JP2012254044A - Method for producing substance using metabolic pathway going through acetyl coa in yeast - Google Patents

Abstract

Provided is a yeast strain capable of producing a metabolite via acetyl-CoA at a high yield.
A yeast strain having enhanced activity of the following enzymes: (a) acetyl CoA synthase (ACS); and (b) acetyl CoA acetyltransferase (ERG10).
[Selection figure] None

Description

Background of the Invention

TECHNICAL FIELD The present invention relates to a method for producing a substance using a metabolic pathway via acetyl CoA in yeast.

Background Art Biofuels are attracting attention as an alternative fuel for oil against the background of the growing environmental problems. Ethanol is well known as a biofuel, but it has a lower energy density than gasoline and it must be mixed with gasoline in a limited concentration range to function as a transportation fuel. There are many drawbacks. Isopropanol has a higher octane number than 1-butanol, and is a highly versatile solvent that is also used as an industrial raw material. Isopropanol is produced by hydration of propylene from naphtha and has never been produced commercially from renewable sources.

  Fermentative production of acetone and butanol by microorganisms was carried out industrially at the beginning of the 20th century. Certain Clostridium bacteria that are absolutely anaerobic bacteria can produce acetone and butanol simultaneously (acetone-butanol fermentation). Acetone is produced using acetyl CoA present in bacterial cells as a raw material. Acetyl CoA is converted to acetoacetyl CoA by acetyl CoA acetyltransferase (thiolase), further converted to acetoacetate by CoA transferase, and further converted to acetone by acetoacetate decarboxylase. Certain Clostridium bacteria also have a secondary alcohol dehydrogenase that produces isopropanol from acetone. Chen et al. Reported that among 52 strains of the genus Clostridium including Clostridium beigerinki, there was a strain that produced 30 mM (1.8 g / L) of isopropanol at the maximum (Non-patent Document 1: Chen, J.-S., and SF Hiu. Acetone-butanol-isopropanol production by Clostridium beijerinckii (synonym, Clostridium butylicum). Biotechnol. Lett. 1986. 8: 371-376). Recently, Hanai et al. And Jojima et al. Introduced Escherichia coli with CoA transferase, acetoacetate decarboxylase, secondary alcohol dehydrogenase derived from the genus Clostridium, and excess endogenous acetyl CoA acetyltransferase. An E. coli strain that can efficiently produce isopropanol by expression is reported (Non-patent Document 2: T. Hanai, S. Atsumi, and JC Liao. Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli. Appl Environ Microbiol, Dec. 2007, p. 7814-7818; Non-Patent Document 3: Toru Jojima & Masayuki Inui & Hideaki Yukawa. Production of isopropanol by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 2008 77: 1219-1224). However, Escherichia coli is often not very resistant to various stresses such as sugar resistance, osmotic pressure resistance, salt resistance, organic solvent resistance, and there is a risk of phage contamination. In addition, low isopropanol resistance means that fermentation of the product is inhibited. In order to avoid fermentation inhibition, a method of continuing fermentation while removing the product from the medium using a gas stripping method or the like is known. However, the use of this method not only complicates the process of fermentation production. , Leading to increased costs. As one means for solving these problems, it is conceivable to produce isopropanol using a microorganism having high resistance to organic solvents.

  Yeast, which is a unicellular eukaryotic microorganism, exhibits high resistance to organic solvents, and, like microorganisms such as Escherichia coli, can be cultured at a lower cost than cells of higher organisms in terms of culture equipment, culture time and culture cost. Has characteristics. In particular, budding yeast is used in the production of alcoholic beverages such as beer and wine and bread, and its safety as a food product has been confirmed.

  In order to produce isopropanol in yeast, it is necessary to introduce exogenous CoA transferase, acetoacetate decarboxylase, and secondary alcohol dehydrogenase into the yeast strain. So far, as an example of production of isopropanol by yeast, it has been reported that isopropanol was produced by introducing genes of isopropyl alcohol producing enzyme group into Saccharomyces cerevisiae and culturing the transformant. (Patent Document 1: JP 2011-97929 A). However, the amount of isopropanol produced by this method is only a small amount of about 100 mg / L as the concentration in the culture solution. Since yeast is a eukaryote and organelles exist, there are multiple pathways for supplying acetyl CoA, which is a substrate for isopropanol synthesis, to the cytoplasm. To date, there has been no investigation of which route is advantageous for isopropanol synthesis by supplying acetyl-CoA to the cytoplasm, or what kind of yeast is superior in such ability.

Candida utilis, as well as Saccharomyces cerevisiae and Saccharomyces fragilis, is certified as an edible yeast by the FDA (Federal Food and Drug Administration). It is an edible yeast that has been approved for safe use. In fact, even today, Candida utilis is produced in various countries around the world, including Japan, Germany and the United States, and is used as feed. Candida utilis has been widely used in the industry as a production strain for microbial production by pentose assimilation, ethyl acetate, L-glutamic acid, glutathione, invertase and the like. In recent years, transformation methods have also been developed in Candida utilis, and techniques for producing various compounds in metabolic engineering have been developed (Non-patent Document 4: Hiroshi Shimada, Keiji Kondo, Paul D. Fraser, Yutaka). Miura, Toshiko Saito, and Norihiko Misawa., Increased Carotenoid Production by the Food Yeast Candida utilis through Metabolic Engineering of the Isoprenoid Pathway., Appl Environ Microbiol, July 1998, p. 2676-2680, Vol. 64, No. 7; Patent Document 5: Yutaka Miura, Keiji Kondo, Toshiko Saito, Hiroshi Shimada, Paul D. Fraser, and Norihiko MisawaProduction of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis., Appl Environ Microbiol, April 1998, .. p 1226-1229, Vol 64, No. 4; non-Patent Document ^{6: Keiji Kondo,, Yutaka Miura} , Hidetaka Sone, Kazuo Kobayashi & Hiroshi lijima., High-level expression of a sweet protein, monellin, in the food yeast Candida utilis., Nature Biotechnology 15, 453-457 (1997); Non-patent document 7: Yutaka Miura, Keiji Kondo, Hiroshi Shimada, Toshiko Saito, Katsumi Nakamura, Norihiko Misawa., Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid., Biotechnology and Bioengineering Volume 58 Issue 2-3, Pages 306-308; Non-Patent Document 8: Shigehito IKUSHIMA, Toshio FUJII, Osamu KOBAYASHI, Satoshi YOSHIDA and Aruto YOSHIDA., Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid., Bioscience, Biotechnology, and Biochemistry Vol. 73 (2009), No. 8 pp.1818-1824 Non-Patent Document 9: Yi-Ren Hong, Ya-Lei Chen, Lynn Farh, Wen-Jen Yang, Chen-Hua Liao and David Shiuan., Recombinant Candida utilis for the production of biotin., Applied Microbiology and Biotechnology., Volume 71 (2006), Number 2, 211-221). Candida utilis, unlike the Saccharomyces genus, is a crabtree-negative yeast, and therefore its respiratory activity does not decrease even in the presence of glucose. Thereby, the production | generation of ethanol can be suppressed by employ | adopting the conditions which fully ventilate. Furthermore, Candida utilis has a high respiratory activity, and once produced ethanol is promptly reanalyzed, it enables efficient production of bacterial cells and metabolites.

When producing a substance such as isopropanol in yeast, the production of ethanol as a by-product is generally a problem. In order to suppress this ethanol by-product, efforts have been made to delete the pyruvate decarboxylase gene ( PDC ), which is an important enzyme for ethanol production (Patent Document 2: WO 2010/095751; Patent Document) 3: International Publication No. 2010/095750; Non-Patent Document 10: Flikweert MT, Van Der Zanden L, Janssen WM, et al. Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12 (1996): 247-257; Non-Patent Document 11: van Maris AJA, Winkler AA, Porro D, et al. Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: Possible consequences of energy-dependent lactate export. Appl Environ Microbiol 70 (2004) : 2898-2905).

Chen, J.-S., and S. F. Hiu.Acetone-butanol-isopropanol production by Clostridium beijerinckii (synonym, Clostridium butylicum). Biotechnol. Lett. 1986. 8: 371-376 T. Hanai, S. Atsumi, and J. C. Liao. Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli. Appl Environ Microbiol, Dec. 2007, p. 7814-7818 Toru Jojima & Masayuki Inui & Hideaki Yukawa. Production of isopropanol by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 2008 77: 1219-1224 Hiroshi Shimada, Keiji Kondo, Paul D. Fraser, Yutaka Miura, Toshiko Saito, and Norihiko Misawa., Increased Carotenoid Production by the Food Yeast Candida utilis through Metabolic Engineering of the Isoprenoid Pathway., Appl Environ Microbiol, July 1998, p. 2676 -2680, Vol. 64, No. 7 Yutaka Miura, Keiji Kondo, Toshiko Saito, Hiroshi Shimada, Paul D. Fraser, and Norihiko MisawaProduction of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis., Appl Environ Microbiol, April 1998, p. 1226- 1229, Vol. 64, No. 4 Keiji Kondo ,, Yutaka Miura, Hidetaka Sone, Kazuo Kobayashi & Hiroshi lijima., High-level expression of a sweet protein, monellin, in the food yeast Candida utilis., Nature Biotechnology 15, 453-457 (1997) Yutaka Miura, Keiji Kondo, Hiroshi Shimada, Toshiko Saito, Katsumi Nakamura, Norihiko Misawa., Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid., Biotechnology and Bioengineering Volume 58 Issue 2-3, Pages 306- 308 Shigehito IKUSHIMA, Toshio FUJII, Osamu KOBAYASHI, Satoshi YOSHIDA and Aruto YOSHIDA., Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid., Bioscience, Biotechnology, and Biochemistry Vol. 73 (2009), No. 8 pp. 1818-1824 Yi-Ren Hong, Ya-Lei Chen, Lynn Farh, Wen-Jen Yang, Chen-Hua Liao and David Shiuan., Recombinant Candida utilis for the production of biotin., Applied Microbiology and Biotechnology., Volume 71 (2006), Number 2, 211-221 Flikweert MT, Van Der Zanden L, Janssen WM, et al. Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose.Yeast 12 (1996): 247-257 van Maris AJA, Winkler AA, Porro D, et al. Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: Possible consequences of energy-dependent lactate export.Appl Environ Microbiol 70 (2004): 2898-2905 JP 2011-97929 A International Publication No. 2010/095751 Pamphlet International Publication No. 2010/095750 Pamphlet

  The present inventors remarkably increase the production amount of substances via acetyl CoA in yeast by enhancing the activities of acetyl CoA synthase (ACS) and acetyl CoA acetyltransferase (ERG10) in yeast. I found out. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide yeast strains with enhanced enzyme activity related to synthesis and metabolism of acetyl CoA and acetoacetyl CoA, which are involved as intermediate substances in the fermentation production of various substances, and substances using the yeast strains. It is to provide a manufacturing method.

And the yeast strain by this invention is the following enzymes:
(A) acetyl CoA synthase (ACS); and (b) acetyl CoA acetyltransferase (ERG10).
Yeast strain with enhanced activity.

  Furthermore, the production method according to the present invention is a method for producing a substance, comprising culturing a yeast strain according to the present invention.

  According to the present invention, the metabolic pathway of yeast involving acetyl CoA and acetoacetyl CoA is activated. Since there are a wide variety of metabolic pathways involving acetyl CoA and acetoacetyl CoA, a wide variety of metabolites are produced. Therefore, according to the present invention, various substances can be produced with high efficiency by culturing yeast strains. Further, depending on the substance to be produced, it is also possible to produce the substance by incorporating the necessary enzyme gene into yeast in a form that can be expressed and culturing the obtained yeast strain.

Detailed description of the invention

  In the yeast strain according to the present invention, the following genes: (a) a gene encoding a polypeptide having an activity of acetyl CoA synthase (ACS); and (b) a poly having an activity of acetyl CoA acetyltransferase (ERG10). Expression of the gene encoding the peptide is enhanced.

  In the present specification, a yeast strain overexpressing four genes for synthesizing isopropanol together with the acetyl CoA synthase (ACS) gene and the acetyl CoA acetyltransferase (ERG10) gene is prepared and cultured. It has been demonstrated that very high concentrations of isopropanol are produced. The pathway for isopropanol production in this yeast strain is as shown in FIG. From the pathway shown in FIG. 1, it is clear that the high production amount of isopropanol is due to the enhancement of the enzyme activity related to the synthesis and metabolism of acetyl CoA and acetoacetyl CoA. Therefore, according to the present invention, not only isopropanol but also various substances that pass through acetyl CoA and acetoacetyl CoA can be produced at high production. As shown in FIG. 1, ACS and ERG10 are enzymes that function in the cytoplasm.

  Acetyl CoA supplied to the cytoplasm is a basic substance of various useful secondary metabolites such as a compound having a flavonoid skeleton, a compound having a terpenoid skeleton, a compound having a sterol skeleton, and a fatty acid. Therefore, if the yeast strain has a synthetic pathway for these secondary metabolites in a natural state or a state in which the gene has been artificially modified, these substances can be produced at high yields by the method of the present invention. .

In general, when a substance is produced by a microorganism, efforts are often made to suppress byproduct production. For example, in the case of producing a substance in yeast, generally, in order to suppress the production of ethanol as a by-product, efforts have been made to delete an enzyme gene involved in ethanol production, such as a pyruvate decarboxylase gene ( PDC ). (International Publication 2010/095751 pamphlet; International Publication 2010/095750 pamphlet; Flikweert MT, Van Der Zanden L, Janssen WM, et al. Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12 ( 1996): 247-257; van Maris AJA, Winkler AA, Porro D, et al. Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: Possible consequences of energy-dependent lactate export. Appl Environ Microbiol 70 (2004): 2898 -2905). However, in the present specification, it has been demonstrated that the yeast strain not deficient in PDC shows a significantly higher isopropanol production amount than the yeast strain deficient in the above PDC. Therefore, the yeast strain according to the present invention is also characterized in that the enzyme gene involved in ethanol production such as PDC is maintained as it is without being deleted, and as a result, the amount of isopropanol produced and thus acetyl CoA and its It is surprising that the production of acetoacetyl CoA, a metabolite, is significantly improved.

  As used herein, “enhanced enzyme activity” means that a stronger enzyme activity is observed in at least one stage of the culturing process compared to yeast that has not been subjected to a means for enhancing activity. To do. Enhancing enzyme activity can be realized by enhancing the expression of the enzyme gene, replacing it with an enzyme gene having a higher specific activity, or modifying the enzyme gene by a protein engineering method. In the present specification, the expression “enhanced” of a gene means that the expression level of the gene in the yeast is increased by genetic modification of the yeast, that is, the gene is overexpressed. Examples of such genetic modification methods include, for example, a method of transforming yeast with the gene (that is, a method of transfecting yeast in such a manner that the gene can be expressed), and a gene existing on the genome of the yeast. Various methods are known, such as a method for enhancing the promoter activity of the gene and a method for enhancing the expression of the gene by replacing the promoter of the gene present on the yeast genome with another promoter, preferably It is a method for transforming yeast with the gene.

Yeast The yeast strain according to the present invention is a yeast genetically modified so that a specific gene is overexpressed. The yeast used for genetic modification is not particularly limited as long as it is a yeast having a metabolic pathway via acetyl CoA or acetoacetyl CoA, but is preferably a crab tree effect-negative yeast with high respiratory activity, more preferably crab. It is called Candida utilis, a tree-negative yeast. Candida utilis strains are various strains known in the art, for example, NBRC0396, NBRC0619, NBRC0988, NBRC0639, NBRC0626, NBRC1086, NBRC10707, ATCC9255, ATCC28955, ATCC44638 Strain, ATCC 96621 strain, ATCC 18201 strain, ATCC 15239 strain and the like, but preferably NBRC1086 strain, ATCC 9255 strain or ATCC 15239 strain.

Enzymes with enhanced activity and their genes Enzymes with enhanced activity in the yeast strains according to the present invention include the following enzymes: (a) acetyl CoA synthase (ACS); and (b) acetyl CoA acetyltransferase (ERG10). ). According to a preferred embodiment of the present invention, the genes of these two enzymes are overexpressed in the yeast strain according to the present invention, that is, the following gene: (a) poly having the activity of acetyl CoA synthase (ACS) Expression of the gene encoding the peptide; and (b) the gene encoding the polypeptide having the activity of acetyl CoA acetyltransferase (ERG10) is enhanced.

  In the yeast strain according to the present invention, a gene encoding a polypeptide having the activity of the following gene: (c) ATP-citrate depletion addition enzyme (ACL); (d) mitochondrial inner membrane carnitine transporter (CRC1) The expression of at least one gene selected from the group consisting of a gene encoding a polypeptide having activity; and (e) a gene encoding a polypeptide having activity of the carnitine acetyltransferase gene (YAT1) is enhanced. Preferably, the expression of all of these genes (c), (d) and (e) is enhanced.

  These enzymes and genes are known to be derived from various yeasts, and their origin is not particularly limited. These enzymes and genes may be endogenous genes of yeast used for breeding and their expression products, or may be derived from other yeasts other than yeast used for breeding. For example, these enzymes and genes can be independently derived from Candida utilis or Yarrowia lipolytica. As the origin of each enzyme, for example, acetyl CoA synthase (ACS) is Candida utilis yeast, acetyl CoA acetyltransferase (ERG10) is Candida utilis yeast, mitochondrial inner membrane carnitine transporter (CRC1) is Candida utilis yeast, carnitine acetyltransferase gene (YAT1) is Candida utilis yeast, ATP-citrate removal and addition enzyme (ACL) is Yarrowia lipolytica yeast (ACL1 and ACLY derived from Yarrowia lipolytica constitute subunits) Gene).

  Among the acetyl CoA synthase (ACS) genes derived from Candida utilis, there are ACS1 gene and ACS2 gene. The coding sequence of ACS1 gene is represented by SEQ ID NO: 11, and the encoded amino acid sequence is represented by SEQ ID NO: 12. The coding sequence of ACS2 gene is represented by SEQ ID NO: 13, and the encoded amino acid sequence is represented by SEQ ID NO: 14. Acetyl CoA synthase (ACS) is an enzyme that converts acetic acid to acetyl CoA. The ACS gene used in the present invention may be either ACS1 or ACS2, but is preferably ACS2. The polypeptide having the activity of acetyl CoA synthase (ACS) is an amino acid in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14. It may be a polypeptide comprising a sequence and having ACS activity.

  The coding sequence of the acetyl CoA acetyltransferase (ERG10) gene derived from Candida utilis is represented by SEQ ID NO: 61, and the encoded amino acid sequence is represented by SEQ ID NO: 62. Acetyl CoA acetyltransferase (ERG10) is an enzyme that converts acetyl CoA to acetoacetyl CoA. The polypeptide having the activity of acetyl-CoA acetyltransferase (ERG10) has an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 62. It may be a polypeptide that contains and has ERG10 activity.

  The coding sequence of the ACL1 gene derived from Yarrowia lipolytica is represented by SEQ ID NO: 31, and the encoded amino acid sequence is represented by SEQ ID NO: 32. The polypeptide having ACL1 activity is a polypeptide having an ACL1 activity, including an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 32. It may be a peptide. Furthermore, the coding sequence of the ACLY gene derived from Yarrowia lipolytica is represented by SEQ ID NO: 33, and the encoded amino acid sequence is represented by SEQ ID NO: 34. The polypeptide having ACLY activity is a polypeptide having an ACLY activity, including an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 34. It may be a peptide. These ATP-citrate removal and addition enzymes (ACL) having ACL1 and ACLY as subunits are enzymes that convert citrate into acetyl CoA.

  The coding sequence of the mitochondrial inner membrane carnitine transporter (CRC1) gene derived from Candida utilis is represented by SEQ ID NO: 23, and the encoded amino acid sequence is represented by SEQ ID NO: 24. Mitochondrial inner membrane carnitine transporter (CRC1) is an enzyme that transports acetylcarnitine from the mitochondria to the cytoplasm. The polypeptide having the activity of the mitochondrial inner membrane carnitine transporter (CRC1) has an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 24. It may be a polypeptide that contains and has CRC1 activity.

  The coding sequence of the carnitine acetyltransferase gene (YAT1) gene derived from Candida utilis is represented by SEQ ID NO: 27, and the encoded amino acid sequence is represented by SEQ ID NO: 28. The carnitine acetyltransferase gene (YAT1) is an enzyme that converts acetylcarnitine into acetyl CoA. The polypeptide having the activity of the carnitine acetyltransferase gene (YAT1) includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 28. And a polypeptide having YAT1 activity.

  Amino acid deletion, substitution, addition, or insertion can be performed by modifying a gene encoding the above polypeptide by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis, such as Mutant-K (Takara Bio Inc.), Mutant-G (Takara Bio Inc.), etc. or using Takara Bio Inc. LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. be able to. The activity of each enzyme described above can be confirmed by a technique known in the art.

  Instead of the above genes, their equivalents can also be used. This equivalent means a gene in which some nucleotide residues are different on the condition that it has a function equivalent to each gene. Such equivalents include homology (sequence identity) of 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more with each nucleotide sequence. And a gene comprising a nucleotide sequence encoding a polypeptide having the activity of each enzyme. The equivalent further includes a gene comprising a nucleotide sequence that hybridizes with each nucleotide sequence or a complementary sequence thereof under stringent conditions and encodes a polypeptide having the activity of each enzyme. The equivalent further includes a nucleotide sequence that includes a sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in each nucleotide sequence, and encodes a polypeptide having the activity of each enzyme. The gene containing is mentioned.

  Here, deletion, substitution, addition, or insertion of a nucleotide residue can be performed by modifying a gene containing the above sequence by a technique known in the art. Mutation can be introduced into a gene by a known method such as Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis may be used. For example, using Mutant-K (Takara Bio) or Mutant-G (Takara Bio) or using Takara Bio's LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. Mutations can be introduced. The activity of each enzyme can be confirmed by a technique known in the art.

  The numerical value (%) indicating the homology (sequence identity) is calculated using default (initial setting) parameters using a base sequence comparison program: eg GENETYX-WIN 7.0.0. That is, each gene on the yeast chromosome may be replaced by a gene encoding a polypeptide that is not identical but has an equivalent function, ie, each activity, through homologous recombination or the like. The activity of each enzyme can be confirmed by a technique known in the art.

  The stringent conditions are, for example, rapid-hyb buffer (manufactured by GE Healthcare Bioscience), preferably at a temperature of 40 to 70 ° C., more preferably 60 ° C. Hybridization conditions. Then, for example, using a general method known to those skilled in the art, washing for 5 minutes with a solution consisting of 2 × SSC and 0.1% (w / v) SDS, followed by 1 × SSC and 0.1% ( w / v) Refers to washing for 10 minutes with a solution consisting of SDS, and further washing for 10 minutes with a solution consisting of 0.1 × SSC and 0.1% (w / v) SDS. However, by setting conditions such as the temperature conditions during hybridization and the salt concentration of the solution used for the subsequent membrane washing, it can be set to a certain level (70%, 80%, 85%, 90%, or 95%). ) DNA containing a base sequence having the above homology can be cloned. The gene thus obtained may be replaced by homologous recombination or the like with a gene that encodes a polypeptide that is not identical in sequence but has an equivalent function, that is, each activity. The activity of each enzyme can be confirmed by a technique known in the art.

Gene for synthesizing a useful substance The yeast strain according to the present invention may be transformed with a gene required for production of a desired useful substance for use in the production of the desired useful substance. For example, in order to produce a metabolite via acetyl CoA by the method of the present invention, the yeast strain according to the present invention may be transformed with a gene for synthesizing a metabolite via acetyl CoA. Necessary genes can be appropriately selected by those skilled in the art depending on the substance to be produced.

  For example, if isopropanol is to be produced, yeast strains can be transformed with an isopropanol synthesis gene as a further genetic modification. As the isopropanol synthesis gene, genes known to those skilled in the art, such as the enzyme gene group described in JP2011-97929A, can be used, for example, CoA transferase subunit A (ctfA), subunit B A combination of four genes, (ctfB), acetoacetate decarboxylase (adc) and secondary alcohol dehydrogenase (sadh) can be used.

  According to one embodiment of the present invention, the yeast strain according to the present invention comprises, as further genetic modifications, CoA transferase subunit A (ctfA), CoA transferase subunit B (ctfB), acetoacetate decarboxylase (Adc) and secondary alcohol dehydrogenase (sadh) are transformed with four genes encoding polypeptides having activity. Such a yeast strain can be used for the production of isopropanol, the production method comprising culturing the yeast strain according to this embodiment.

  These genes are known to be derived from various organisms, and their origin is not particularly limited. For example, these genes can be independently derived from Clostridium acetobutylicum or Clostridium begerinki. As the origin of each enzyme, for example, CoA transferase gene subunit A (ctfA) and subunit B (ctfB) are preferably Clostridium acetobutylicum, acetoacetate decarboxylase (adc) and secondary alcohol dehydrogenase (Sadh) is preferably Clostridium Begelinki.

  The amino acid sequence of CoA transferase subunit A (ctfA) derived from Clostridium acetobutylicum is represented by SEQ ID NO: 2. CoA transferase subunit A (ctfA) is an enzyme that, together with subunit B (ctfB), converts acetoacetyl-CoA to acetoacetate. The gene sequence encoding the amino acid sequence represented by SEQ ID NO: 2 can be appropriately selected by those skilled in the art, such as the sequence of the same gene derived from Clostridium acetobutylicum, and is not particularly limited, but preferably according to the present invention. The sequence uses a codon that is frequently used in the yeast strain species. For example, the nucleotide sequence represented by SEQ ID NO: 1 is an example of a gene sequence that matches the codon frequency in Candida utilis. The polypeptide having the activity of CoA transferase subunit A (ctfA) includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 2, And a polypeptide having ctfA activity.

  The amino acid sequence of CoA transferase subunit B (ctfB) derived from Clostridium acetobutylicum is represented by SEQ ID NO: 4. CoA transferase subunit B (ctfB) is an enzyme that, together with the subunit A (ctfA), converts acetoacetyl-CoA to acetoacetate. The gene sequence encoding the amino acid sequence represented by SEQ ID NO: 4 can be appropriately selected by those skilled in the art, such as the sequence of the same gene derived from Clostridium acetobutylicum, and is not particularly limited, but preferably according to the present invention. The sequence uses a codon that is frequently used in the yeast strain species. For example, a nucleotide sequence represented by SEQ ID NO: 3 is an example of a gene sequence that matches the codon frequency in Candida utilis. The polypeptide having the activity of CoA transferase subunit B (ctfB) includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 4, And a polypeptide having ctfB activity.

  The amino acid sequence of acetoacetate decarboxylase (adc) derived from Clostridium begerinki is represented by SEQ ID NO: 6. Acetoacetic acid decarboxylase (adc) is an enzyme that converts acetoacetic acid to acetone. The gene sequence encoding the amino acid sequence represented by SEQ ID NO: 6 is not particularly limited because it can be appropriately selected by those skilled in the art, such as the sequence of the same gene derived from Clostridium begerinki, but is preferably according to the present invention. The sequence uses a codon that is frequently used in the yeast strain species. For example, a nucleotide sequence represented by SEQ ID NO: 5 is an example of a gene sequence that matches the codon frequency in Candida utilis. A polypeptide having an activity of acetoacetate decarboxylase (adc) includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 6, and It may be a polypeptide having adc activity.

  The amino acid sequence of secondary alcohol dehydrogenase (sadh) derived from Clostridium begerinki is represented by SEQ ID NO: 8. Secondary alcohol dehydrogenase (sadh) is an enzyme that converts acetone to isopropanol. The gene sequence encoding the amino acid sequence represented by SEQ ID NO: 8 is not particularly limited because it can be appropriately selected by those skilled in the art, such as the sequence of the same gene derived from Clostridium begerinki, but is preferably according to the present invention. The sequence uses a codon that is frequently used in the yeast strain species. For example, a nucleotide sequence represented by SEQ ID NO: 7 is an example of a gene sequence that matches the codon frequency in Candida utilis. A polypeptide having secondary alcohol dehydrogenase (sadh) activity comprises an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 8, And a polypeptide having a sadh activity.

  Amino acid deletion, substitution, addition, or insertion can be performed by modifying a gene encoding the above polypeptide by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis, such as Mutant-K (Takara Bio Inc.), Mutant-G (Takara Bio Inc.), etc. or using Takara Bio Inc. LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. be able to. The activity of each enzyme described above can be confirmed by a technique known in the art.

  Instead of the above genes, their equivalents can also be used. This equivalent means a gene in which some nucleotide residues are different on the condition that it has a function equivalent to each gene. Such equivalents include homology (sequence identity) of 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more with each nucleotide sequence. And a gene comprising a nucleotide sequence encoding a polypeptide having the activity of each enzyme. The equivalent further includes a gene comprising a nucleotide sequence that hybridizes with each nucleotide sequence or a complementary sequence thereof under stringent conditions and encodes a polypeptide having the activity of each enzyme. The equivalent further includes a nucleotide sequence that includes a sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in each nucleotide sequence, and encodes a polypeptide having the activity of each enzyme. The gene containing is mentioned.

  Here, deletion, substitution, addition, or insertion of a nucleotide residue can be performed by modifying a gene containing the above sequence by a technique known in the art. Mutation can be introduced into a gene by a known method such as Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis may be used. For example, using Mutant-K (Takara Bio) or Mutant-G (Takara Bio) or using Takara Bio's LA PCR in vitro Mutagenesis series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. Mutations can be introduced. The activity of each enzyme can be confirmed by a technique known in the art.

  The numerical value (%) indicating the homology (sequence identity) is calculated using default (initial setting) parameters using a base sequence comparison program: eg GENETYX-WIN 7.0.0. That is, each gene on the yeast chromosome may be replaced by a gene encoding a polypeptide that is not identical but has an equivalent function, ie, each activity, through homologous recombination or the like. The activity of each enzyme can be confirmed by a technique known in the art.

  The stringent conditions are, for example, rapid-hyb buffer (manufactured by GE Healthcare Bioscience), preferably at a temperature of 40 to 70 ° C., more preferably 60 ° C. Hybridization conditions. Then, for example, using a general method known to those skilled in the art, washing for 5 minutes with a solution consisting of 2 × SSC and 0.1% (w / v) SDS, followed by 1 × SSC and 0.1% ( w / v) Refers to washing for 10 minutes with a solution consisting of SDS, and further washing for 10 minutes with a solution consisting of 0.1 × SSC and 0.1% (w / v) SDS. However, by setting conditions such as the temperature conditions during hybridization and the salt concentration of the solution used for the subsequent membrane washing, it can be set to a certain level (70%, 80%, 85%, 90%, or 95%). ) DNA containing a base sequence having the above homology can be cloned. The gene thus obtained may be replaced by homologous recombination or the like with a gene that encodes a polypeptide that is not identical in sequence but has an equivalent function, that is, each activity. The activity of each enzyme can be confirmed by a technique known in the art.

In addition to isopropanol, it is possible to construct yeast strains that produce substances such as specific carotenoids such as lycopene, squalene or specific sterols produced from squalene. For example, Candida utilis that expresses the GGPP synthase gene, phytoene synthase gene, phytoene desaturase gene from Erwinia uredovora and overexpresses the endogenous HMG-CoA reductase gene ( HMG1 ) increases lycopene. (Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N. Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway.Appl Environ Microbiol. 1998 Jul ; 64 (7): 2676-80). Also, Saccharomyces cerevisiae, which is deficient in the expression of the zymostosterol-24-methyltransferase gene and ergosta 5,7,24, (28) -trienol-22-dehydrogenase gene and engineered to overexpress HMG1. Recombinant strains are known to accumulate high concentrations of squalene (Japanese Patent Application Laid-Open No. 5-192184). By using these techniques, it is possible to construct a yeast strain that produces lycopene and squalene.

Molecular Breeding of Yeast Strain Molecular breeding of a yeast strain according to the present invention is performed by applying genetic modification for overexpression of the above gene group to the yeast strain in a state where the above gene group can be expressed, for example. It can be done by introducing. These gene groups are preferably integrated on the yeast chromosome by techniques such as homologous recombination. Selection of a gene sequence for homologous recombination to realize such integration on a chromosome is well known to those skilled in the art, and those skilled in the art can select an appropriate gene sequence for homologous recombination as necessary. Thus, a DNA fragment for homologous recombination can be constructed.

The position on the chromosome where the above gene group is integrated into the yeast genome is not particularly limited. For example, the orotidine 5 ′ phosphate decarboxylase gene ( URA3 ) locus, the alpha amino adipate reductase gene ( LYS2 ) locus Etc. can be selected. In addition, these genes may be placed under the control of a promoter originally present in the incorporated locus, such as the URA3 gene promoter, the LYS2 gene promoter, etc., but glyceraldehyde-3-phosphate dehydrogenase ( GAP ) may be placed under the control of another promoter such as a gene promoter.

  According to a preferred embodiment of the present invention, the yeast used for breeding the yeast strain according to the present invention is Candida utilis. Candida utilis is highly ploidy and does not form spores. When trying to introduce a mutation into a gene of a highly polyploid strain, it is necessary to add the mutation more severely than in a haploid strain. It is considered that the possibility of mutation is increased. Therefore, when introducing a mutation into the gene of Candida utilis, it is preferable to use a technique that can efficiently add multiple mutations only to the target gene. As a method for transforming Candida utilis, there is a technique described in JP-A No. 2003-144185. In this document, as a usable vector, a sequence homologous to the chromosomal DNA of Candida utilis and a selection marker gene are included, and the heterologous gene can be incorporated into the chromosomal DNA of Candida utilis by homologous recombination. A DNA sequence that can be transformed into Candida utilis, or a DNA sequence having an autonomous replication function in Candida utilis and a selectable marker gene, has been developed.

Examples of selectable marker genes that can be used in the transformation system include drug resistance markers that can function in yeast, preferably cycloheximide-resistant L41 gene, genes that confer geneticin (G418) resistance, or genes that confer hygromycin B resistance. There is. A gene that confers geneticin (G418) resistance or a gene that confers hygromycin B resistance is a sequence that does not exist in wild yeast, and is therefore considered to have a high probability of being incorporated into a target locus. In addition, these drug resistance genes are considered to have little effect on the host traits (Baganz F et al., 13 (16): 1563-73., 1997) (Cordero Otero R et al., Appl Microbiol Biotechnol, 46 (2): 143-8., 1996).

Another transformation system is the Cre-loxP system derived from bacteriophage P1. This is a site-specific recombination system between the loxP sequences of the two 34 bp, this recombination is catalyzed by Cre recombinase which Cre gene. This system has also been reported to function in yeast cells such as Saccharomyces cerevisiae, and it is known that the selectable marker gene placed between two loxP sequences is removed by recombination between loxP sequences. (Guldener, U. et al., Nucleic Acids Res., 24, 2519-24., 1996). This system has been utilized in several yeast species such as Kluyveromyces lactis (Steinsma, HY et al., Yeast, 18, 469-72., 2001).

Method for Producing Substance A substance (for example, isopropanol) can be produced in a culture by culturing the yeast strain according to the present invention in the presence of an appropriate carbon source. According to the method for producing a substance (for example, isopropanol) according to the present invention, the substance (for example, isopropanol) can be obtained by performing a step of separating the substance (for example, isopropanol) from the culture system. In the present invention, the culture includes cultured cells or microbial cells, cells or disrupted microbial cells, in addition to the culture supernatant.

In culturing the yeast strain according to the present invention, a culture method and culture conditions can be selected according to the kind of yeast. Examples of the culture method include a liquid culture method using a test tube, a flask, or a jar fermenter, and a culture format such as batch culture or semi-batch culture can be employed. The shaking speed and the aeration amount are generally 50 to 200 rpm, preferably 80 to 150 rpm, or an aeration amount corresponding to this shaking speed. The OD ₆₀₀ as the initial cell mass is generally 0.1 to 30, preferably 0.5 to 10. Further, in culturing the yeast strain according to the present invention, it is preferable that the shaking speed is fast (that is, the amount of aeration is preferably large), and the initial OD ₆₀₀ is preferably high. For example, in culture in a test tube or flask, the shaking speed is preferably about 120 rpm or more and the initial OD ₆₀₀ is about 10.

  In the method for producing a substance (for example, isopropanol) according to the present invention, the composition of the medium is not particularly limited as long as it is a composition containing various nutrients that allow yeast to grow and produce the target substance. As the assimilating carbon source contained in the medium, for example, xylose or sucrose can be used in addition to glucose as long as it can be assimilated. According to a preferred embodiment of the invention, the carbon source comprises glucose. A person skilled in the art can appropriately adjust the glucose concentration in the medium while confirming the production efficiency of the target substance. In particular, in the culture of the yeast strain according to the present invention, it is preferable to keep the glucose concentration in the medium at a relatively low concentration. For example, in the culture for 192 hours, the initial concentration of glucose is set to 100 g / L, and the initial concentration of glucose is set to 50 g / L compared to the case where glucose solid is added so that the final concentration becomes 100 g / L after 72 hours of culture. The production efficiency of the substance is higher when the glucose solid is added so that the final concentration becomes 50 g / L after 48 hours, 96 hours and 144 hours.

  As a nutrient source contained in the medium, for example, yeast extract, peptone, whey and the like are used, but a medium obtained by adding the above-mentioned activated carbon source to YP (10 g / L yeast extract, 20 g / L peptone), for example, YPD5 It is convenient to use a medium such as (50 g / L glucose, 10 g / L yeast extract, 20 g / L peptone), and the pH of which is appropriately adjusted.

  The fermentation temperature can be selected within the range in which the yeast to be used can grow. The fermentation temperature can be, for example, about 15 ° C to 45 ° C, more preferably 20 to 40 ° C, still more preferably 25 to 35 ° C, and most preferably about 30 ° C. Moreover, it is preferable to hold | maintain the pH of the culture medium in a fermentation process at 3-8, More preferably, it is pH 4-7, A pH regulator can be used as needed. Examples of the pH adjuster to be used include calcium carbonate, sodium hydroxide, potassium hydroxide and the like, and calcium carbonate is preferable.

  The reaction time required for the production of the substance (for example, isopropanol) is not particularly limited, and the reaction is performed for any reaction time as long as the effect of the present invention is recognized. Those skilled in the art can easily optimize these conditions.

  In the method for producing a substance (for example, isopropanol) according to the present invention, the substance (for example, isopropanol) produced in this way is separated and collected from the culture medium. The specific procedure for this is the substance to be separated and collected. Depending on the situation, those skilled in the art can easily select.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated to a more specific example, these Examples do not restrict | limit the technical scope of this invention.

Example 1: Selection of Candida utilis strains exhibiting high isopropanol resistance The ATCC 9206 strain, ATCC 96621 strain, ATCC 18201 strain, and ATCC 15239 strain were inoculated into YPD2 (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) and cultured at 30 ° C. Steady-phase yeast culture solution was serially diluted every 10 times, spotted on YPD2 agar medium containing 10 to 100 g / L isopropanol, and cultured at 30 ° C. The culture was continued for 3 days and the formation of colonies was observed. The results are listed in Table 1.

  From the above results, it was revealed that among the strains subjected to the assay, the NBRC1086 strain, the ATCC9255 strain, and the ATCC15239 strain showed the highest 65 g / L IPA resistance.

Example 2: Construction of Candida utilis ATCC15239 strain in which pyruvate decarboxylase gene was disrupted In Candida utilis ATCC15239 strain, its endogenous gene pyruvate decarboxylase gene ( PDC ) was disrupted. This gene disruption was carried out using the Cre-loxP system in the same manner as in International Publication No. 2010/095750 pamphlet. A strain (dPDC1 strain) in which the PDC1 allele was completely deleted was constructed by two disruptions. Although the dPDC1 strain was not completely deficient in ethanol production ability, the ethanol production ability was remarkably reduced.

Example 3: Construction of a vector expressing a CoA transferase subunit A gene, a subunit B gene derived from Clostridium acetobutylicum, an acetoacetate decarboxylase gene derived from Clostridium begerinki and a secondary alcohol dehydrogenase gene four genes involved in the synthesis, CoA transferase gene subunit a from Clostridium acetobutylicum (ctfA), the subunit B (ctfB), acetoacetic acid decarboxylase from Clostridium beijerinckii (adcs) and secondary alcohol dehydrogenase In order to efficiently express the enzyme ( sadh ) in Candida utilis, a novel gene sequence that does not exist in nature was designed and synthesized using the following items as indicators:
(1) Use of codons frequently used in Candida utilis;
(2) elimination of inappropriate restriction sites for gene cloning; and (3) for introducing the chromosomal integration type expression vectors, applying the BamH I site in the 'Xba I site and 3 at the end' terminal 5.

  The nucleotide sequences of the synthesized CoA transferase gene subunit A gene, subunit B gene, acetoacetate decarboxylase gene and secondary alcohol dehydrogenase gene are shown in SEQ ID NOs: 1, 3, 5 and 7, respectively. In addition, the amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NOs: 2, 4, 6 and 8, respectively. The plasmids having these genes were named GA09142-ctfA, GA09142-ctfB, GA09142-adc, and GA09142-sad, respectively.

pVT92 is an integration vector into the orotidine 5 ′ acid decarboxylase gene ( URA3 ) locus of Candida utilis and has a hygromycin resistance gene as a selection marker (WO 2010/095750 pamphlet). pVT92 can be chromosomally introduced by treating with BglII to give linear DNA. In order to express a foreign gene, pVT92 has a glyceraldehyde dehydrogenase gene promoter and a glycerate 3′-phosphate kinase gene terminator outside the Xba I / BamH I gap, respectively. In addition, pVT92 has an Nhe I site and a Spe I site on the 5 ′ end side of the promoter and the 3 ′ end side of the terminator, respectively.

pVT239 is an integration vector into the alpha amino adipate reductase gene ( LYS2 ) locus of Candida utilis, and has a geneticin resistance gene as a selection marker (WO 2010/095750 pamphlet). pVT239 can be chromosomally introduced by Not I treatment to give linear DNA. Moreover, pVT239 has a Spe I site on the 5 ′ end side of the geneticin resistance gene.

Four vectors GA09142-ctfA containing each isopropanol synthase gene, GA09142-ctfB, GA09142-adc , and GA09142-sadh, treated Xba I and BamH I, after purification by agarose gel extraction, with the same restriction enzymes Ligation to the treated pVT92 yielded pVT296, pVT298, pVT300 and pVT302. pVT296 and pVT300 were treated with Spe I and dephosphorylated. Further, the pVT298 and pVT302 after Nhe I, the Spe I treatment, followed by purification of the DNA fragment containing ctfB expression cassette, the sadh expression cassettes respectively, coupled to each pVT296 and PVT300, to obtain a pVT321 and PVT325. pVT325 was treated with Nhe I and Spe I and subjected to purification of the DNA fragment containing the adc expression cassette and sadh expression cassette, and introduced into Spe I site PVT239, was obtained PVT346. Finally obtained pVT321 have respective expression cassette and a hygromycin resistance gene ctfA and ctfB, a vector to be incorporated into URA3 locus of Candida utilis, PVT346 is a respective expression cassettes adc and sadh This vector has a geneticin resistance gene and is integrated into the LYS2 locus of Candida utilis.

Example 4: Construction of a Candida utilis strain having four isopropanol synthesis genes pVT321 was digested with BglII and concentrated by ethanol precipitation. Using the obtained DNA fragment, the C. utilis ATCC15239 strain and the dPDC1 strain obtained in Example 2 were transformed, smeared on a YPD medium containing 600 μg / mL hygromycin, and cultured at 30 ° C. for 2 days. . A strain having all the introduced genes was selected.

PVT346 was further digested with Not I and concentrated by ethanol precipitation. Each of the obtained DNA fragments was used to transform the two strains obtained above, smeared on a YPD medium containing 600 μg / mL hygromycin and 200 μg / mL geneticin, and cultured at 30 ° C. for 2 days. A strain having all the introduced genes was selected, and the isopropanol producing strain derived from the ATCC15239 strain was designated as TMS272 and the isopropanol producing strain derived from the dPDC strain was designated as the TMS290 strain.

Example 5: Isopropanol production test The isopropanol production ability of the transformant obtained in Example 4 was evaluated as shown below.

  Various transformants were inoculated into a 2 mL YPD liquid medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) / 14 mL test tube, and cultured with shaking at 140 rpm and 30 ° C. for 24 hours. The obtained pre-cultured solution was 100 mL YPD10 (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose) / 200 mL Erlenmeyer flask or YPD10C (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose, 45 g / L calcium carbonate) / 200 mL Erlenmeyer flask was inoculated, and cultured with shaking at 30 ° C. at an initial OD600 = 1, 100 rpm. After sampling from the culture solution over time and filtering with a 0.2 μm filter, quantitative analysis of various components was performed. Glucose, acetic acid, ethanol and glycerol were quantified using a high performance liquid chromatography (manufactured by Shimadzu Corporation), an ISep-ION300 column (manufactured by Tokyo Chemical Industry Co., Ltd.) and a suggested refractometer. Isopropanol and acetone were quantified using Gas Chromatography GC-2010 Plus (manufactured by Shimadzu Corporation).

  In addition, the same experiment was performed using YPD10C (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose, 45 g / L calcium carbonate) medium as a simple neutralization culture in a flask.

  The results of the culture test under non-neutralized conditions are shown in FIG. 2, and the results of the culture test under neutralized conditions are shown in FIG. The data is shown as an average of 3 repeated trials.

  According to FIG. 2, production of isopropanol was not observed in the parent strains ATCC15239 and dPDC1, whereas in the TMS272 and TMS290 strains into which the isopropanol synthesis gene was introduced, the maximum was 0.16 g / L and 0, respectively. Production of .13 g / L isopropanol was observed. Moreover, in the strain in which the PDC1 gene was disrupted, the sugar consumption rate and the growth ability were significantly reduced. In the TMS272 strain, glucose was completely consumed in 24 hours of culture and ethanol accumulation was observed, but the ethanol concentration decreased with the continuation of the subsequent culture. Since the production of isopropanol was observed while the ethanol concentration was decreasing, it was considered that the TMS272 strain produced isopropanol by secondary consumption of ethanol once produced. In the TMS290 strain derived from the dPDC1 strain, the production rate of isopropanol decreased, suggesting that the pathway through which cytoplasmic acetyl-CoA as a substrate is supplied from ethanol is the most important in isopropanol production in yeast. Since ethanol assimilation is an oxidative reaction, these results suggest that crabtree-negative yeasts with high respiratory activity, such as Candida utilis, may be suitable for isopropanol production by yeasts.

  According to FIG. 3, by adding calcium carbonate to the medium, the TMS272 strain produced 1.1 g / L of isopropanol, and the production amount increased from the non-neutralized condition. The TMS272 strain accumulated 16.2 g / L of acetic acid, and it was considered that acetic acid was accumulated for some reason, resulting in a decrease in pH and inhibition of isopropanol production. Also in the TMS290 strain, the isopropanol production increased to 0.9 g / L under neutralization conditions, while a high concentration of glycerol of 37.4 g / L was accumulated.

Example 6: Selection of genes that improve isopropanol productivity by overexpression A gene that improves isopropanol productivity by overexpression was selected. The metabolic pathway catalyzed by the candidate gene is described in FIG.

Candida utilis endogenous acetyl CoA hydrolase gene ( ACH1 ; nucleotide sequence: SEQ ID NO: 9; amino acid sequence: SEQ ID NO: 10), acetyl CoA synthase gene ( ACS1 ; nucleotide sequence: SEQ ID NO: 11; amino acid sequence: sequence) No. 12, ACS2 ; nucleotide sequence: SEQ ID NO: 13; amino acid sequence: SEQ ID NO: 14), cytoplasmic localized ethanol dehydrogenase gene ( ADH2 ; nucleotide sequence: SEQ ID NO: 15; amino acid sequence: SEQ ID NO: 16), mitochondrial localization Type ethanol dehydrogenase gene ( ADH3 ; nucleotide sequence: SEQ ID NO: 17; amino acid sequence: SEQ ID NO: 18), cytoplasmic localized acetaldehyde dehydrogenase gene ( ALD1 ; nucleotide sequence: SEQ ID NO: 19; amino acid sequence: SEQ ID NO: 20) , Tokondoria localized acetaldehyde dehydrogenase gene (ALD2; nucleotide sequence: SEQ ID NO: 21; amino acid sequence: SEQ ID NO: 22), the inner mitochondrial membrane carnitine transporter (CRC1; nucleotide sequence: SEQ ID NO: 23; amino acid sequence: SEQ ID NO: 24) , Mitochondrial localized carnitine acetyl-CoA transferase ( CAT2 ; nucleotide sequence: SEQ ID NO: 25; amino acid sequence: SEQ ID NO: 26), carnitine acetyltransferase gene ( YAT1 ; nucleotide sequence: SEQ ID NO: 27; amino acid sequence: SEQ ID NO: 28) YAT2; nucleotide sequence: SEQ ID NO: 29; amino acid sequence: SEQ ID NO: 30), acetyl-CoA acetyltransferase (ERG10; nucleotide sequence: SEQ ID NO: 61; amino acid sequence: SEQ ID NO: 62), Yarrowia Ripori It mosquito origin of ATP- citrate remove additional gene (ACL1; nucleotide sequence: SEQ ID NO: 31; amino acid sequence: Configuration SEQ ID NO: 34 subunit: SEQ ID NO: 32, ACLy;:; nucleotide sequence amino acid sequence SEQ ID NO: 33 Gene) was amplified by PCR using each genomic DNA as a template. The primers used for PCR are as follows: ( ACH1 ; SEQ ID NO: 35 and SEQ ID NO: 36), ( ACS1 ; SEQ ID NO: 37 and SEQ ID NO: 38), ( ACS2 ; SEQ ID NO: 39 and SEQ ID NO: 40), ( ADH2 SEQ ID NO: 41 and SEQ ID NO: 42), ( ADH3 ; SEQ ID NO: 43 and SEQ ID NO: 44), ( ALD1 ; SEQ ID NO: 45 and SEQ ID NO: 46), ( ALD2 ; SEQ ID NO: 47 and SEQ ID NO: 48), ( CAT2 : Sequence; No. 49 and SEQ ID NO: 50), (CRC1; SEQ ID NO: 51 and SEQ ID NO: 52), (YAT1; SEQ ID NO: 53 and SEQ ID NO: 54), (YAT2; SEQ ID NO: 55 and SEQ ID NO: 56), (ACL1; SEQ ID NO: 57 And SEQ ID NO: 58), ( ACLY ; SEQ ID NO: 59 and SEQ ID NO: 60), ( ERG10 ; SEQ ID NO: 63 and SEQ ID NO: 64).

The resulting PCR product was treated with Xba I / BamH I and treated with the same restriction enzymes. PCU155 (Keiji Kondo, Yutaka Miura, Hidetaka Sone, Kazuo Kobayashi & Hiroshi lijima., High-level expression of a sweet protein, monellin , in the food yeast Candida utilis., Nature Biotechnology 15, 453-457 (1997)). pCU155 has a cycloheximide resistance gene as a selection marker, is a vector that is multicopy-integrated at the URA3 locus, and has the same terminator as the glyceraldehyde dehydrogenase gene promoter outside the Xba I / BamHI gap, respectively. ing. Although the URA3 locus of isopropanol production strains are constructed so far is incorporated isopropanol synthase gene, C.Utilis is higher polyploid, since the URA3 locus is present multiple copies, more URA3 locus It is possible to introduce a gene. In addition, since the isopropanol synthesis gene is inserted into the URA3 locus by two-point recombination, the introduction of pCU155 does not cause substitution of the introduced gene. The plasmid from which the candidate gene was cloned was digested with BglII and concentrated by ethanol precipitation. C. utilis TMS272 strain was transformed with the obtained DNA fragment, smeared on YPD medium containing 600 μg / mL hygromycin, 200 μg / mL geneticin and 20 μg / mL cycloheximide, and cultured at 30 ° C. for 4 days. ACL1 and ACLY DNA were mixed and transformed at the same time. Fermentation tests were performed on each of the obtained 4 clones, and isopropanol productivity was investigated. The results are shown in FIG.

According to FIG. 4, ACS1, ACS2, CRC1, YAT1, ERG10 or overexpression of ACL,, the amount of isopropanol production was found to be a 1.5 to 4.0-fold. ACS1, ACS2, YAT1, and each gene of ACL is an enzyme genes involved in the synthesis of acetyl CoA, CRC1 gene is the carrier protein gene exported to the cytoplasm precursors of acetyl-CoA synthesis from mitochondria, ERG10 gene acetyl CoA Is a gene that synthesizes acetoacetyl-CoA from These results indicate that it is important to up-regulate the expression or activity of an enzyme gene for efficient production of cytoplasmic acetyl-CoA for high production of isopropanol by yeast.

Example 7: Construction and culture test of isopropanol production strain overexpressing ACL1 / ACLY, ACS2, ERG10, CRC1 and YAT1 simultaneously
ACL1, ACLy, the ACS2, ERG10, CRC1 and each gene YAT1 was cloned plasmid was digested with Bgll II, and concentrated by ethanol precipitation. Each of the obtained 5 μg DNA fragments was mixed, and this mixture was used to transform the TMS272 strain. The mixture was smeared on a YPD medium containing 600 μg / mL hygromycin, 200 μg / mL geneticin and 10 μg / mL cycloheximide, at 30 ° C. Cultured for 4 days. The obtained 80 clones were subjected to a fermentation test, and the strain with the highest isopropanol production was selected and designated TMS378 strain.

  The results of the culture test of TMS378 strain are shown in FIG. The TMS272 strain produced 1.3 g / L of isopropanol, whereas the TMS378 strain showed 9.8 g / L, an approximately 7.5-fold increase in isopropanol production. In addition, acetic acid accumulated 18.1 g / L in the TMS272 strain, whereas 4.5 g / L in the TMS378 strain, acetic acid as a main by-product was reduced to about a quarter.

Example 8: Influence of initial OD ₆₀₀ and shaking speed in isopropanol production of TMS378 strain The culture conditions of TMS378 strain suitable for isopropanol production were examined. The TMS378 strain was inoculated into a 2 mL YPD liquid medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) / 14 mL test tube, and cultured with shaking at 140 rpm at 30 ° C. for 24 hours. The obtained preculture solution was inoculated into a 100 mL YPD10C (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose, 45 g / L calcium carbonate) / 300 mL Erlenmeyer flask, and the initial OD ₆₀₀ was set to 1 or 10. The culture was performed with shaking at 100 rpm or 120 rpm at 30 ° C. Glucose solid was added to the culture solution to a final concentration of 100 g / L after 72 hours of culture, and the culture was further continued for 144 hours. It sampled with time and filtered with the 0.2 micrometer filter. Various quantitative analyzes were performed according to the method described in Example 5. The data is shown as an average of 3 repeated trials. The results are shown in FIG.

  According to FIG. 6, the highest isopropanol production was observed when cultured at an initial OD600 = 10 and 120 rpm, and a maximum of 18.5 g / L of isopropanol was produced after 144 hours of culture. The sugar yield at that time was 30.8%. From this, it became clear that the faster the shaking speed (the greater the air flow rate) and the higher the initial OD, the better for isopropanol production.

Example 9: Influence of added glucose concentration on isopropanol production of TMS378 strain The culture conditions of TMS378 strain suitable for isopropanol production were examined. The TMS378 strain was inoculated into a 2 mL YPD liquid medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) / 14 mL test tube, and cultured with shaking at 140 rpm at 30 ° C. for 24 hours. The obtained preculture solution was added to 100 mL YPD10C (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose, 45 g / L calcium carbonate) / 300 mL Erlenmeyer flask or YPD5C (20 g / L peptone, 10 g / L yeast extract). , 50 g / L glucose, 45 g / L calcium carbonate) / 300 mL Erlenmeyer flask and inoculated in an initial OD600 = 1, 120 rpm, 30 ° C. with shaking. For the sample cultured with YPD10C, glucose solid was added to a final concentration of 100 g / L after 72 hours of culture, and the culture was further continued for 192 hours. Moreover, about the sample which culture | cultivated by YPD5C, the glucose solid was added so that it might become a final concentration of 50 g / L 3 times every 48 hours of culture | cultivation, and also it culture | cultivated to 192 hours. In both conditions, the final glucose concentration is 200 g / L. It sampled with time and filtered with the 0.2 micrometer filter. Various quantitative analyzes were performed according to the method described in Example 5. The data is shown as an average of 3 repeated trials. The results are shown in FIG.

  According to FIG. 7, when culturing with YPD10C and adding 100 g / L glucose after 72 hours of culture, a maximum of 19.2 g / L of isopropanol was produced after 122 hours of culture. However, thereafter, no increase in isopropanol production was observed and acetic acid accumulated (27.2 g / L). The maximum sugar yield in the production of isopropanol was 29.0%. On the other hand, when culturing with YPD5C and adding 50 g / L of glucose three times every 48 hours, an increase in isopropanol was observed until 192 hours of culture, and a maximum of 27.4 g / L of isopropanol was produced after 192 hours of culture. It was done. In addition, acetic acid accumulation was hardly observed. The maximum sugar yield in the production of isopropanol under these conditions was 41.5%. From this, it became clear that it is preferable in isopropanol production to keep the concentration of added glucose at a relatively low concentration.

Example 10: Identification of genes that greatly contribute to the improvement in isopropanol production In order to identify genes that greatly contribute to the improvement in isopropanol production by simultaneously overexpressing, (i) overexpression of a combination of ACL1, ACLY and ACS2 (Ii) a strain overexpressing a combination of ACL1, ACLY and ERG10, (iii) a strain overexpressing a combination of ACS2 and ERG10, (iv) a strain overexpressing a combination of ACL1, ACLY, ACS2 and ERG10 Were respectively constructed, and isopropanol production was compared between a strain overexpressing a combination of ACL1, ACLY, ACS2, CRC1, ERG10, and YAT1 (TMS378 strain) and an overexpression strain of each single gene. The recombinant strain was constructed using the same method as in Example 6 and Example 7. The gene DNA to be incorporated was mixed and transformed at the same time. The obtained 32 clones were subjected to a fermentation test, and strains showing average isopropanol production were selected for each transgene combination. Each constructed strain was inoculated into a 2 mL YPD liquid medium (20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose) / 14 mL test tube, and cultured with shaking at 140 rpm at 30 ° C. for 24 hours. The obtained preculture was inoculated into 3 mL YPD10C (20 g / L peptone, 10 g / L yeast extract, 100 g / L glucose, 45 g / L calcium carbonate) / glass test tube, and initial OD600 = 1, 100 rpm, 30 ° C. Cultured with shaking. Sampling was performed over time, and isopropanol production was evaluated by high performance liquid chromatography and gas chromatography GC-2010. The data is shown as an average of 3 repeated trials.

  According to FIG. 8, it became clear that the amount of isopropanol produced was 2.2 to 2.7 times due to overexpression of ACS1, ACS2 and ERG10 alone. The isopropanol production of (i) a strain overexpressing a combination of ACL1, ACLY and ACS2 and (ii) a strain overexpressing a combination of ACL1, ACLY and ERG10 is the same as the strain overexpressing ACS2 and ERG10, respectively. There was no significant difference. On the other hand, (iii) in the strain overexpressing the combination of ACS2 and ERG10, a dramatic improvement in isopropanol production was observed, and the production amount was improved 11.6 times. In addition, (iii) a strain that overexpresses a combination of ACS2 and ERG10, (iv) a strain that overexpresses a combination of ACL1, ACLY, ACS2, and ERG10, a TMS378 strain (a combination of ACL1, ACLY, ACS2, CRC1, ERG10, and YAT1) No significant difference was observed in the amount of isopropanol produced among the strains of the strains overexpressing the.

  From these results, when overexpressing at the same time, overexpression of ACL, CRC1 and YAT1 contributes little to the improvement of isopropanol productivity, and in order to improve isopropanol productivity, ACS2 and ERG10 must be overexpressed simultaneously. Was shown to be the most important. From the above results, it is important to enhance the expression or activity of enzyme genes involved in the metabolic pathway for synthesizing acetyl CoA from ethanol, that is, the metabolic pathway for synthesizing acetyl CoA from acetic acid, for high isopropanol production by yeast. It is shown that.

FIG. 1 is a diagram showing an isopropanol synthesis pathway by yeast. FIG. 2 is a diagram showing the results of a fermentation test under a non-neutralizing condition of a strain into which an isopropanol production gene was introduced, and the isopropanol concentration (panel A), glucose concentration (panel B), and ethanol concentration (panel) in the culture solution. C), changes over time in acetic acid concentration (panel D), glycerol concentration (panel E) and OD600 (panel F). In each panel, black circles indicate the results for ATCC15239 strain, white circles indicate the results for TMS272 strain (ATCC15239 strain + IPA synthetic gene), black triangles indicate the results for the PDC1 disruption strain, and white triangles indicate the TMS290 strain (PDC disruption strain + IPA). The result of (synthetic gene) is shown. FIG. 3 is a diagram showing the results of a fermentation test under neutralization conditions of a strain into which an isopropanol production gene has been introduced. The concentration of isopropanol in the culture solution (panel A), glucose concentration (panel B), and ethanol concentration (panel C). ), Acetic acid concentration (panel D) and glycerol concentration (panel E) over time. In each panel, black circles indicate the results for ATCC15239 strain, white circles indicate the results for TMS272 strain (ATCC15239 strain + IPA synthetic gene), black triangles indicate the results for the PDC1 disruption strain, and white triangles indicate the TMS290 strain (PDC disruption strain + IPA). The result of (synthetic gene) is shown. FIG. 4 is a graph showing the amount of isopropanol produced by overexpressing various genes in the TMS272 strain as a relative amount with respect to the amount of isopropanol produced by the TMS272 strain. FIG. 5 is a diagram showing the results of a fermentation test of a strain overexpressing all genes that improve isopropanol bioacidity, and the isopropanol concentration (panel A), glucose concentration (panel B), and ethanol concentration (panel) in the culture solution. C), time-dependent changes in acetic acid concentration (panel D), glycerol concentration (panel E) and acetone concentration (panel F). In each panel, solid circle shows the results of the TMS272 strain, white circles TMS378 strain (TMS272 shares + ACL1: ACLY: ACS2: ERG10 : CRC1: YAT1) shows the results of. FIG. 6 is a diagram showing the results of a fermentation test (influence of initial OD ₆₀₀ and shaking speed) at 30 ° C. of TMS378 strain overexpressing all genes that improve isopropanol productivity, and the concentration of isopropanol in the culture solution (Panel A), glucose concentration (panel B), ethanol concentration (panel C), acetic acid concentration (panel D), glycerol concentration (panel E), and acetone concentration (panel F) over time. In each panel, the black circle indicates the result under the condition of initial OD600 = 1 and the number of stirring = 100 rpm, the black triangle indicates the result under the condition of initial OD600 = 1 and the number of stirring = 120 rpm, and the white circle indicates the initial OD600 = The result under the condition of 10 and the number of stirring = 100 rpm is shown, and the white triangle shows the result under the condition of the initial OD600 = 10 and the number of stirring = 120 rpm. FIG. 7 is a diagram showing the results of a fermentation test (influence of fed-batch glucose concentration) at 30 ° C. of the TMS378 strain overexpressing all genes that improve isopropanol productivity. Panel A shows the result of culturing for 192 hours, with an initial glucose concentration of 100 g / L, fed with 100 g / l of glucose after 72 hours of culture. Panel B shows the result of culturing for 192 hours, with an initial glucose concentration of 50 g / L, fed with 50 g / l glucose after 48 hours, 96 hours and 144 hours, respectively. In each panel, time-dependent changes in isopropanol concentration (black triangle), glucose concentration (black circle), ethanol concentration (white circle), and acetic acid concentration (white triangle) are shown. FIG. 8 is a graph showing the amount of isopropanol produced when various genes are overexpressed in the TMS272 strain.

Claims (15)

The following enzymes:
(A) acetyl CoA synthase (ACS); and (b) acetyl CoA acetyltransferase (ERG10).
Yeast strains with enhanced activity. The following genes:
(A) a gene encoding a polypeptide having an activity of acetyl CoA synthase (ACS); and (b) an expression of a gene encoding a polypeptide having an activity of acetyl CoA acetyltransferase (ERG10) is enhanced. The yeast strain according to claim 1. A polypeptide having ACS activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14, or (ii) one or several amino acids deleted in the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14, The yeast strain according to claim 2, which is a polypeptide having an amino acid sequence substituted, added or inserted and having ACS activity. A polypeptide having ERG10 activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 62, or (ii) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 62 The yeast strain according to claim 2 or 3, which is a polypeptide comprising a sequence and having ERG10 activity. In addition, the following genes:
(C) a gene encoding a polypeptide having the activity of ATP-citrate depletion additive enzyme (ACL);
(D) a gene encoding a polypeptide having the activity of the mitochondrial inner membrane carnitine transporter (CRC1); and (e) a gene encoding a polypeptide having the activity of the carnitine acetyltransferase gene (YAT1). The yeast strain according to any one of claims 1 to 4, wherein expression of at least one kind of gene is enhanced. The polypeptide having ACL activity consists of a polypeptide having ACL1 activity and a polypeptide having ACLY activity,
A polypeptide having ACL1 activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32, or (ii) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 32 A polypeptide comprising a sequence and having ACL1 activity;
A polypeptide having ACLY activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 34, or (ii) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 34 The yeast strain according to claim 5, which is a polypeptide comprising a sequence and having an ACLY activity. A polypeptide having CRC1 activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 24, or (ii) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 24 The yeast strain according to claim 5 or 6, which is a polypeptide comprising a sequence and having CRC1 activity. A polypeptide having YAT1 activity is
(I) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 28, or (ii) an amino acid wherein one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 28 The yeast strain according to any one of claims 5 to 7, which is a polypeptide comprising a sequence and having YAT1 activity.   The yeast strain according to any one of claims 1 to 8, which is a crab tree effect negative yeast.   The yeast strain according to any one of claims 1 to 9, which is Candida utilis.   The yeast strain according to any one of claims 1 to 10, which has been transformed with a gene for synthesizing a metabolite via acetyl CoA.   Coding a polypeptide having the activity of CoA transferase subunit A (ctfA), CoA transferase subunit B (ctfB), acetoacetate decarboxylase (adc) and secondary alcohol dehydrogenase (sadh) 4 The yeast strain according to claim 11, which is transformed with a gene of a species.   A method for producing a substance, comprising culturing the yeast strain according to any one of claims 1 to 12.   The method according to claim 13, wherein the substance is a metabolite via acetyl CoA.   15. A method according to claim 13 or 14, wherein the substance is isopropanol.

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