discloses a yeast in which a xylose reductase gene and a xylitol dehydrogenase gene derived from Pichia stipitis and a xylulokinase gene derived from S. cerevisiae have been integrated into a chromosome.
  • Non-Patent Documents 1 and 2 disclose yeast into which a xylose assimilation gene has been introduced significantly inhibits ethanol fermentation using xylose as a sugar source.
  • fermented mash obtained by saccharifying cellulosic biomass with cellulase mainly contains unfermented residues, poorly fermented residues, enzymes and microorganisms for fermentation.
  • the fermentation microorganisms can be reused by using the reaction liquid containing moromi for the next fermentation, the amount of new fermentation microorganisms to be input can be reduced, and the cost can be reduced.
  • acetic acid contained in the mash is also brought in at the same time, and as a result, the concentration of acetic acid contained in the fermentation medium increases, which may hinder ethanol fermentation.
  • Another method of avoiding fermentation inhibition by acetic acid is to metabolize acetic acid in the medium simultaneously with ethanol fermentation.
  • acetic acid metabolism is an aerobic reaction and overlaps the ethanol metabolic pathway. Therefore, when fermentation is performed under aerobic conditions, acetic acid may be metabolized, but at the same time, ethanol, which is a target substance, is metabolized.
  • acetaldehyde dehydrogenase In order to metabolize acetic acid under anaerobic conditions in which ethanol is not metabolized, a gene encoding acetaldehyde dehydrogenase (EC 1.2.1.10) was introduced into the Saccharomyces cerevisiae strain in which the GPD1 and GPD2 genes of the glycerin production pathway were disrupted. Thus, there are reports that acetic acid has been assimilated (Non-Patent Documents 5 and 6 and Patent Documents 2 to 4). In addition, acetaldehyde dehydrogenase catalyzes the following reversible reaction.
  • the glycerin production pathway by the GPD1 and GPD2 genes is a pathway that oxidizes surplus coenzyme NADH generated by metabolism to NAD + as shown in the following formula.
  • this reaction pathway is disrupted by disrupting the GPD1 and GPD2 genes, and surplus coenzyme NADH is supplied by introducing acetaldehyde dehydrogenase, and the following reaction proceeds.
  • acetyl coenzyme A is synthesized from acetic acid by acetyl-CoA synthase, and acetaldehyde is converted into ethanol. Accordingingly, the following reaction formula is finally obtained. Is also metabolized.
  • Non-Patent Document 7 xylose reductase (XR) and xylitol dehydrogenase (XDH) in the xylose metabolic pathway instead of disrupting the GPD1 and GPD2 genes, the intracellular redox state due to the difference in the coenzyme between XR and XDH is reduced. There is a report that a surplus coenzyme NADH is supplied by causing imbalance (Non-Patent Document 7).
  • XR uses NAD + as a coenzyme when converting xylose to xylitol, and mainly uses NADPH to convert it to NADP +
  • XDH uses NAD + as a coenzyme when converting xylitol to xylulose.
  • NADH NADP +
  • XDH NAD + as a coenzyme when converting xylitol to xylulose.
  • NADH To convert to NADH.
  • This imbalance of requirement for coenzymes of both enzymes results in accumulation of NADH.
  • the ethanol fermentation from xylose by the XR / XDH-introduced yeast accumulates an intermediate metabolite xylitol. As a result, acetic acid is metabolized, but is not practical because the ethanol yield is poor.
  • Non-Patent Document 8 There is also a report in which acetaldehyde dehydrogenase has been introduced into non-disrupted strains of the GPD1 gene and the GPD2 gene.
  • Non-Patent Document 8 does not report that the amount of acetic acid in the medium is reduced although the production of acetic acid is reduced by the introduction of acetaldehyde dehydrogenase.
  • Non-Patent Document 8 is also not a report on xylose-utilizing yeast.
  • chaperones are proteins that have a function of repairing proteins or the like whose three-dimensional structure is not properly taken, and play an important role in protein quality control.
  • a typical protein that functions as a chaperone is known as a heat shock protein (HSP).
  • HSP heat shock protein
  • Molecular chaperones are composed of many members and are conserved in all species, from E. coli to yeast and mammals. Representative members include the HSP40 family, HSP60 family, HSP70 family, HSP90 family, HSP100 family, and the like, depending on their molecular weight.
  • Non-Patent Document 9 an example of improving the activity of other heterologous proteins by introducing a heterologous molecular chaperone is the HSP60 family of eubacteria-derived ribulose-1,5-bisphosphate carboxylase.
  • GroL derived from Escherichia coli and its subunit GroS are expressed (Non-Patent Document 9).
  • the present invention provides a recombinant yeast having improved acetic acid assimilation ability without disrupting the glycerin production pathway in which ethanol fermentation is reduced, and a method for producing ethanol using the recombinant yeast.
  • the purpose is to provide.
  • recombinant yeast into which a predetermined chaperone (synonymous to molecular chaperone and intramolecular chaperone) has been introduced can be used for cultivation such as ethanol fermentation. At this time, they found that acetic acid utilization could be improved, and completed the present invention.
  • the present invention includes the following.
  • the homologous gene encodes a protein selected from the group consisting of SSA1, SSA2, SSA3, SSA4, SSB1, SSB2, SSE1, SSE2, SSZ1, KAR2, LHS1, SSQ1, SSQ1, and ECM10 in Saccharomyces cerevisiae.
  • Homologous genes are YDJ1, XDJ1, APJ1, SIS1, DJP1, ZUO1, SWA2, JJJ1, JJJ2, JJJ3, CAJ1, CWC1, CWC23, MDJ1, MDJ2, PAM18, JAC1, JAC1 in Saccharomyces cerevisiae.
  • the recombinant yeast according to (5) which is a gene encoding one kind of protein selected from the group consisting of JEM1, SEC63 and ERJ5.
  • the recombinant yeast according to (8), wherein the acetaldehyde dehydrogenase derived from Escherichia coli is a protein of the following (a) or (b).
  • the group of enzymes constituting the non-oxidation process pathway in the pentose phosphate pathway is ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase. )).
  • a method for producing ethanol comprising a step of culturing the recombinant yeast according to any one of (1) to (16) in a medium containing glucose and / or xylose to perform ethanol fermentation.
  • the acetic acid concentration in the medium can be reduced during culturing such as ethanol fermentation. Therefore, by using the recombinant yeast according to the present invention, fermentation inhibition caused by acetic acid in the medium can be effectively avoided. As a result, in the method for producing ethanol using the recombinant yeast according to the present invention, the efficiency of ethanol fermentation can be kept high, and an excellent ethanol yield can be achieved.
  • the method for producing ethanol using the recombinant yeast according to the present invention for example, when the recombinant yeast is reused or used for continuous culture, the amount of acetic acid brought in can be reduced, and the method is excellent. Ethanol yield can be maintained.
  • the recombinant yeast according to the present invention is a recombinant yeast into which an acetaldehyde dehydrogenase gene and a molecular chaperone gene belonging to the HSP70 family or the HSP40 family have been introduced.
  • the recombinant yeast according to the present invention is characterized in that it can metabolize acetic acid contained in a medium, and the acetic acid concentration in the medium decreases with culturing such as ethanol fermentation.
  • the molecular chaperone gene belonging to the HSP70 family encodes one of molecular chaperones that are involved in the structure formation and function expression of many types of proteins using the energy of ATP hydrolysis.
  • the molecular chaperone gene belonging to the HSP70 family to be introduced into yeast is not particularly limited, and a gene derived from any organism may be used.
  • the molecular chaperone gene belonging to the HSP70 family is not particularly limited, and examples thereof include a gene encoding a molecular chaperone called DnaK in Escherichia coli (DnaK gene) or a homologous gene thereof (DnaK homologous gene).
  • the nucleotide sequence of the DnaK gene and the amino acid sequence of the protein encoded by the DnaK gene are shown in SEQ ID NOs: 1 and 2, respectively.
  • As the DnaK homologous gene SSA1, SSA2, SSA3, SSA4, SSB1, SSB2, SSE1, SSE2, SSZ1, SZ1, KAR2, LHS1, SSC1, SSQ1 in Saccharomyces cerevisiae, and a kind of protein selected from the group consisting of ECM10 Genes that can be used.
  • the nucleotide sequence and amino acid sequence of the DnaK homologous gene in Saccharomyces cerevisiae can be obtained from databases such as Saccharomyces GENOME DATABASE and GenBank.
  • the DnaK gene is not limited to those specified in SEQ ID NOs: 1 and 2, and if it is a molecular chaperone belonging to the HSP70 family, the base sequence and amino acid sequence are different, but the relationship of paralogs or the relationship of homologs in a narrow sense is different. May be the gene in the above.
  • the molecular chaperone genes belonging to the HSP70 family are not limited to those specified by SEQ ID NOs: 1 and 2, and may be, for example, 70% or more, preferably 80% or more, based on the amino acid sequence of SEQ ID NO: 2. It may be composed of an amino acid sequence having preferably 90% or more, most preferably 95% or more sequence similarity or identity, and may encode a molecular chaperone belonging to the HSP70 family. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings).
  • sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions. It is calculated as the ratio of the total number in all amino acid residues. Note that the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • the molecular chaperone genes belonging to the HSP70 family are not limited to those specified by SEQ ID NOS: 1 and 2, and for example, one or several amino acids may be substituted or deleted from the amino acid sequence of SEQ ID NO: 2. It may have a lost, inserted or added amino acid sequence and may encode a molecular chaperone belonging to the HSP70 family.
  • the term “several” means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • molecular chaperone genes belonging to the HSP70 family are not limited to those specified in SEQ ID NOs: 1 and 2, and include, for example, all or part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 1.
  • it may hybridize under stringent conditions and encode a molecular chaperone belonging to the HSP70 family.
  • ⁇ stringent conditions '' as used herein means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a nucleotide sequence different from SEQ ID NO: 1 or a gene encoding an amino acid sequence different from SEQ ID NO: 2 functions as a molecular chaperone gene belonging to the HSP70 family is determined by determining whether the gene is suitable. If an expression vector integrated between a promoter and a terminator is prepared, a host such as Escherichia coli is transformed using the expression vector, and it is evaluated whether the expressed protein functions as a molecular chaperone belonging to the HSP70 family. Good. Whether the protein functions as a molecular chaperone belonging to the HSP70 family is evaluated, for example, by the method described in the column of Chaperone Activity Assay in Sophia Diamant et al., J. Biol. Chem. 275, 21107-21113 (2000). be able to.
  • the molecular chaperone gene belonging to the HSP40 family encodes a cochaperone that controls the functional expression of the HSP40 family molecular chaperone without depending on ATP.
  • the molecular chaperone gene belonging to the HSP40 family to be introduced into yeast is not particularly limited, and a gene derived from any organism may be used. Further, the molecular chaperone gene belonging to the HSP40 family, organisms other than fungi such as yeast, for example, bacteria and animals, plants, insects, when using genes derived from algae, the nucleotide sequence according to the codon usage in the yeast to be introduced It is preferable to use a gene in which is modified.
  • the molecular chaperone gene belonging to the HSP40 family is not particularly limited, and examples thereof include a gene encoding a molecular chaperone called DnaJ in Escherichia coli (DnaJ gene) or a homologous gene thereof (DnaJ homologous gene).
  • the nucleotide sequence of the DnaJ gene and the amino acid sequence of the protein encoded by the DnaJ gene are shown in SEQ ID NOs: 3 and 4, respectively. Further, as DnaJ homologous genes, YDJ1, XDJ1, APJ1, SIS1, DJP1, ZUO1, SWA2, JJJ1, JJJ2, JJJ3, CAJ1, CWC23, MDJ1, MDJ2, PAM18, JAC1, JID1, SCJ1, HLJ1 in Saccharomyces cerevisiae Examples include a gene encoding one kind of protein selected from the group consisting of JEM1, SEC63 and ERJ5. The nucleotide sequence and amino acid sequence relating to the DnaJ homologous gene in Saccharomyces cerevisiae can be obtained from databases such as Saccharomyces GENOME DATABASE and GenBank.
  • the DnaJ gene is not limited to those specified in SEQ ID NOs: 3 and 4, and if it is a molecular chaperone belonging to the HSP40 family, the base sequence and amino acid sequence are different, but the relationship of paralogs or the relationship of homologs in a narrow sense is different. May be the gene in the above.
  • the molecular chaperone genes belonging to the HSP40 family are not limited to those specified by SEQ ID NOs: 3 and 4, and may be, for example, 70% or more, preferably 80% or more, based on the amino acid sequence of SEQ ID NO: 4. It may consist of an amino acid sequence having preferably 90% or more, most preferably 95% or more sequence similarity or identity, and may encode a molecular chaperone belonging to the HSP40 family. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings).
  • sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions. It is calculated as the ratio of the total number in all amino acid residues. Note that the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • the molecular chaperone genes belonging to the HSP40 family are not limited to those specified by SEQ ID NOS: 3 and 4, and for example, one or several amino acids may be substituted or deleted from the amino acid sequence of SEQ ID NO: 4. It may have a lost, inserted or added amino acid sequence and encode a molecular chaperone belonging to the HSP40 family.
  • the term “several” means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • the molecular chaperone genes belonging to the HSP40 family are not limited to those specified by SEQ ID NOs: 3 and 4, and include, for example, all or a part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 3.
  • it may hybridize under stringent conditions and encode a molecular chaperone belonging to the HSP40 family.
  • ⁇ stringent conditions '' as used herein means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a nucleotide sequence different from SEQ ID NO: 3 or a gene encoding an amino acid sequence different from SEQ ID NO: 4 functions as a molecular chaperone gene belonging to the HSP40 family depends on whether the gene is appropriate.
  • a host such as Escherichia coli is transformed using this expression vector, and it is evaluated whether the expressed protein functions as a molecular chaperone belonging to the HSP40 family. Good. Whether the protein functions as a molecular chaperone belonging to the HSP40 family is evaluated, for example, by the method described in the section of Chaperone Activity Assay in Sophia Diamant et al., J. Biol. Chem. 275, 21107-21113 (2000). be able to.
  • the recombinant yeast according to the present invention is a recombinant yeast into which not only the molecular chaperone gene belonging to the HSP70 family or the HSP40 family described above but also the acetaldehyde dehydrogenase gene has been introduced.
  • the acetaldehyde dehydrogenase gene to be introduced is not particularly limited, and a gene derived from any organism may be used.
  • the acetaldehyde dehydrogenase gene when using genes derived from organisms other than fungi such as yeast, for example, bacteria, animals, plants, insects, and algae, modify the nucleotide sequence according to the codon usage in the yeast to be introduced. It is preferable to use a gene which has been prepared.
  • the acetaldehyde dehydrogenase gene the mhpF gene in Escherichia coli, ALDH1 in Entamoeba histolytica as disclosed in Applied and Environmental Microbiology, May 2004, p. 2892-2897, Vol. 70, No. 5 Genes can be used.
  • the nucleotide sequence of the mhpF gene in Escherichia coli and the amino acid sequence of the protein encoded by the mhpF gene are shown in SEQ ID NOS: 5 and 6, respectively.
  • the acetaldehyde dehydrogenase gene is not limited to those specified in SEQ ID NOS: 5 and 6, and the enzyme defined in EC No. 1.2.1.10 has a different base sequence and amino acid sequence but a paralog. It may be a gene having a relation or a homolog relation in a narrow sense.
  • Examples of the acetaldehyde dehydrogenase gene include the adhE gene and eutE gene in Escherichia coli, the acetaldehyde dehydrogenase gene derived from Clostridium beijerinckii, and the acetaldehyde dehydrogenase gene derived from Chlamydomonas reinhardtii.
  • nucleotide sequence of the adhE gene in Escherichia coli and the amino acid sequence of the protein encoded by the adhE gene are shown in SEQ ID NOs: 7 and 8, respectively.
  • the nucleotide sequence of the eutE gene in Escherichia coli and the amino acid sequence of the protein encoded by the eutE gene are shown in SEQ ID NOS: 9 and 10, respectively.
  • the nucleotide sequence of the acetaldehyde dehydrogenase gene derived from Clostridium beijerinckii and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOS: 11 and 12, respectively.
  • the nucleotide sequence of the acetaldehyde dehydrogenase gene derived from Chlamydomonas onareinhardtii and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs: 13 and 14, respectively.
  • the acetaldehyde dehydrogenase gene is not limited to those specified by SEQ ID NOS: 5 and 6, 7 and 8, 9 and 10, 11 and 12, and 13 and 14, and for example, SEQ ID NOs: 6, 8, An amino acid sequence having an amino acid sequence having a sequence similarity or identity of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with respect to the amino acid sequence of 10, 12 or 14; It may encode a protein having dehydrogenase activity. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings).
  • sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions. It is calculated as the ratio of the total number in all amino acid residues. Note that the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • acetaldehyde dehydrogenase genes are not limited to those specified by SEQ ID NOS: 5 and 6, 7 and 8, 9 and 10, 11 and 12, and 13 and 14, and for example, SEQ ID NOs: 6, 8,
  • the protein may have an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added to the amino acid sequence of 10, 12 or 14, and may encode a protein having acetaldehyde dehydrogenase activity.
  • severeal means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • acetaldehyde dehydrogenase genes are not limited to those specified by SEQ ID NOS: 5 and 6, 7 and 8, 9 and 10, 11 and 12, and 13 and 14, and for example, SEQ ID NOs: 5, 7 , 9, 11 or 13 may encode a protein that hybridizes to all or a part of the complementary strand of DNA under stringent conditions and has acetaldehyde dehydrogenase activity.
  • ⁇ stringent conditions '' means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a nucleotide sequence different from SEQ ID NO: 5, 7, 9, 11, or 13 or a gene encoding an amino acid sequence different from SEQ ID NO: 6, 8, 10, 12, or 14 is acetaldehyde dehydrogenated. Whether it functions as an enzyme gene, an expression vector is prepared by incorporating the gene between an appropriate promoter and terminator, etc., and a host such as Escherichia coli is transformed using this expression vector to express the protein to be expressed. The acetaldehyde dehydrogenase activity may be measured.
  • Acetaldehyde dehydrogenase activity is determined by preparing a solution containing acetaldehyde, CoA and NAD + as a substrate, allowing the protein to be tested to act at an appropriate temperature, and converting the produced acetyl-CoA to acetyl phosphate by the action of phosphoacetyltransferase. Can be measured after conversion to NADH, or the generated NADH can be measured spectrophotometrically.
  • the yeast into which the molecular chaperone gene and the acetaldehyde dehydrogenase gene belonging to the HSP70 family or the HSP40 family described above are preferably yeast having a xylose metabolism ability.
  • a yeast having xylose metabolism ability is a yeast to which xylose metabolism ability has been imparted by introducing a xylose isomerase gene to a yeast originally having no xylose metabolism ability, originally a xylose metabolism. It is meant to include both yeast having xylose metabolism ability by introducing a xylose isomerase gene and other xylose metabolism-related genes into yeast having no ability, and yeast originally having xylose metabolism ability.
  • a yeast having a xylose metabolism ability can produce ethanol by utilizing xylose contained in a medium.
  • the xylose contained in the medium may be obtained by a process of saccharifying xylan, hemicellulose, or the like having xylose as a constituent sugar, or xylan or hemicellulose contained in the medium may be saccharified by a saccharifying enzyme. May be supplied to the medium. The latter case means a so-called simultaneous saccharification and fermentation system.
  • the xylose isomerase gene (XI gene) is not particularly limited, and a gene derived from any species may be used.
  • a plurality of xylose isomerase genes derived from termite intestinal protists disclosed in JP-A-2011-147445 can be used without particular limitation.
  • the xylose isomerase gene is derived from the anaerobic mold Piromyces (spiromyces) sp. E2 species (Japanese Unexamined Patent Publication No. 2005-514951), the anaerobic mold Syllamyces aberensis (Cyllamyces aberensis), and bacteria. It is also possible to use genes derived from a certain Bacteroides ⁇ ⁇ taiotaomicron, from a bacterium, Clostridium phytofermentus, or from a Streptomyces murinas cluster.
  • xylose isomerase gene it is preferable to use a xylose isomerase gene derived from an intestinal protist of the termite (Reticulitermes speratus) intestine.
  • SEQ ID NOS: 15 and 16 show the nucleotide sequence of the coding region of the xylose isomerase gene derived from the intestinal protist of the termite (Reticulitermes @ speratus) and the amino acid sequence of the protein encoded by the gene, respectively.
  • the xylose isomerase gene is not limited to those specified in SEQ ID NOS: 15 and 16, and may be a gene having a different base sequence or amino acid sequence but having a paralog relationship or a homolog relationship in a narrow sense.
  • the xylose isomerase gene is not limited to those specified by SEQ ID NOS: 15 and 16, and is, for example, 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence of SEQ ID NO: 16. As described above, it may most preferably consist of an amino acid sequence having 95% or more sequence similarity or identity, and may encode a protein having xylose isomerase activity. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings). The value of sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions.
  • the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • the xylose isomerase gene is not limited to those specified in SEQ ID NOs: 15 and 16, and for example, one or several amino acids may be substituted, deleted, inserted, or substituted for the amino acid sequence of SEQ ID NO: 16. It may be a protein having an added amino acid sequence and encoding a protein having xylose isomerase activity.
  • severe means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • the xylose isomerase gene is not limited to those specified by SEQ ID NOs: 15 and 16, and may be, for example, a stringent DNA with respect to all or a part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 15. It may be a protein that hybridizes under various conditions and encodes a protein having xylose isomerase activity.
  • ⁇ stringent conditions '' as used herein means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a nucleotide sequence different from SEQ ID NO: 15 or a gene encoding an amino acid sequence different from SEQ ID NO: 16 functions as a xylose isomerase gene depends on whether the gene has an appropriate promoter and terminator.
  • An expression vector integrated between the above steps may be prepared, a host such as Escherichia coli may be transformed with the expression vector, and the xylose isomerase activity of the protein to be expressed may be measured.
  • the xylose isomerase activity means an activity of isomerizing xylose to xylulose.
  • xylose isomerase activity can be evaluated by preparing a solution containing xylose as a substrate, allowing the protein to be tested to act at an appropriate temperature, and measuring the amount of reduced xylose and / or the amount of xylulose produced.
  • xylose isomerase gene a gene encoding a mutant xylose isomerase having an amino acid sequence obtained by introducing a specific mutation to a specific amino acid residue in the amino acid sequence shown in SEQ ID NO: 16 and having improved xylose isomerase activity is used. It is preferred to use.
  • examples of the gene encoding the mutant xylose isomerase include a gene encoding an amino acid sequence in which the asparagine at position 337 in the amino acid sequence shown in SEQ ID NO: 16 has been substituted with cysteine.
  • Xylose isomerase consisting of an amino acid sequence in which the asparagine at position 337 in the amino acid sequence shown in SEQ ID NO: 16 has been substituted with cysteine has excellent xylose isomerase activity as compared with wild-type xylose isomerase.
  • the mutant xylose isomerase is not limited to the 337th asparagine substituted with cysteine, and may be the 337th asparagine substituted with an amino acid other than cysteine, or in addition to the 337th asparagine. Further, a different amino acid residue may be substituted with another amino acid, or another amino acid residue other than the above-mentioned 337th asparagine may be substituted.
  • a xylose metabolism-related gene other than the xylose isomerase gene is a xylose reductase gene encoding xylose reductase that converts xylose to xylitol, a xylitol dehydrogenase gene encoding xylitol dehydrogenase that converts xylitol to xylulose, and phosphorylating xylulose. It is meant to include the xylulokinase gene encoding xylulokinase that produces xylulose 5-phosphate. In addition, xylulose 5-phosphate generated by xylulokinase enters the pentose phosphate pathway and is metabolized.
  • the xylose metabolism-related gene is not particularly limited, and examples thereof include a xylose reductase gene derived from Pichia stipitis and a xylitol dehydrogenase gene, and a xylulokinase gene derived from Saccharomyces cerevisiae (Eliasson A. et al., Appl. Environ. Microbiol. , 66: 3381-3386 and Toivari MN et al., Metab. Eng. 3: 236-249).
  • a xylose reductase gene derived from Candida tropicalis or Candida parapsilosis can be used as the xylose reductase gene.
  • xylitol dehydrogenase gene a xylitol dehydrogenase gene derived from Candida tropicalis or Candida parapsilosis can be used.
  • a xylulokinase gene derived from Pichia stipitis can also be used as the xylulokinase gene.
  • the yeast originally having xylose metabolism ability is not particularly limited, and examples thereof include Pichia stipitis, Candida tropicalis, and Candida parapsilosis.
  • the recombinant yeast according to the present invention may be a yeast into which another gene has been introduced.
  • the other gene is not particularly limited, but may be, for example, a gene into which a gene involved in sugar metabolism such as glucose is introduced.
  • the recombinant yeast can be a yeast having ⁇ -glucosidase activity by introducing a ⁇ -glucosidase gene.
  • the ⁇ -glucosidase activity means an activity of catalyzing a reaction of hydrolyzing a ⁇ -glycosidic bond of a sugar. That is, ⁇ -glucosidase can degrade cellooligosaccharides such as cellobiose into glucose.
  • the ⁇ -glucosidase gene can also be introduced as a cell surface display type gene.
  • the cell surface display type gene is a gene modified so that the protein encoded by the gene is expressed so as to be displayed on the cell surface.
  • the cell surface display type ⁇ -glucosidase gene is a gene obtained by fusing a ⁇ -glucosidase gene and a cell surface localization protein gene.
  • the cell surface-localized protein refers to a protein that is fixed to the yeast cell surface and is present on the cell surface.
  • ⁇ - or a-agglutinin which is an aggregating protein, FLO protein and the like can be mentioned.
  • a cell surface localized protein has a secretory signal sequence on the N-terminal side and a GPI anchor attachment recognition signal on the C-terminal side. Although they have a secretory signal, they are common to secretory proteins, but they differ from secretory proteins in that cell surface localization proteins are fixed to the cell membrane and transported via GPI anchors.
  • the cell surface localization protein When passing through the cell membrane, the cell surface localization protein selectively cleaves the GPI anchor attachment recognition signal sequence, binds to the GPI anchor at the newly protruding C-terminal portion, and is fixed to the cell membrane. Thereafter, the base of the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC). Next, the protein separated from the cell membrane is incorporated into the cell wall, fixed to the cell surface, and localized on the cell surface (for example, see JP-A-2006-174767).
  • PI-PLC phosphatidylinositol-dependent phospholipase C
  • the ⁇ -glucosidase gene is not particularly limited, and examples thereof include a ⁇ -glucosidase gene derived from Aspergillus aculeatus (Murai et al., Appl. Environ. Microbiol. 64: 4857-4861).
  • ⁇ -glucosidase gene ⁇ -glucosidase gene derived from Aspergillus oryzae
  • ⁇ -glucosidase gene derived from Clostridium cellulovorans ⁇ -glucosidase gene derived from Saccharomycopsis fibuligera, and the like can be used.
  • the recombinant yeast according to the present invention may be one into which a gene encoding another enzyme constituting cellulase has been introduced in addition to the ⁇ -glucosidase gene or in addition to the ⁇ -glucosidase gene.
  • enzymes that constitute cellulase include exo-type cellobiohydrolases (CBH1 and CBH2) that release cellobiose from the ends of crystalline cellulose, non-crystalline cellulose (amorphous cellulose) chains that cannot degrade crystalline cellulose.
  • An end-type endoglucanase (EG) that cuts at random can be mentioned.
  • genes to be introduced into the recombinant yeast include an alcohol dehydrogenase gene having an activity of converting acetaldehyde to ethanol (ADH1 gene) and an acetyl-CoA synthase gene having an activity of converting acetic acid to acetyl-CoA. (ACS1 gene) and genes having an activity of converting acetaldehyde to acetic acid (ALD4 gene, ALD5 gene and ALD6 gene).
  • ADH4 gene genes having an activity of converting acetaldehyde to acetic acid
  • ALD4 gene genes having an activity of converting acetaldehyde to acetic acid
  • ALD4 gene genes having an activity of converting acetaldehyde to acetic acid
  • ALD4 gene genes having an activity of converting acetaldehyde to acetic acid
  • ALD4 gene genes having an activity of converting acetaldehyde to acetic acid
  • ALD6 gene genes having an
  • the recombinant yeast according to the present invention has a feature of highly expressing an alcohol dehydrogenase gene (ADH1 gene) having an activity of converting acetaldehyde to ethanol.
  • Methods for high expression of the gene include a method of substituting an endogenous promoter of the gene with a promoter for high expression and a method of introducing an expression vector capable of expressing the gene into yeast.
  • the nucleotide sequence of the ADH1 gene of ⁇ Saccharomyces ⁇ cerevisiae and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOS: 17 and 18, respectively.
  • the alcohol dehydrogenase gene to be highly expressed is not limited to those specified in SEQ ID NOs: 17 and 18, and is a gene having a different base sequence or amino acid sequence but having a paralog relationship or a narrow homolog relationship. It may be.
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOS: 17 and 18, and is, for example, 70% or more, preferably 80% or more, more preferably the amino acid sequence of SEQ ID NO: 14. It may consist of an amino acid sequence having 90% or more, most preferably 95% or more sequence similarity or identity, and may encode a protein having alcohol dehydrogenase activity. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings). The value of sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions.
  • the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOS: 17 and 18, and for example, substitution or deletion of one or several amino acids with respect to the amino acid sequence of SEQ ID NO: 18 It may consist of an inserted or added amino acid sequence and may encode a protein having alcohol dehydrogenase activity.
  • the term “several” means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOS: 17 and 18, and for example, for all or a part of the complementary strand of DNA consisting of the base sequence of SEQ ID NO: 17, It may hybridize under stringent conditions and encode a protein having alcohol dehydrogenase activity.
  • ⁇ stringent conditions '' as used herein means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a nucleotide sequence different from SEQ ID NO: 17 or a gene encoding an amino acid sequence different from SEQ ID NO: 18 functions as an alcohol dehydrogenase gene having an activity of converting acetaldehyde to ethanol
  • an expression vector in which the gene is inserted between an appropriate promoter and a terminator is prepared, and a host such as yeast is transformed with the expression vector, and the alcohol dehydrogenase activity of the expressed protein is measured. do it.
  • Alcohol dehydrogenase activity for converting acetaldehyde to ethanol is prepared by preparing a solution containing an aldehyde and NADH or NADPH as a substrate, allowing the protein to be tested to act at an appropriate temperature, and measuring the alcohol produced, or NAD + Alternatively, NADP + can be measured spectrophotometrically.
  • the recombinant yeast according to the present invention has a feature in which the expression level of the alcohol dehydrogenase gene (ADH2 gene) having an activity of converting ethanol to acetaldehyde is reduced.
  • Methods for reducing the expression level of the gene include modifying the endogenous promoter of the gene or deleting the gene. When the gene is deleted, one of the pair of ADH2 genes present in the diploid recombinant yeast may be deleted, or both may be deleted.
  • Methods for suppressing gene expression include so-called transposon method, transgene method, post-transcriptional gene silencing method, RNAi method, nonsense-mediated decay (Nonsense mediated decay, NMD) method, ribozyme method, antisense method, miRNA ( micro-RNA), siRNA (small-interfering-RNA), and the like.
  • the nucleotide sequence of the ADH2 gene of ⁇ Saccharomyces ⁇ cerevisiae and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 19 and 20, respectively.
  • the alcohol dehydrogenase gene of interest is not limited to those specified in SEQ ID NOs: 19 and 20, and is a gene having a different base sequence or amino acid sequence but having a paralog relationship or a narrow sense homolog relationship. May be.
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOs: 19 and 20, and may be, for example, 70% or more, preferably 80% or more, more preferably, the amino acid sequence of SEQ ID NO: 20. It may have an amino acid sequence having a sequence similarity or identity of 90% or more, most preferably 95% or more, and may encode a protein having alcohol dehydrogenase activity. Sequence similarity and identity values can be calculated by the BLASTN or BLASTX programs implementing the BLAST algorithm (default settings). The value of sequence similarity was calculated by calculating the sum of amino acid residues that completely match when a pair of amino acid sequences were subjected to pairwise alignment analysis and amino acid residues having physicochemically similar functions.
  • the identity value is calculated as a ratio of the number of the amino acid residues in all the compared amino acid residues by calculating an amino acid residue that completely matches when a pair of amino acid sequences are subjected to pairwise alignment analysis. .
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOs: 19 and 20, and for example, substitution or deletion of one or several amino acids with respect to the amino acid sequence of SEQ ID NO: 20 It may consist of an inserted or added amino acid sequence and may encode a protein having alcohol dehydrogenase activity.
  • the term “several” means, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and most preferably 2 to 5.
  • the alcohol dehydrogenase gene is not limited to those specified by SEQ ID NOs: 19 and 20, and for example, for all or a part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 19, It may hybridize under stringent conditions and encode a protein having alcohol dehydrogenase activity.
  • ⁇ stringent conditions '' as used herein means conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Laboratory Manual (Third Edition). can do.
  • the stringency can be set according to the temperature at the time of Southern hybridization or the salt concentration contained in the solution, and the temperature at the washing step of Southern hybridization or the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate) is at a temperature of 42 ° C.
  • a gene having a base sequence different from SEQ ID NO: 19 or a gene encoding an amino acid sequence different from SEQ ID NO: 20 functions as an alcohol dehydrogenase gene having an activity of converting ethanol to acetaldehyde
  • an expression vector in which the gene is inserted between an appropriate promoter and a terminator is prepared, and a host such as yeast is transformed with the expression vector, and the alcohol dehydrogenase activity of the expressed protein is measured. do it.
  • Alcohol dehydrogenase activity which converts ethanol to acetaldehyde, can be determined by preparing a solution containing alcohol and NAD + or NADP + as a substrate, allowing the protein to be tested to act at an appropriate temperature, and measuring the aldehydes produced, or measuring NADH.
  • NADPH can be measured by spectroscopic measurement.
  • genes to be introduced into the recombinant yeast include genes involved in the metabolic pathway of L-arabinose, a 5-carbon sugar contained in hemicellulose constituting biomass.
  • genes include, for example, prokaryotic L-arabinose isomerase gene, L-librokinase gene, L-ribulose-5-phosphate 4-epimerase gene, and eukaryotic L-arabitol-4-dehydrogenase.
  • L-xylose reductase gene examples include, for example, prokaryotic L-arabinose isomerase gene, L-librokinase gene, L-ribulose-5-phosphate 4-epimerase gene, and eukaryotic L-arabitol-4-dehydrogenase.
  • L-xylose reductase gene include, for example, prokaryotic L-arabinose isomerase gene, L-librokinase gene, L-ribulose-5-phosphate 4-epimerase gene, and e
  • genes to be introduced into the recombinant yeast include genes capable of promoting the use of xylose in the medium.
  • genes capable of promoting the use of xylose in the medium include a gene encoding xylulokinase having an activity of generating xylulose-5-phosphate using xylulose as a substrate.
  • the recombinant yeast can introduce a gene encoding an enzyme selected from a group of enzymes constituting a non-oxidation process pathway in the pentose phosphate pathway.
  • the enzymes constituting the non-oxidation process pathway in the pentose phosphate pathway include ribose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and transaldolase. It is preferable to introduce one or more genes encoding these enzymes. Further, it is more preferable to introduce two or more of these genes in combination, it is more preferable to introduce three or more of them in combination, and it is most preferable to introduce all kinds of genes.
  • the xylulokinase (XK) gene can be used without any particular limitation on the origin organism.
  • the XK gene is carried by many microorganisms such as bacteria and yeast that utilize xylulose. Information on the XK gene can be appropriately obtained by searching the NCBI website and the like.
  • XK genes derived from yeast, lactic acid bacteria, Escherichia coli, plants and the like are mentioned.
  • Examples of the XK gene include XKS1 (GenBank: Z72979), which is an XK gene derived from S. cerevisiae strain S288C (the nucleotide sequence and amino acid sequence of the coding region of CDS).
  • the transaldolase (TAL) gene, the transketolase (TKL) gene, the ribulose-5-phosphate epimerase (RPE) gene, and the ribose-5-phosphate ketoisomerase (RKI) gene are particularly Can be used without limitation.
  • Many genes having the pentose phosphate pathway carry these genes.
  • general-purpose yeasts such as S. cerevisiae also carry these genes. Information on these genes can be obtained as appropriate by accessing HP such as NCBI.
  • each gene is derived from the same genus as the host eukaryotic cell, such as eukaryotic cells or yeast, and more preferably from the same species as the host eukaryotic cell.
  • the TAL gene can be preferably used as the TAL gene
  • the TKL1 and TKL2 genes can be used as the TKL gene
  • the RPE1 gene can be used as the RPE gene
  • the RKI1 gene can be preferably used as the RKI gene.
  • these genes TAL1 gene derived from S. cerevisiae S288 strain (GenBank: U19102), TKL1 gene derived from S. cerevisiae S288 strain (GenBank: X73224), RPE1 gene derived from S. cerevisiae S288 strain (GenBank: X83571) and RKI1 gene (GenBank: Z75003) derived from S. cerevisiae ⁇ S288 ⁇ strain.
  • the recombinant yeast according to the present invention can be produced by introducing the above-mentioned acetaldehyde dehydrogenase gene into the genome of a yeast serving as a host and controlling the enzyme involved in trehalose accumulation by the yeast.
  • the acetaldehyde dehydrogenase gene may be introduced into a yeast that does not have xylose metabolism, may be introduced into a yeast that originally has xylose metabolism, or may be introduced into a yeast that does not have xylose metabolism. It may be introduced together with a xylose metabolism-related gene.
  • all the genes may be introduced simultaneously, or they may be introduced sequentially using different expression vectors.
  • yeasts that can be used as hosts include, but are not limited to, yeasts such as Candida Shehetae, Pichia stipitis, Pachysolen tanninophilus, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, with Saccharomyces cerevisiae being particularly preferred.
  • yeast may be an experimental strain used for convenience in experiments, or an industrial strain (practical strain) used for usefulness in practical use. Examples of industrial strains include yeast strains used for making wine, sake, and shochu.
  • yeast having homothallic properties As a yeast to be a host, it is preferable to use a yeast having homothallic properties. According to the technique disclosed in Japanese Patent Application Laid-Open No. 2009-34036, the use of yeast having homothallic properties makes it possible to easily introduce a multicopy gene into the genome.
  • the homothallic yeast has the same meaning as the homothallic yeast.
  • the yeast having homothallic properties is not particularly limited, and any yeast can be used. Examples of yeast having homothallic properties include, but are not limited to, Saccharomyces cerevisiae OC-2 strain (NBRC2260).
  • Other yeasts with homothallic properties include alcohol yeast (Taken No. 396, NBRC0216) (Source: "Properties of alcohol yeast", Sakeken Kaiho, No.
  • yeast having a heterothallic phenotype can be used as a homothallic yeast by introducing the HO gene in an expressible manner. That is, in the present invention, the yeast having homothallic properties includes a yeast into which the HO gene has been introduced so that it can be expressed.
  • the Saccharomyces cerevisiae OC-2 strain is preferable because it is a strain that has been used in wine brewing and has been confirmed to be safe. Further, the Saccharomyces cerevisiae OC-2 strain is preferable because it is a strain having excellent promoter activity under high sugar concentration conditions, as shown in Examples described later. In particular, the Saccharomyces cerevisiae OC-2 strain is preferable because the pyruvate decarboxylase gene (PDC1) promoter activity is high under high sugar concentration conditions.
  • PDC1 pyruvate decarboxylase gene
  • the promoter of the gene to be introduced is not particularly limited, for example, a glyceraldehyde 3-phosphate dehydrogenase gene (TDH3) promoter, a 3-phosphoglycerate kinase gene (PGK1) promoter, a hyperosmotic response 7 gene ( HOR7) promoter can be used.
  • TDH3 glyceraldehyde 3-phosphate dehydrogenase gene
  • PGK1 3-phosphoglycerate kinase gene
  • HOR7 hyperosmotic response 7 gene
  • the promoter of the pyruvate decarboxylase gene (PDC1) is preferable because of its high ability to highly express the downstream target gene.
  • the above-described gene may be introduced into the yeast genome together with a promoter for controlling expression and other expression control regions.
  • the above-described gene may be introduced such that its expression is controlled by the promoter of the gene originally present in the genome of the yeast as a host or other expression control regions.
  • any conventionally known method known as a yeast transformation method can be applied. Specifically, for example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”, lithium acetate method “J. Bacteriology, 153, p163 (1983)”, Proc. Natl. Acad. Sci. It can be implemented, but is not limited to this.
  • the method for producing ethanol according to the present invention is a method for synthesizing ethanol from a sugar source contained in a medium using the above-mentioned recombinant yeast.
  • the method for producing ethanol according to the present invention is characterized in that acetic acid contained in a medium can be metabolized by the above-mentioned recombinant yeast, and the concentration of acetic acid in the medium decreases with ethanol fermentation.
  • ethanol fermentation culture is performed in a medium containing at least glucose and / or xylose. That is, the medium for ethanol fermentation contains at least glucose and / or xylose as a carbon source.
  • the medium may contain another carbon source.
  • glucose and / or xylose contained in a medium used for ethanol fermentation can be derived from cellulosic biomass.
  • the cellulosic biomass may be one subjected to a conventionally known pretreatment.
  • the pretreatment is not particularly limited, and examples thereof include a treatment for decomposing lignin by a microorganism and a pulverization treatment for cellulosic biomass.
  • a treatment of immersing the pulverized cellulosic biomass in a dilute sulfuric acid solution, an alkali solution, or an ionic liquid, a hydrothermal treatment, or a pulverization treatment may be applied.
  • the medium may have a composition containing cellulose such as cellulosic biomass and cellulase.
  • the medium contains glucose produced by cellulase acting on cellulose.
  • the medium may have a composition containing cellulosic biomass and hemicellulase that saccharifies hemicellulose contained in the cellulosic biomass to produce xylose.
  • the medium contains xylose produced by the action of hemicellulase on hemicellulose.
  • a saccharified solution obtained by saccharifying cellulosic biomass may be added to a medium used for ethanol fermentation.
  • the saccharified solution contains remaining cellulose and cellulase and generated glucose, and remaining hemicellulose and hemicellulase and generated xylose.
  • the method for producing ethanol according to the present invention includes at least the step of ethanol fermentation using glucose and / or xylose as a sugar source.
  • the method for producing ethanol according to the present invention can produce ethanol by ethanol fermentation using glucose and / or xylose as a sugar source.
  • ethanol is recovered from the medium after ethanol fermentation.
  • the method for recovering ethanol is not particularly limited, and any conventionally known method can be applied. For example, after the above-mentioned ethanol fermentation is completed, a liquid layer containing ethanol and a solid layer containing recombinant yeast and solid components are separated by a solid-liquid separation operation. Thereafter, by separating and purifying the ethanol contained in the liquid layer by a distillation method, highly pure ethanol can be recovered. In addition, the purification degree of ethanol can be appropriately adjusted according to the purpose of use of ethanol.
  • fermentation inhibitors such as acetic acid and furfural may be produced in the above-mentioned pretreatment or saccharification treatment.
  • acetic acid is known to inhibit the growth and growth of yeast and reduce the efficiency of ethanol fermentation using xylose as a sugar source.
  • the method for producing ethanol according to the present invention can achieve an excellent ethanol yield as compared with the case where yeast which does not introduce an acetaldehyde dehydrogenase gene and a molecular chaperone gene belonging to the HSP70 family or the HSP40 family is used. .
  • the concentration of acetic acid in the medium is low.
  • the amount of acetic acid brought in can be reduced.
  • the amount of acetic acid brought in can be reduced for the same reason even when the cells are collected and reused after the end of the ethanol fermentation step.
  • the step of saccharifying cellulose contained in the culture medium with cellulase and the step of ethanol fermentation using xylose and glucose generated by saccharification as a sugar source proceed simultaneously, so-called, Simultaneous saccharification and fermentation may be used.
  • the simultaneous saccharification and fermentation treatment means a treatment that is simultaneously performed without discriminating between the step of saccharifying the cellulosic biomass and the step of ethanol fermentation.
  • the saccharification method is not particularly limited, and examples thereof include an enzymatic method using a cellulase preparation such as cellulase and hemicellulase.
  • the cellulase preparation contains a plurality of enzymes involved in the degradation of cellulose chains and hemicellulose chains, and exhibits a plurality of activities such as endoglucanase activity, endoxylanase activity, cellobiohydrolase activity, glucosidase activity, and xylosidase activity.
  • Examples of the cellulase preparation include, but are not particularly limited to, cellulases produced by Trichoderma reesei, Acremonium cellulolyticus, and the like. A commercially available cellulase preparation may be used.
  • a cellulase preparation and the above-mentioned recombinant yeast are added to a medium containing cellulosic biomass (which may be after pretreatment), and the recombinant yeast is cultured in a predetermined temperature range.
  • the cultivation temperature is not particularly limited, but may be 25 to 45 ° C., preferably 30 to 40 ° C. in consideration of the efficiency of ethanol fermentation.
  • the pH of the culture solution is preferably set to 4 to 6. In the culture, stirring and shaking may be performed.
  • Example 1 a recombinant yeast into which an acetaldehyde dehydrogenase gene was introduced and into which a molecular chaperone gene belonging to the HSP70 family or HSP40 family was introduced was prepared, and the acetic acid metabolizing ability of this recombinant yeast was evaluated.
  • AdhE acetaldehyde dehydrogenase gene
  • the ADH1 gene with the addition of the TDH3 promoter and the adhE gene with the addition of the HOR7 promoter and the DIT1 terminator (NCBI access No. 945837, the full length was changed to match the codon usage in yeast on the 5 'side) Sequence), a homologous recombination region on the yeast genome, a gene sequence (5U_ADH2) of a region about 700 bp upstream from the 5 'end of the ADH2 gene, and a gene sequence downstream from the 3' end of the ADH2 gene.
  • 2777477 full-length synthesized sequence of codon-converted to match the codon usage of yeast
  • a fusion product of an intron sequence contained in the COX5B gene of Saccharomyces cerevisiae BY4742 was used.
  • the marker gene and the Cre gene are sandwiched between two LoxP sequences, and by expressing the Cre gene, it is possible to remove the marker and the Cre gene simultaneously.
  • each DNA sequence can be amplified by PCR using the primers in Table 1.
  • a primer is added with a DNA sequence so as to overlap the adjacent DNA sequence by about 15 bp, and using them, a DNA fragment of interest is prepared using the Saccharomyces cerevisiae BY4742 genome or adhE synthetic gene DNA as a template.
  • Amplification DNA fragments were sequentially linked using In-Fusion (registered trademark) ⁇ HD ⁇ Cloning Kit, and the like, and cloned into plasmid pUC19 to prepare a final target plasmid.
  • This plasmid contains the ADH1 gene with a TDH3 promoter added to the 5 'end, the eutE gene with a HOR7 promoter and a DIT1 terminator added (NCBI access No. 946943, Sequence), a homologous recombination region on the yeast genome, a gene sequence (5U_ADH2) of a region about 700 bp upstream from the 5 'end of the ADH2 gene, and a gene sequence downstream from the 3' end of the ADH2 gene.
  • An 800 bp DNA sequence (3U_ADH2) was constructed so as to include a nat marker and a DNA recombinase Cre gene that performs a recombination reaction in a loxP sequence site-specific manner as a marker.
  • the marker gene and the Cre gene are sandwiched between two LoxP sequences, and by expressing the Cre gene, the marker and the Cre gene can be simultaneously removed.
  • each DNA sequence can be amplified by PCR using the primers in Table 1.
  • a primer is added with a DNA sequence so that the adjacent DNA sequence overlaps by about 15 bp, and using them, plasmid pUC-5U_ADH2-P_TDH3-ADH1-T_ADH1-T_DIT1-adhE-P_HOR7- LoxP-P_TEF1-SAT-T_LEU2-P_GAL1-CRE- T_CYC1-LoxP -3U_ADH2 or eutE Synthetic gene DNA as a template to amplify the target DNA fragment, In-Fusion (registered trademark) HD Cloning Kit, etc. After ligation and cloning into plasmid pUC19, the final target plasmid was prepared.
  • DnaJ gene transfer plasmid A plasmid containing a sequence necessary for introducing the DnaJ gene of a molecular chaperone derived from E. coli into yeast upstream of the SUC2 locus, pUC-5U850_SUC2-P_FBA1-dnaJ-T_RPL3-LoxP66- P_TEF1-SAT-T_LEU2-P_GAL1-Cre-T_CYC1-LoxP71-5U_SUC2 was produced.
  • This plasmid contains the DnaJ gene (NCBI access No.944753, full-length synthesized sequence of codons converted to match the codon usage frequency of yeast) with the addition of the FBA1 promoter and RPL3 terminator.
  • a gene sequence (5U850_SUC2) in a region of about 850 to 1450 bp upstream from the 5 ′ end of the SUC2 gene, a DNA sequence (5U_SUC2) in a region of about 850 b upstream from the 5 ′ end, and nat as a marker It was constructed so as to include a marker and a DNA recombinase Cre gene that performs a recombination reaction in a loxP sequence site-specific manner. The marker gene and the Cre gene are sandwiched between two LoxP sequences, and by expressing the Cre gene, the marker and the Cre gene can be simultaneously removed.
  • each DNA sequence can be amplified by PCR using the primers in Table 1.
  • a primer is added with a DNA sequence so that the adjacent DNA sequence overlaps by about 15 bp, and using them, plasmid pUC-5U_ADH2-P_TDH3-ADH1-T_ADH1-T_DIT1-adhE-P_HOR7- LoxP-P_TEF1-SAT-T_LEU2-P_GAL1-CRE- T_CYC1-LoxP -3U_ADH2, Saccharomyces cerevisiae BY4742 strain genome or DnaJ synthetic gene DNA as a template to amplify target DNA fragment, In-Fusion (registered trademark) HD Cloning Kit, etc.
  • the DNA fragments were sequentially ligated and cloned into plasmid pUC19 to prepare the final target plasmid.
  • DnaK gene transfer plasmid A plasmid containing a sequence necessary for introducing the DnaK gene of a molecular chaperone derived from E. coli into yeast upstream of the SUC2 locus, pUC-5U850_SUC2-P_HOR7-dnaK-T_DIT1-LoxP66- P_TEF1-SAT-T_LEU2-P_GAL1-Cre-T_CYC1-LoxP71-5U_SUC2 was produced.
  • This plasmid contains the DnaK gene (NCBI Access No. 944750, a full-length sequence obtained by converting codons in accordance with the codon usage frequency of yeast) with the addition of the HOR7 promoter and DIT terminator on the 5 'side.
  • a gene sequence of a region of about 850 to 1450 bp upstream from the 5 ′ end of the SUC2 gene (5U850_SUC2), and a DNA sequence of a region of about 850 b upstream of the 5 ′ end (5U_SUC2)
  • the marker was constructed to include the nat marker and the Cre gene, a DNA recombinase that performs recombination reaction in a loxP sequence site-specific manner.
  • each DNA sequence can be amplified by PCR using the primers in Table 1.
  • a primer is added with a DNA sequence so as to overlap the adjacent DNA sequence by about 15 bp, and using them, plasmid pUC-5U850_SUC2-P_FBA1-dnaJ-T_RPL3-LoxP66- P_TEF1-SAT- Amplify the target DNA fragment using T_LEU2-P_GAL1-Cre-T_CYC1-SLoxP71-5U_SUC2 or DnaK synthetic gene DNA as a template, sequentially bind DNA fragments using In-Fusion (registered trademark) HD Cloning Kit, etc., and clone into plasmid pUC19 Thus, a final target plasmid was prepared.
  • In-Fusion registered trademark
  • ⁇ Preparation of vector-introduced yeast strain The diploid yeast Saccharomyces cerevisiae OC-2 strain (NBRC2260) was used as a host, and the yeast was transformed using Frozen-EZ Yeast Transformation II (ZYMO RESEARCH) according to the attached protocol.
  • NBRC2260 diploid yeast Saccharomyces cerevisiae OC-2 strain
  • ZYMO RESEARCH Frozen-EZ Yeast Transformation II
  • the grown colonies were purified. Each of the purified selected strains was sporulated on a sporulation medium (1 g / L potassium phosphate, 1 g / L yeast extract, 0.5 g / L glucose, 20 g / L agar), and homosolic. Doubling was performed. A strain was obtained in which the ADH1 gene and the adhE gene or the ADH1 gene and the eutE gene were integrated into the ADH2 locus region of the diploid chromosome, and the ADH2 gene was disrupted.
  • a sporulation medium (1 g / L potassium phosphate, 1 g / L yeast extract, 0.5 g / L glucose, 20 g / L agar
  • Doubling was performed.
  • a strain was obtained in which the ADH1 gene and the adhE gene or the ADH1 gene and the eutE gene were integrated into the ADH2 locus region of the diploid chromosome, and the ADH2 gene was disrupted.
  • Each strain was further cultured in a YPGa a (10 g / L yeast extract, 20 g / L peptone, 20 g / L galactose) medium to induce the expression of the Cre gene, and a nat marker was obtained by a Cre / LoxP site-specific recombination reaction. And the Cre gene was removed.
  • YPGa a (10 g / L yeast extract, 20 g / L peptone, 20 g / L galactose) medium to induce the expression of the Cre gene, and a nat marker was obtained by a Cre / LoxP site-specific recombination reaction. And the Cre gene was removed.
  • the purified selected strains derived from the Uz2405dSm strain were named Uz2805 and Uz2801, respectively, and the selected strains derived from the Uz2233dSm strain were named Uz2806 and Uz2802, respectively. It was confirmed that each strain had a heterologous (one copy) recombination.
  • the genotypes of the produced strains are summarized in Table 2 below.
  • the test strain was inoculated into a 100 ml baffled flask into which 20 ml of a YPD liquid medium having a glucose concentration of 20 g / L (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) was dispensed, and the mixture was heated at 30 ° C. and 120 rpm. For 24 hours.
  • the cells were inoculated into a 24-well deep well plate into which 4.9 ml of a medium for ethanol production (glucose 225 g / L, yeast extract 10 g / L, peptone 20 g / L, acetic acid 2.0 g / L) was dispensed.
  • a fermentation test was performed at 0.3 g of dry cells / L), shaking culture (230 rpm, amplitude 25 mm, 30 ° C.) and a temperature of 31 ° C.
  • the 24-well deep well plate was covered with a silicon lid with a check valve in each treatment zone, and the generated carbon dioxide gas was released to the outside air, but oxygen was not allowed to enter from outside. Was kept anaerobic.
  • Glucose, acetic acid, and ethanol in the fermentation broth were measured using HPLC (Prominence; Shimadzu) under the following conditions.
  • the DnaK gene which is a molecular chaperone gene of the HSP70 family

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Abstract

La présente invention améliore la capacité d'assimilation d'acide acétique sans impliquer la destruction de la voie de production de glycérine par laquelle la fermentation de l'éthanol est dégradée. Un gène d'acétaldéhyde déshydrogénase et un gène chaperon moléculaire appartenant à la famille HSP 70 ou à la famille HSP 40 sont introduits.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023285282A1 (fr) * 2021-07-12 2023-01-19 Dsm Ip Assets B.V. Cellule de levure recombinée

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