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WO2011043401A1 - Levure productrice d'alcool isopropylique et procédé de production d'alcool isopropylique - Google Patents

Levure productrice d'alcool isopropylique et procédé de production d'alcool isopropylique Download PDF

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Publication number
WO2011043401A1
WO2011043401A1 PCT/JP2010/067596 JP2010067596W WO2011043401A1 WO 2011043401 A1 WO2011043401 A1 WO 2011043401A1 JP 2010067596 W JP2010067596 W JP 2010067596W WO 2011043401 A1 WO2011043401 A1 WO 2011043401A1
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Prior art keywords
isopropyl alcohol
producing
enzyme
gene
yeast
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Japanese (ja)
Inventor
佳子 松本
のぞみ 竹林
均 高橋
光史 和田
昭彦 近藤
純 石井
貴志 近藤
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Mitsui Chemicals Inc
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Mitsui Chemicals Inc
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to an isopropyl alcohol-producing yeast and an isopropyl alcohol production method using the same.
  • Isopropyl alcohol produced from plant-derived materials is promising as a carbon-neutral propylene material because it can be converted to propylene through a dehydration process.
  • it is obliged to reduce carbon dioxide emissions by 5% compared to 1990 levels throughout the developed countries between 2008 and 2012 according to the Kyoto Protocol.
  • Carbon neutral propylene is extremely important for the global environment due to its versatility. It is.
  • Bacteria that produce isopropyl alcohol by assimilating plant-derived raw materials are already known (see, for example, International Publication No. 2009/008377, Chinese Patent Application Publication No. CN1043956A, and JP-A-61-67493). ). Among them, Escherichia coli described in International Publication No. 2009/008377 is capable of producing isopropyl alcohol at high production using glucose as a raw material, and is excellent as a biocatalyst for industrial production of isopropyl alcohol.
  • Fermentation methods which are substance production methods by culturing microorganisms, can be classified mainly into batch fermentation methods and continuous fermentation methods.
  • the batch fermentation method has an advantage that the culture is completed in a short time and damage caused by contamination with bacteria is small. Over time, the concentration of the product in the fermentation broth increases, and the productivity and yield decrease due to the influence of the inhibition of the product, etc., so stable, high yield and high productivity over a long period of time Is difficult to keep.
  • microorganisms are filtered through a separation membrane, etc., the product is recovered from the filtrate, and the filtered microorganisms are retained or refluxed in the fermentation broth, thereby being stable for a long time. It has been reported that high substance productivity can be obtained (see JP 2007-252367 A).
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a microorganism that produces isopropyl alcohol that can be used repeatedly, and a method for producing isopropyl alcohol using the microorganism.
  • the present invention is as follows. [1] The activity of an isopropyl alcohol-producing enzyme group consisting of isopropyl alcohol dehydrogenase, acetoacetate decarboxylase, CoA transferase, and thiolase is imparted or enhanced, and the enzyme activity of isopropyl alcohol dehydrogenase is derived from a gene introduced as a gene variant. Acid resistant isopropyl alcohol-producing yeast. [2] The isopropyl alcohol-producing yeast according to [1], wherein the yeast is Saccharomyces cerevisiae.
  • the isopropyl alcohol-producing yeast according to any one of [1] or [2], wherein the genetic variant is a genetic variant modified based on a codon usage frequency of a host.
  • Enzyme genes encoding the enzymes in the isopropyl alcohol producing enzyme group consisting of isopropyl alcohol dehydrogenase, acetoacetate decarboxylase, CoA transferase, and thiolase are independently selected from the genus Clostridium, Bacillus, and Escherichia.
  • the isopropyl alcohol-producing yeast according to any one of [1] to [3], wherein the yeast is derived from an enzyme gene selected from the group consisting of yeasts belonging to the genus Acetabria or Saccharomyces.
  • yeast is derived from an enzyme gene selected from the group consisting of yeasts belonging to the genus Acetabria or Saccharomyces.
  • the isopropyl alcohol-producing yeast according to 1. [7] The isopropyl alcohol-producing yeast according to any one of [1] to [6], wherein the enzyme activity of acetoacetate decarboxylase, CoA transferase, and thiolase is derived from a gene introduced as a gene variant.
  • Any one of the enzyme genes encoding the respective enzymes in the isopropyl alcohol-producing enzyme group including the gene modification satisfies at least one of the following amino acid codon usage frequencies [1] to [7 ]
  • the isopropyl alcohol-producing yeast according to any one of (1) The ratio of codons encoding alanine [GCT: GCC: GCA: GCG] [35 to 45:15 to 25:25 to 40: 0 to 15], (2) The ratio of codon [AAT: AAC] encoding asparagine is [45 to 70: 30 to 55], (3) The ratio of codons [TGT: TGC] encoding cysteine is [50 or more and 80 or less: 20 or more and 50 or less], (4) The ratio of codons [GGT: GGC: GGA: GGG] encoding glycine [45 to 55:15 to 25:10 to 25: 0 to 15], (5) The ratio of codons [GAA: GAG] encoding glutamic acid [60
  • the method for producing isopropyl alcohol according to [9] comprising treating the isopropyl alcohol-producing yeast with an acid having a pH of 4 or less before or after producing isopropyl alcohol.
  • the activity of an isopropyl alcohol-producing enzyme group consisting of isopropyl alcohol dehydrogenase, acetoacetate decarboxylase, CoA transferase, and thiolase is imparted or enhanced, and the activity of isopropyl alcohol dehydrogenase is introduced as a gene variant.
  • the isopropyl alcohol production method of the present invention includes producing isopropyl alcohol using the isopropyl alcohol-producing yeast.
  • the isopropyl alcohol-producing yeast of the present invention is an acid-resistant yeast.
  • the enzyme gene encoding at least one enzyme selected from the group of enzymes to which the activity of the above-mentioned isopropyl alcohol-producing enzyme group has been imparted or strengthened has been introduced as a genetic modification, Even yeast species that do not have alcohol-producing ability can produce isopropyl alcohol.
  • the isopropyl alcohol-producing yeast of the present invention has acid resistance, it can be reused by preventing acid contamination by acid treatment.
  • “Granting” or “enhancement” of activity in the present specification means that the expression level of an enzyme gene (regardless of origin) in the host is increased based on the gene introduced from outside the cell into the host.
  • the host yeast enhances the promoter activity of the enzyme gene held on the genome or replaces it with another promoter. Or by strengthening the enzyme activity as a result of reducing or inactivating the repressor activity of the enzyme gene.
  • the “host” means the yeast that becomes the isopropyl alcohol-producing yeast of the present invention as a result of introduction of one or more genes from outside the cell.
  • the term “process” is not limited to an independent process, and is included in this term if the intended action of this process is achieved even when it cannot be clearly distinguished from other processes. .
  • a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively. The present invention will be described below.
  • the isopropyl alcohol-producing yeast of the present invention is an acid-resistant isopropyl alcohol-producing yeast in which the activity of the above isopropyl alcohol-producing enzyme group is imparted or enhanced, and the activity of isopropyl alcohol dehydrogenase is derived from a gene introduced as a gene variant. is there.
  • the host in the present invention is an acid-resistant yeast.
  • the term “acid resistance” means that the acid used for the recycling treatment is resistant to the extent that the ability to produce isopropyl alcohol is not lost. Specifically, yeast that does not lose its ability to produce isopropyl alcohol under pH conditions of 4 or less is included.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • the acetoacetate decarboxylase in the present invention is classified into enzyme number 4.1.1.4 based on the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and produces acetone from acetoacetate.
  • the generic name of the enzyme that catalyzes Examples thereof include Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii (Clostridium beijerinckii) and other abacterial bacterium (Bacillus polymyxa), And those derived from the genus Acettabria.
  • the gene encoding acetoacetate decarboxylase to be introduced into the host yeast of the present invention is based on DNA having the base sequence of the gene encoding acetoacetate decarboxylase obtained from each of the aforementioned derived organisms or a known base sequence thereof. Synthetic DNA sequences synthesized in this way can be used. Preferable examples include those derived from Clostridium bacteria or Bacillus bacteria, and examples thereof include DNA having a nucleotide sequence of a gene derived from Clostridium acetobutylicum or Bacillus polymixa. Particularly preferred is DNA having a base sequence of a gene derived from Clostridium acetobutylicum.
  • the isopropyl alcohol dehydrogenase in the present invention is classified into enzyme number 1.1.1.180 according to the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and a reaction for producing isopropyl alcohol from acetone. This is a general term for enzymes that catalyze. As such a thing, the thing derived from Clostridium bacteria, such as Clostridium beijerinki, is mentioned, for example.
  • the gene encoding isopropyl alcohol dehydrogenase introduced into the host yeast of the present invention is synthesized based on DNA having the base sequence of the gene encoding isopropyl alcohol dehydrogenase obtained from each of the above-mentioned derived organisms or a known base sequence thereof.
  • the synthesized synthetic DNA sequence can be used. Preferable examples include those derived from Clostridium bacteria, for example, DNA having a base sequence of a gene derived from Clostridium begerinki.
  • the CoA transferase in the present invention is classified into enzyme number 2.8.3.8 based on the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and generates acetoacetate from acetoacetyl CoA.
  • the generic name of the enzyme that catalyzes As such, for example, Clostridium bacteria such as Clostridium acetobutylicum and Clostridium beigerinki, Roseberia bacteria such as Roseburia intestinalis, Faecalibacteria prainziz, etc. Examples include bacteria derived from bacteria, Coprococcus, Trypanosoma brucei, and other Escherichia bacteria such as Escherichia coli (Escherichia coli).
  • the gene encoding the CoA transferase introduced into the host yeast of the present invention was synthesized based on the DNA having the base sequence of the gene encoding the CoA transferase obtained from each of the above-mentioned derived organisms or a known base sequence thereof. Synthetic DNA sequences can be utilized. Suitable examples include Clostridium bacteria such as Clostridium acetobutylicum, Roseburia bacteria such as Roseburia intestinalis, Facalibacteria bacteria such as Fakaribacterium plausents, Coprococcus bacteria, Trypanosoma brucei, etc.
  • DNA having the base sequence of a gene derived from a bacterium belonging to the genus Escherichia such as Trypanosoma cerevisiae and Escherichia coli is exemplified. More preferable examples include those derived from Clostridium bacteria or Escherichia bacteria, and particularly preferred is DNA having a base sequence of a gene derived from Clostridium acetobutylicum or Escherichia coli.
  • the thiolase in the present invention is classified into enzyme number 2.3.1.9 based on the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and a reaction for producing acetoacetyl CoA from acetyl CoA.
  • This is a general term for enzymes that catalyze. Examples thereof include yeasts such as Saccharomyces cerevisiae, Clostridium bacteria such as Clostridium acetobutylicum and Clostridium begerinki, Escherichia bacteria such as Escherichia coli, and Halobacterium species (Halobacterium species). sp.) Bacteria, Zoogloa ramigera and other Zogroa bacteria, Rhizobium sp.
  • Candida tropicalis such as Candida tropicalis
  • Caulobacter bacteria such as Streptomyces Colinas (Streptomyces collinus) genus Streptomyces bacteria such as, those derived from the genus Enterococcus bacteria such as Enterococcus Fakarisu (Enterococcus faecalis), and the like.
  • a DNA having a base sequence of a gene encoding a thiolase obtained from each of the above-described organisms or a synthetic DNA synthesized based on the known base sequence Arrays can be used.
  • Suitable examples include yeasts such as Saccharomyces cerevisiae, Clostridium acetobutylicum, Clostridium bacteria such as Clostridium begerinki, Escherichia bacteria such as Escherichia coli, Zogroa such as Halobacterium lamiguera, etc.
  • Genus bacteria Rhizobium species bacteria, Bradyrizobium bacteria such as Bradyrizobium japonicacum, Candida bacteria such as Candida tropicalis, Caulobacter bacteria such as Caulobacter crescentus, Streptomyces corinas etc.
  • Examples thereof include DNA having a base sequence of a gene derived from Enterococcus bacteria such as Streptomyces bacteria and Enterococcus facaris. More preferable examples include those derived from Clostridium bacteria or Escherichia bacteria, and particularly preferably a DNA having a base sequence of a gene derived from Saccharomyces cerevisiae, Clostridium acetobutylicum or Escherichia coli. It is.
  • the four types of enzymes are preferably derived from at least one selected from the group consisting of Clostridium bacteria, Bacillus bacteria, and Escherichia bacteria, from the viewpoint of enzyme activity.
  • Acetate decarboxylase and isopropyl alcohol dehydrogenase are derived from Clostridium bacteria
  • CoA transferase activity is derived from Clostridium bacteria or Escherichia bacteria
  • thiolase activity is derived from Saccharomyces, or these four kinds More preferably, the enzymes are all derived from Clostridium bacteria.
  • the activity of at least one enzyme selected from the group consisting of isopropyl alcohol dehydrogenase, acetoacetate decarboxylase and CoA transferase is derived from the transgene, and the thiolase activity is an endogenous gene. From the viewpoint of the ability to produce isopropyl alcohol, it is preferably derived from the enhanced expression of.
  • the four types of enzymes according to the present invention are preferably derived from any one of Clostridium acetobutylicum, Clostridium bayerinki, Escherichia coli or Saccharomyces cerevisiae from the viewpoint of enzyme activity.
  • Carboxylase is an enzyme derived from Clostridium acetobutylicum
  • CoA transferase is an enzyme derived from Clostridium acetobutylicum or Escherichia coli
  • thiolase is an enzyme derived from Saccharomyces cerevisiae, Clostridium acetobutylicum or Escherichia coli, isopropyl
  • the alcohol dehydrogenase is an enzyme derived from Clostridium begerinki, and the four types of enzymes described above are enzyme activities.
  • the acetoacetate decarboxylase activity is derived from Clostridium acetobutylicum
  • the isopropyl alcohol dehydrogenase activity is derived from Clostridium begerinki
  • the CoA transferase activity is derived from Clostridium acetobutylicum or Escherichia coli
  • the thiolase activity is saccharoma. It is particularly preferred that it is derived from Isis cerevisiae.
  • the four types of enzymes according to the present invention are preferably derived from Clostridium acetobutylicum, Clostridium beigelinki, Escherichia coli, and Saccharomyces cerevisiae from the viewpoint of enzyme activity.
  • the enzyme activity of isopropyl alcohol dehydrogenase among the isopropyl alcohol-producing enzymes is derived from a gene introduced as a genetic modification.
  • the “gene variant” includes all the nucleotide sequences of the enzyme gene that have been modified such as deletion, substitution, addition and the like. Specifically, among the base sequence of the enzyme gene, only the codon is changed, and the amino acid sequence synthesized based on the base sequence obtained by changing only the codon is not changed, or the enzyme gene The amino acid sequence synthesized based on the base sequence in which only the promoter region is changed and only the promoter region is changed is exemplified.
  • the enzyme gene to be modified may be an original gene of the host or may be an enzyme gene derived from various yeasts. Only the enzyme gene encoding isopropyl alcohol dehydrogenase may be genetically modified, and the enzyme gene encoding an enzyme other than isopropyl alcohol dehydrogenase may be genetically modified at the same time. Specifically, the codon of the enzyme gene encoding CoA transferase and the codon of the enzyme gene encoding acetoacetate decarboxylase, the promoter region of the enzyme gene encoding thiolase, and CoA transferase are encoded. And the codons of the enzyme gene encoding acetoacetate decarboxylase are modified.
  • any modification may be applied as long as the enzyme activity of the corresponding enzyme is imparted or enhanced to the host as a result of the genetic modification.
  • the genetically modified product is a modified gene obtained by modifying the used codon based on the codon usage frequency of yeast.
  • to modify the codon used means to modify a codon that is a sequence of 3 bases corresponding to each amino acid on the gene sequence that defines the amino acid sequence.
  • Codon modification means that only the base sequence is modified without changing the amino acid sequence.
  • the codon usage frequency is greatly different from that of the host when the host is yeast, for example.
  • the codons encoding alanine are GCT, GCC, GCA, and GCG.
  • GCA is used at a frequency of 50% and GCC is used at a rate of 0%.
  • GCA usage frequency bias can be prevented and the expression level of the enzyme in the host can be increased.
  • Tables 1 to 6 show codon usage frequencies of typical amino acids in the present invention. In the table, amino acids defined by one codon were excluded. Table 1 shows codon usage frequency (original) of isopropyl alcohol dehydrogenase (IPAdh), codon usage frequency of isopropyl alcohol dehydrogenase (# 1, SEQ ID NO: 35, # 2 SEQ ID NO: 52) after codon modification in the present invention, and Table 2 shows acetoacetic acid Decarboxylase (Adc) codon usage (original), codon usage after codon modification in the present invention (SEQ ID NO: 58), Tables 3 to 6 are CoA transferases (ctfA, ctfB, atoD, atoA) ) Codon usage (original), CoA transferase after modification of the codon in the present invention (modified ctfA SEQ ID NO: 62, modified ctfB SEQ ID NO: 66, modified atoD SEQ ID NO: 70, modified atoA SEQ
  • codon modification for eliminating codon bias of isopropyl alcohol dehydrogenase, acetoacetate decarboxylase, and CoA transferase.
  • Further preferred amino acid usage frequencies (%) are shown below. However, this was excluded when the number of corresponding amino acids in the entire sequence was 2 or less.
  • the codon usage frequency used for reference is the database of the Kazusa DNA Research Institute: http: // www. kazusa. or. Data of Saccharomyces cerevisiae described in jp / codon were used. In the data, it is expressed as uracil (U) on RNA, but in the text, it was expressed as thymidine (T) following the DNA sequence.
  • the codon usage frequency in the data was summed up with the codon usage frequency corresponding to each amino acid, and the value was set to 100, and the usage frequency corresponding to the codon of each amino acid was calculated.
  • the frequency of lysine use is described as an example.
  • the above calculation was calculated for codons corresponding to all amino acids, and the calculated codon usage (%) of Saccharomyces cerevisiae was an ideal value.
  • the frequency of use of the alanine codon GCT: GCC: GCA: GCG is changed from 35 to 45:15 to 25:25 to 40: 0 to 15. It is preferable to change the frequency of use of the codon AAT: AAC of asparagine from 45 to 70: 30 to 55. It is preferable to change the use frequency of the glycine codons GGT: GGC: GGA: GGG to 45 or more and 55 or less: 15 or more and 25 or less: 10 or more and 25 or less: 0 or more and 15 or less. It is preferable to change the usage frequency of the codon GAA: GAG of glutamic acid from 60 to 75:25 to 40.
  • TGT TGC of cysteine
  • ATT ATC: ATA of isoleucine from 45 to 60:20 to 30:20 to 30.
  • the leucine codon TTA: TTG: CTT: CTC: CTA: CTG use frequency is preferably changed from 25 to 40: 25 to 40: 5 to 15: 0:10 to 15: 5 to 15 .
  • the frequency of use of the lysine codon AAA: AAG is preferably changed from 50 to 65:35 to 50.
  • the frequency of use of the codon TTT: TTC of phenylalanine is preferably changed from 55 to 75:25 to 45.
  • the frequency of use of the proline codon CCT: CCC: CCA: CCG is changed from 30 to 40:10 to 40:35 to 50: 0 to 15.
  • the tyrosine codon TAT: TAC usage frequency is changed from 55 to 70: 30 to 45. It is preferable to change the usage frequency of the valine codons GTT: GTC: GTA: GTG from 40 to 50:15 to 25:15 to 25:15 to 20.
  • any one amino acid is satisfied for each of the tables, and more preferably two or more amino acids are satisfied. Most preferably, all of the above are satisfied.
  • the activity of these enzymes is imparted or enhanced by introducing a genetically modified product from outside the cell, enhancing the promoter activity of the enzyme gene possessed by the host yeast on the genome, or other Although it can be realized by substituting with a promoter or the like, it is not limited to these methods.
  • the activity of an enzyme is imparted or enhanced when the number of copies of an enzyme gene encoding the enzyme increases, or when the activity of each enzyme is measured by a known method. In other words, the expression level of the enzyme in the host is increased, or the enzyme concentration in the host is increased.
  • Examples of a method for imparting or enhancing enzyme activity include a method in which enzyme genes encoding these four types of enzymes are introduced into cells from outside the host yeast cells using genetic recombination techniques. It is done. At this time, the introduced enzyme gene may be the same or different from the host cell.
  • Preparation of genomic DNA necessary for gene introduction from outside the cell DNA cleavage and ligation, transformation, PCR (Polymerase Chain Reaction), design of oligonucleotides used as primers, synthesis, etc. It can be carried out in the usual way well known to the traders. These methods are described in Sambrook, J. et al. , Et. al. , “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989).
  • Any promoter that can be expressed in the host yeast may be used as a promoter used to enhance promoter activity or to strongly express an enzyme gene.
  • ADH1 promoter alcohol dehydrogenase (ADH1) promoter, phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter, glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter, metallothionein (CUP1) promoter, Promoters derived from host yeast such as repressible acid phosphatase (PHO5) promoter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter are used.
  • a foreign promoter such as a cytomegalovirus (CMV) promoter, may be used. These can be appropriately selected depending on the origin and type of the enzyme used.
  • CMV cytomegalovirus
  • any of those that can be expressed in the host may be used.
  • alcohol dehydrogenase (ADH1) promoter phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter, glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter, metallothionein (CUP1) promoter , Promoters such as repressible acid phosphatase (PHO5) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and cytomegalovirus (CMV) promoter should be used to replace the promoter of thiolase derived from host yeast Can do.
  • ADH1 promoter phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter, glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter,
  • promoters may be introduced into a host yeast cell according to a usual method so that the target enzyme gene can be expressed.
  • the promoter is linked to the same vector as the target enzyme gene, and the host together with the enzyme gene is hosted. What is necessary is just to introduce
  • yeast is used as a host from the viewpoint of reuse efficiency and isopropyl alcohol production ability, and the use codon of the enzyme gene encoding isopropyl alcohol dehydrogenase is modified based on the codon usage frequency of the yeast. It is preferably an isopropyl alcohol-producing yeast introduced with a modified gene. Furthermore, it is more preferred that the use codon of the enzyme gene encoding CoA transferase and the use codon of the enzyme gene encoding acetoacetate decarboxylase are modified.
  • the promoter region of the enzyme gene encoding thiolase, CoA transferase Most preferred are those in which the use codon of the encoding enzyme gene and the use codon of the enzyme gene encoding acetoacetate decarboxylase are modified.
  • the method for producing isopropyl alcohol of the present invention includes producing isopropyl alcohol using the isopropyl alcohol-producing yeast of the present invention described above. Thereby, according to this invention, isopropyl alcohol can be continuously produced by processing yeast with an acid.
  • the isopropyl alcohol-producing yeast may be brought into contact with a plant-derived raw material.
  • the plant-derived raw material is a carbon source obtained from a plant, and is not particularly limited as long as yeast is metabolized and can be converted into isopropyl alcohol.
  • it refers to organs such as roots, stems, trunks, branches, leaves, flowers, seeds, plants containing them, degradation products of these plant organs, and further from plant bodies, plant organs, or degradation products thereof.
  • the obtained carbon sources those that yeast can use as a carbon source in culture are also included in plant-derived materials.
  • Carbon sources included in such plant-derived materials generally include sugars such as starch, glucose, fructose, sucrose, xylose, arabinose, or vegetative degradation products and cellulose hydrolysis that contain a large amount of these components. Examples can be given. Furthermore, vegetable oil-derived glycerin and fatty acids also correspond to the carbon source in the present invention. As plant-derived materials in the present invention, crops such as cereals, corn, rice, wheat, soybeans, sugar cane, beet, cotton and the like can be preferably used. There is no particular limitation on the pulverized product. Moreover, the form of only the above-mentioned carbon source may be sufficient.
  • the isopropyl alcohol-producing yeast of the present invention only needs to have an ability to produce isopropyl alcohol from these plant-derived raw materials.
  • plant-derived raw materials are assimilated by culturing and secreted isopropyl alcohol into the culture solution after a certain time.
  • the method for producing isopropyl alcohol according to the present invention includes culturing isopropyl alcohol-producing yeast in a mixture containing isopropyl alcohol-producing yeast and plant-derived materials (culturing step), and purifying isopropyl alcohol from the culture solution ( Purification step).
  • the mixture in the method for producing isopropyl alcohol may be any mixture as long as it is mainly composed of a basic medium generally used for culturing yeasts, and any medium that is usually used according to the type of isopropyl alcohol-producing yeast can be used. can do.
  • a basic medium is not particularly limited as long as it is a medium containing a carbon source, a nitrogen source, inorganic ions, and other trace components as required.
  • a carbon source sugars such as glucose, fructose and molasses, organic acids such as fumaric acid, citric acid and succinic acid, alcohols such as methanol, ethanol and glycerol, and others are appropriately used.
  • organic ammonium salts, inorganic ammonium salts, nitrate nitrogen, ammonia gas, aqueous ammonia, inorganic and organic nitrogen sources such as protein hydrolysates, and the like are appropriately used.
  • inorganic ions magnesium ions, phosphate ions, potassium ions, iron ions, manganese ions, and others are appropriately used as necessary.
  • organic trace components vitamins, amino acids and the like, yeast extracts containing these, peptone, polypeptone, corn steep liquor, casein degradation products, and others are used as appropriate.
  • the amount of plant-derived raw material in the mixture varies depending on the type and activity and number of isopropyl alcohol-producing yeast contained in the mixture, but generally the initial sugar concentration in terms of glucose is 20% by mass or less based on the total mass of the mixture. Preferably, from the viewpoint of sugar resistance of yeast, the initial sugar concentration can be 15% by mass or less.
  • Each of these other components may be added in an amount usually added to the yeast medium, and is not particularly limited.
  • the other additive component normally added to the culture medium of yeast may be included in the amount usually used.
  • the culture medium used in the present invention include a liquid culture medium based on an aqueous medium and a solid culture medium based on a solid phase such as agar, but the liquid culture medium is considered in view of use for industrial production. Is preferred.
  • the aqueous medium constituting the liquid medium commonly used ones such as distilled water and buffer solution can be used.
  • the culture conditions are not particularly limited.
  • the culture can be performed while appropriately controlling the pH and temperature.
  • isopropyl alcohol in the culture solution is recovered.
  • the method for recovering the isopropyl alcohol accumulated in the culture solution is not particularly limited.
  • the isopropyl alcohol is collected by an ordinary separation method such as distillation, membrane separation, or extraction.
  • a method of separating alcohol can be employed.
  • the method for producing isopropyl alcohol of the present invention includes a pre-culture step for bringing the isopropyl alcohol-producing yeast to be used into an appropriate number of bacteria or a moderately active state before the culture step for producing isopropyl alcohol. May be.
  • the pre-culture process may be a culture under the culture conditions that are normally used according to the type of isopropyl alcohol-producing yeast.
  • the method for producing isopropyl alcohol of the present invention preferably includes a culture step of culturing the isopropyl alcohol-producing yeast while supplying gas to the mixture containing the isopropyl alcohol-producing yeast and the plant-derived raw material.
  • the production yeast is cultured while supplying gas to the mixture (aeration culture).
  • the isopropyl alcohol-producing yeast and the plant-derived raw material can be mixed well, and the gas supplied into the mixture can be efficiently distributed throughout the mixture.
  • the duration of the culturing step varies depending on the number and activity of isopropyl alcohol-producing yeast in the mixture and the amount of plant-derived raw material, but from the start of cultivation until the plant-derived raw material in the mixture is consumed, preferably 10 hours or more It may be sufficient, and even after the activity of the isopropyl alcohol-producing yeast is lowered, it can be further continued by performing acid treatment. Moreover, the culture period can be continued indefinitely by re-inputting plant-derived materials.
  • the isopropyl alcohol production method of the present invention includes an acid treatment of isopropyl alcohol-producing yeast at a pH of 4 or less before the culturing step or after the isopropyl alcohol recovery step.
  • isopropyl alcohol-producing yeast can be washed to prevent virus infection and the like. Since the isopropyl alcohol-producing yeast in the present invention is acid resistant, the ability to produce isopropyl alcohol is not lost by this acid treatment, and then isopropyl alcohol is repeatedly produced by culturing in a production medium. As a result, it is possible to reuse (recycle) the isopropyl alcohol-producing yeast once used.
  • an acid having a pH of 4 or less, more preferably an acid having a pH of 2.6 to 3.4 is more preferable from the viewpoint of preventing contamination.
  • the acid treatment may be performed by adding an amount of acid necessary for pH effective in preventing infection to a culture solution contained in isopropyl alcohol-producing yeast. It is appropriately set depending on the number of alcohol-producing yeasts.
  • an inorganic acid, an organic acid, a combination thereof, or a buffer solution thereof can be used.
  • inorganic acid sulfuric acid, hydrochloric acid, phosphoric acid
  • organic acid citric acid, phthalic acid, succinate Acid, acetic acid, other glycine, and the like
  • citric acid citric acid
  • phthalic acid phthalic acid
  • succinate Acid acetic acid
  • acetic acid other glycine, and the like
  • the acid treatment may be after recovering isopropyl alcohol or before producing isopropyl alcohol. Before the production of isopropyl alcohol, it is preferable to isolate and wash the treated isopropyl alcohol-producing yeast and then transfer it to the production medium.
  • the treatment time may be 6 hours or more, and is preferably 6 hours or more and several days from the viewpoint of preventing contamination and maintaining the fungus body.
  • Example 1 ⁇ Construction of Clostridium Bacteria-Derived Isopropyl Alcohol Gene Enzyme Gene Group Expression Vector and the Expression Vector Transformant>
  • the amino acid sequences of the four isopropyl alcohol-producing enzymes of the genus Clostridium and the nucleotide sequences of the genes have already been reported. That is, the thiolase is described in complementary strands 3005963 to 3007364 of the genome sequence described in GenBank accession number AE001437.
  • CoA transferase is described in GenBank accession number X72831, acetoacetate decarboxylase is described in GenBank accession number M55392, and isopropyl alcohol dehydrogenase is described in GenBank accession number AF157307. Furthermore, the base sequence of the promoter necessary for expressing these four genes is the alcohol dehydrogenase derived from Saccharomyces cerevisiae (hereinafter referred to as ADH1) described in 113-612 in the base sequence information of GenBank accession number Z25479.
  • ADH1 alcohol dehydrogenase derived from Saccharomyces cerevisiae
  • the terminator base sequence is the cytochrome C (hereinafter referred to as CYC1) terminator sequence derived from Saccharomyces cerevisiae described in 581-780 in the base sequence information of GenBank accession number L26347. can do.
  • CYC1 cytochrome C
  • ACAATCAACTATCTCATATACAATGAAAGAAAAGTGTGATATAGCTA SEQ ID NO: 1
  • ACAAGGAAAAGGGGCCGTTCTAGCACTTTTCTAGCAATH SEQ ID NO: 2
  • the obtained three DNA fragments were amplified by PCR using GCTCTAGAACTTGTAGCCCTAGACTTG (SEQ ID NO: 3) and GCTCTAGAGATGAGAGTGTAAACTGCGAA (SEQ ID NO: 6), and digested with the restriction enzyme XbaI to contain DNA sequences encoding the ADH1 promoter and CYC1 terminator.
  • a 1.9 kbp thiolase fragment was obtained.
  • the obtained two DNA fragments were amplified by PCR using AACTGCAGACTGTTAGCCCTAGTAGACTTG (SEQ ID NO: 9) and ACGCGTCGAACTTATGCAGGCTCCTCTCACTA (SEQ ID NO: 8), and the resulting DNA fragment was digested with restriction enzymes PstI and SalI to encode the ADH1 promoter An approximately 1.2 kbp CoA transferase A fragment containing the sequence was obtained.
  • the obtained four DNA fragments were amplified by the PCR method with ACGCGTCCGACCGGAATTTCTTTATGATTTAGA (SEQ ID NO: 15) and GCTCTGAGATAGGAGGTGTAAACTGCGAA (SEQ ID NO: 6), and the obtained DNA fragments were digested with restriction enzymes SalI and XbaI, ADH1 promoter, ADH1 promoter, ADH1 promoter An approximately 1.6 kbp CoA transferase B fragment containing the DNA sequence encoding the CYC1 terminator was obtained.
  • ACAATCAACTATCTCATATACAATGTTAAAGGGATGAAGGTAATTA SEQ ID NO: 18
  • ACAAAGGAAAAGGGGCCTGTTTACTTAAGATAATACATATAAC SEQ ID NO: 19
  • the four DNA fragments obtained were amplified by PCR using GGAATTCACTGTAGCCCTAGAGACTTG (SEQ ID NO: 20) and GGGGTACCGATGAGAGTGTAAACTGCGAA (SEQ ID NO: 23), and the resulting DNA fragments were digested with restriction enzymes EcoRI and KpnI to thereby generate ADH1 promoter and CYC1 terminator. An approximately 1.6 kbp acetoacetate decarboxylase fragment containing a DNA sequence encoding was obtained.
  • Clostridium acetobutylicum (Clostridium acetobutylicum ATCC824) and Saccharomyces cerevisiae X2180 strain ATCC 26786 can be obtained from the American Type Culture Collection, a cell, microorganism, and gene bank.
  • the genomic DNA of Clostridium begerinki NRRL B-593 was used as a template, and ACAATCAACTATTCTCATATACAATGAAAGGGTTTGCAATGCTAG (SEQ ID NO: 24) and ACAAGAGAAAAGGGGCTTAGTATATATATATATA PCRATTATA
  • the genomic DNA of Saccharomyces cerevisiae X2180 strain was used as a template, and GGAATTCACTTGTAGCCCTAGACTTG (SEQ ID NO: 20) and CTAGCATTGCAAAACACTTTCATTGTATATAGGATAGAGTG
  • TTGT SEQ ID NO: 26
  • the genomic DNA of Saccharomyces cerevisiae X2180 strain was used as a template, TTAAAGCAGGTAGTTATATATAAAACAGGCCCCTTTTCCTTGTGT (SEQ ID NO: 26)
  • the three DNA fragments obtained were amplified by PCR using GGAATTCACTGTTAGCCCTAGAGACTTG (SEQ ID NO: 20) and GGAATTCGATGAGAGTGTAAACTGCGAA (SEQ ID NO: 28), and the resulting DNA fragment was digested with the restriction enzyme EcoRI to thereby obtain an ADH1 alcohol dehydrogenase promoter and a CYC1 terminator. An approximately 2.1 kbp isopropyl alcohol dehydrogenase fragment containing the DNA sequence encoding was obtained.
  • Clostridium beijerinckii NRRL B-593 can be obtained from the VTT Culture Collection, a cell and microorganism bank.
  • the fragments obtained by digesting with XbaI were mixed and ligated using ligase, then transformed into Escherichia coli DH5 ⁇ competent cells, and LB Broth, Miller (Difco 244520) agar containing 100 ⁇ g / mL of ampicillin. A transformant growing on the plate was obtained. The obtained colonies were cultured overnight at 37 ° C.
  • YEplac195 (ATCC 87589) can be obtained from the American Type Culture Collection, a cell / microorganism bank.
  • the above-mentioned thiolase fragment was mixed with the fragment obtained by digesting the plasmid pYEplacKAN (a plasmid incorporating the kanMX4 gene amplified by PCR into the marker gene region of YEplac195) with the restriction enzyme XbaI, and ligated using ligase. Thereafter, the cells were transformed into Escherichia coli DH5 ⁇ strain competent cells to obtain transformants that grew on LB Broth, Miller medium agar plates containing 100 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C.
  • kanMX4 gene was obtained from Wach, A .; Et. al. (1994) Yeast 10, 1793-1808.
  • the above acetoacetate decarboxylase fragment and the fragment obtained by digesting plasmid pTHI with restriction enzymes EcoRI and KpnI were mixed, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant was obtained that grew on LB Broth, Miller broth agar plates containing ampicillin 100 ⁇ g / mL. The obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 100 ⁇ g / mL of ampicillin, and the plasmid pADC-THI was recovered from the obtained cells.
  • the plasmids pIPA-CTFAB and pADC-THI are transformed into Saccharomyces cerevisiae X2180 strain and cultured overnight at 30 ° C. on YPD Broth (Difco 242720) agar plate containing 200 ⁇ g / mL geneticin and 200 ⁇ g / mL nadosricin.
  • YPD Broth Difco 242720
  • agar plate containing 200 ⁇ g / mL geneticin and 200 ⁇ g / mL nadosricin.
  • an isopropyl alcohol-producing enzyme gene group expression vector transformant [pIPA-CTFAB and pADC-THI / X2180 strain] was obtained.
  • Example 2 ⁇ Isopropyl alcohol production by Saccharomyces cerevisiae [pIPA-CTFAB and pADC-THI / X2180 strain] (comparative example)>
  • YPD culture solution containing 200 ⁇ g / mL geneticin and 200 ⁇ g / mL nadosricin was placed in a 14 mL plastic tube (FALCON 2057), and the Saccharomyces cerevisiae obtained in Example 1 pADC-THI / X2180 strain] was inoculated and cultured overnight with shaking at 30 ° C. and 120 rpm.
  • Saccharomyces cerevisiae [pYEplacNAT and YEplacKAN / X2180 strain] was cultured with shaking under the same medium composition and culture conditions.
  • the total amount of each preculture was transferred to a 100 mL Erlenmeyer flask containing 40 mL of YPD Broth liquid medium containing Geneticin 200 ⁇ g / mL and Noaceosricin 200 ⁇ g / mL.
  • the culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • the cell culture solution was sampled, the cells were removed by centrifugation, and the amount of isopropyl alcohol accumulated in the obtained culture supernatant was measured by HPLC according to a standard method.
  • Saccharomyces cerevisiae [pIPA-CTFAB and pADC-THI / X2180 strain] and Saccharomyces cerevisiae [pYEplNAT and YElacKAN / X2180 strain], there was no accumulation of isopropyl alcohol.
  • Example 3 ⁇ Construction of Saccharomyces cerevisiae-derived thiolase gene, Escherichia coli-derived thiolase gene, Escherichia coli-derived CoA transferase gene, Clostridium bacterium-derived isopropyl alcohol-producing enzyme gene group expression vector and the expression vector transformant>
  • the amino acid sequences of Saccharomyces cerevisiae thiolase, Escherichia coli thiolase, and Escherichia coli CoA transferase have already been reported. That is, the gene encoding Saccharomyces cerevisiae thiolase is described in GenBank accession number EF059447.
  • a gene encoding a thiolase of Escherichia coli is described in 2324131 to 2325315 of the Escherichia coli MG1655 strain described in GenBank accession number U00096.
  • a gene encoding CoA transferase is described in the above-mentioned Escherichia coli MG1655 strain genomic sequence 232469 to 2322781.
  • the nucleotide sequence of the promoter necessary for expressing these four genes is a phosphoglycerate kinase derived from Saccharomyces cerevisiae (hereinafter referred to as PGK1) described in 121-781 in the nucleotide sequence information of GenBank accession number FJ415226.
  • PGK1 phosphoglycerate kinase derived from Saccharomyces cerevisiae
  • the terminator base sequence the PGK1 terminator sequence derived from Saccharomyces cerevisiae described in 138991-139272 in the base sequence information of GenBank accession number X59720 can be used.
  • TTTCTCGAGAAAGATGCCGATTTGGCGCC (SEQ ID NO: 29) and GCCCGCTAGCGTTTTATTTTTGTTGTTAAA (SEQ ID NO: 29) were obtained using the genomic DNA of Saccharomyces cerevisiae S288C strain (ATCC 26108) as a template to obtain the PGK1 promoter.
  • the fragment was digested with restriction enzymes XhoI and NheI to obtain a DNA fragment corresponding to the PGK1 promoter of about 700 bp.
  • a DNA fragment obtained by annealing the DNA fragment B obtained by digesting with the enzyme B, which is obtained by digesting with the enzyme B, which is obtained by digesting with the enzyme B, which is digested with the multipartite I which is obtained by digesting the DNA with TTTTGCTACGCGTCGACACCTAGTGGATCCCCCGGGTCCTAGAGATATTCAGATCCT (SEQ ID NO: 31) The sequence was obtained.
  • Saccharomyces cerevisiae S288C strain genomic DNA was used as a template, and the DNA obtained by PCR was amplified by the PCR method using GCCCAGATTCTGAAAATAATTGAATTGAATT (SEQ ID NO: 33) and TTTTGCCGCCGCAGCTTTAACGAACGCCAGA (SEQ ID NO: 34).
  • a DNA fragment corresponding to the PGK1 promoter of about 300 bp was obtained by digestion with restriction enzymes BglII and NotI.
  • 2 ⁇ m is Kanai et al. : Appl. Microbiol. Biotechnol. Vol. 44, p. 759-765, (1996) is used, and details of pRS402 + 2 ⁇ m, pRS403 + 2 ⁇ m, pRS404 + 2 ⁇ m, pRS405 + 2 ⁇ m, and pRS406 + 2 ⁇ m are described in Ishii, J. et al. Et al. : J. Biochemistry. Vol. 145, No. 6, p. 701-708, (2009).
  • Plasmids pRS402 (ATCC 87477), pRS403 (ATCC 87514), pRS404 (ATCC 87515), pRS405 (ATCC 87516), and pRS406 (ATCC 87517) can be obtained from the American Type Culture Collection, which is a cell / microorganism / gene bank.
  • bases 1060 to 1062 are stop codons. Also, the first and last 6 bases do not encode an enzyme.
  • the vector in which the above DNA fragment was inserted was digested with restriction enzymes NheI and BglII to obtain an isopropyl alcohol dehydrogenase fragment having a codon modified of about 1.1 kbp.
  • the obtained DNA fragment and the previously prepared plasmid pGK426 were mixed with the fragments obtained by digesting with restriction enzymes NheI and BglII, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pIPAc1 was recovered from the obtained cells.
  • CCCCGCTAGCATGAAAATTTGTGTCATCGTCAGTGCGGGTA SEQ ID NO: 36
  • GGGTCCCGGGTTAATTCAACCGTCCATCPCR were obtained by using the genomic DNA of Escherichia coli MG1655 as a template.
  • the resulting DNA fragment was mixed with the previously prepared plasmid pGK422 by digestion with restriction enzymes NheI and XmaI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colonies were cultured overnight at 37 ° C. in LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pATOB was recovered from the obtained cells.
  • the resulting DNA fragment was mixed with the previously prepared plasmid pGK422 by digestion with restriction enzymes NheI and XmaI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pERG10 was recovered from the obtained cells.
  • CCCCGCTAGCATGATAGAGATGTAGATAATATAGTAAGTCCTTTTAACTCTPCRACTCCPCR41 was obtained by using the genomic DNA of Clostridium acetobutylicum (ATCC824) as a template.
  • the fragment was digested with restriction enzymes NheI and XmaI to obtain a thiolase fragment of about 1.2 kbp.
  • the resulting DNA fragment was mixed with the previously prepared plasmid pGK422 by digestion with restriction enzymes NheI and XmaI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained. The obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pTHI2 was recovered from the resulting cells.
  • the resulting DNA fragment was mixed with the previously prepared plasmid pGK423 by digestion with restriction enzymes SalI and EcoRI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth, Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pATOA was recovered from the obtained cells.
  • the obtained DNA fragment and the previously prepared plasmid pGK424 were mixed with the fragments obtained by digesting with restriction enzymes NheI and BglII, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth, Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pATOD was recovered from the resulting cells.
  • CCCCCTCGAGATGAACTCTAAAAATAATTTAGATTTGGAAAAT SEQ ID NO: 46
  • GGGGGAATTCTATTGCAGGCTCTCTTACTATATATT PCR SEQ ID NO: 46
  • the obtained DNA fragment was mixed with the previously prepared plasmid pGK423 by digestion with restriction enzymes SalI and EcoRI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth, Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pCTFA was recovered from the obtained cells.
  • a 600 bp CoA transferase B subunit fragment was obtained.
  • the resulting DNA fragment was mixed with the previously prepared plasmid pGK424 by digestion with restriction enzymes NheI and BglII, ligated with ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd.) Company DNA-903) was transformed to obtain transformants that grow on LB Broth, Miller agar plates containing 50 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pCTFB was recovered from the obtained cells.
  • acetoacetate decarboxylase gene the genomic DNA of Clostridium acetobutylicum was used as a template, and CCCCGCTAGCATGTTAAAGGATGGAAGTAATTTAAAACAAATT (SEQ ID NO: 50) and GGGGGGGATCCTTACACTTAAGATAATCATATAACTACTCAGC (SEQ ID NO: 51) were obtained by PCR and amplified by the PCR method. Digestion with restriction enzymes NheI and BamHI gave an acetoacetate decarboxylase fragment of about 700 bp.
  • the obtained DNA fragment and the previously prepared plasmid pGK425 were mixed with the fragments obtained by digesting with restriction enzymes NheI and BglII, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on LB Broth Miller agar plates containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pADC was recovered from the obtained cells.
  • PIPAc1 containing a codon-modified isopropyl alcohol dehydrogenase gene, pAdc containing an acetoacetate decarboxylase gene, pATOD and pATOA containing a CoA transferase gene derived from Escherichia coli, pCTFA and pCTFB containing a CoA transferase gene derived from Clostridium bacteria, and saccharomysis PERG10 containing a thiolase gene derived from cerevisiae, pATOB containing a thiolase gene derived from Escherichia coli, and pTHI containing a thiolase gene derived from Clostridium bacterium into a Saccharomyces cerevisiae YPH499 strain, and then adenine, histidine, tryptophan, leucine , SD medium without uracil (0.67% Yeast Nitrogen base w / O Amino Acids
  • an isopropyl alcohol-producing enzyme gene group expression vector transformant [pIPAc1 pADC pCTFA pCTFB pTHI2 / YPH499 Strain], [pIPAc1 pADC pATOA pATOD pTHI2 / YPH499 strain], [pIPAc1 pADC pCTFA pCTFB pATOB / YPH499 strain], [pIPAc1 pADC pATOA pATOD pATOB pATOpTp pATOA pATOD pERG10 / YPH499 strain].
  • the Escherichia coli MG1655 strain (ATCC 47076), Clostridium acetobutyricum ATCC 824, Saccharomyces cerevisiae S288C strain (ATCC 26108), and Saccharomyces cerevisiae YPH499 strain (ATCC 204679) are the cell / microorganism type Microorganism / Bank Microorganisms / American Microorganisms / American Culture Type It can be obtained from the collection.
  • Example 4 ⁇ Saccharomyces cerevisiae [pIPAc1 pADC pCTFA pCTFB pTHI2 / YPH499 strain], [pIPAc1 pADC pATOA pATAD pTHI2 / PHH499 strain], [pIPAc1 pADC pATA pCFA pCTFA pCTFA pCTFA pCTAp Isopropyl alcohol production by [pIPAc1 pADC pCTFA pCTFB pERG10 / YPH499 strain], [pIPAc1 pADC pATOA pATOD pERG10 / YPH499 strain] As a preculture, 5 mL of SD culture solution containing no adenine, histidine, tryptophan, leucine, or uracil was placed in a 14 mL plastic tube (FALCON 2057).
  • Saccharomyces cerevisiae [pGK422, 423, 424, 425, pGK426 / YPH499 strain] was cultured with shaking under the same medium composition and culture conditions. The total amount of each preculture was transferred to a 100 mL Erlenmeyer flask containing 40 mL of SD culture solution containing no adenine, histidine, tryptophan, leucine, or uracil, and cultured. The culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • the cell culture solution was sampled 96 hours after the start of the culture, and after removing the cells by centrifugation, the amount of isopropyl alcohol accumulated in the obtained culture supernatant was measured by GC-MS according to a conventional method.
  • the results are shown in Table 7.
  • Saccharomyces cerevisiae [pGK422, 423, 424, 425, pGK426 / YPH499 strain]
  • isopropyl alcohol can be produced in yeast for the first time by using codon-modified IPAdh in yeast.
  • Example 5 ⁇ Saccharomyces cerevisiae-derived thiolase gene, codon-modified Escherichia coli-type CoA transferase gene, codon-modified Clostridium genus bacterial CoA transferase gene, codon-modified Clostridium genus isopropyl alcohol-producing enzyme gene group expression vector and the expression vector transformation Body Construction>
  • Saccharomyces cerevisiae X cerevisiae X Is used as a template to amplify the DNA fragments by GGAATTCACTGTTAGCCCTAGACTTG (SEQ ID NO: 20) and CCAACATTGCAAAACCTTTCATTGTATATGAGATAGTTGATTGT (SEQ ID NO: 55), and the two DNA fragments obtained are GGAATTCACTGTTAGCCCTTAGACTTG No.
  • CGGGATCCTTATAAGATCACTACGCGCTTTA SEQ ID NO: 54
  • the obtained DNA fragment is digested with the restriction enzymes EcoRI and BamHI, thereby containing about 2.1 kbp of isopropyl alcohol containing the DNA sequence encoding the ADH1 promoter A dehydrogenase fragment was obtained.
  • the genomic DNA of the Saccharomyces cerevisiae X2180 strain was used as a template, and was amplified by the PCR method using CGGGATCCACAGGCCCCTTTTCCTTTGT (SEQ ID NO: 56) and GGAATTCGATGAGAGTGTATAACTGCGAA (SEQ ID NO: 57).
  • the obtained DNA fragment was digested with restriction enzymes BamHI and EcoRI to obtain a DNA sequence encoding a cytochrome C terminator.
  • the obtained two DNA fragments and the fragment obtained by digesting plasmid pUC119 with restriction enzyme EcoRI were mixed, ligated with ligase, transformed into Escherichia coli DH5 ⁇ competent cell, and 50 ⁇ g / ml of ampicillin / Transformants that grew on LB Broth, Miller agar plates containing mL were obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pUC119-IPAc2 was recovered from the obtained cells.
  • GGAATTCACTGTTAGCCCTAGACTTG SEQ ID NO: 20
  • TTAATAACTACTCATCTTTAACATTGTAATGAGATAGTAGTGATTGT SEQ ID NO: 61
  • Isopropyl alcohol dehydrogenase fragment was obtained.
  • the genomic DNA of Saccharomyces cerevisiae X2180 strain was used as a template, and was amplified by the PCR method using CGGGATCCACAGCCCCCTTTTCCTTTGT (SEQ ID NO: 56) and GGGGTACCCATGAGAGTGTAAACTGCGAA (SEQ ID NO: 23).
  • the obtained DNA fragment was digested with restriction enzymes BamHI and KpnI to obtain a DNA sequence encoding a cytochrome C terminator.
  • the obtained two DNA fragments and the fragment obtained by digesting plasmid pYEplacKAN with restriction enzymes EcoRI and KpnI were mixed, ligated with ligase, transformed into Escherichia coli DH5 ⁇ competent cell, and ampicillin A transformant that grew on an LB Broth, Miller agar plate containing 50 ⁇ g / mL was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pADCc was recovered from the obtained cells.
  • Saccharomyces cerevisiae X 180 cerevisiae X cerevisiae strain X cerevisiae X Amplification was performed by PCR using AACTGCAGACTGTTAGCCCTAGACTTG (SEQ ID NO: 9) and AATTCATTATTTTTAGAGTTCATTGTATATGAGATAGTTTATTGT (SEQ ID NO: 65).
  • the obtained two DNA fragments were amplified by PCR using AACTGCAGACTGTTAGCCCTAGTAGACTTG (SEQ ID NO: 9) and ACGCGTCGAACTTATGCAGGCTCCTCTCACTA (SEQ ID NO: 64), and the resulting DNA fragment was digested with restriction enzymes PstI and SalI to encode the ADH1 promoter
  • An approximately 1.2 kbp codon modified Clostridial CoA transferase fragment (codon modified ctfA) containing the sequence was obtained.
  • the ACAATCAACTATCTCATATACACATGATTAATGATAAAAAACCTAGC SEQ ID NO: 67
  • CGGGATCCTCAAACAGCCATGGGTCTAAG SEQ ID NO: 68
  • a template is used to amplify the ADH1 terminator by the PCR method using TTTTATGTCCTCAGAGAGACAAACTGTTAGCCCTTAGACTTGATA (SEQ ID NO: 13) and GCTAGGTTTTTATCATTAATCATGTTATAGGATAGTAGTTGTATTGT (SEQ ID NO: 69).
  • ATCCGTCGAGCGCGAATTTCTTATGATTTTATGA SEQ ID NO: 15
  • TATCAAGTCTAGGGCTACAGTTTGTCCTCTGAGGGACATAAAA SEQ ID NO: 16
  • CGGGATCTCCAAACAGCCATGGGTCTAAG SEQ ID NO: 68
  • the genomic DNA of the Saccharomyces cerevisiae X2180 strain was used as a template, and CGGGATCCCAGGGCCCTTTTCCTTTGT (SEQ ID NO: 56) and GCTCTTAGAGATGAGAGTGTATAACTGCGAA (SEQ ID NO: 6) were amplified by the PCR method.
  • the obtained DNA fragment was digested with restriction enzymes BamHI and XbaI to obtain a DNA sequence encoding a CYC1 terminator.
  • Saccharomyces cerevisiae X cerevisiae X cerevisiae X cerevisiae X cerevisiae X cerevisiae X cerevisiae X Amplification was performed by PCR using AACTGCAGACTGTAGCCCTAGACTTG (SEQ ID NO: 9) and ATGTCATCAACTTTGTTTTTCATTGTATATGAGATAGTTGATTGT (SEQ ID NO: 73).
  • the ADH1 terminator is used to obtain the ADH1 terminator by using the template to amplify by the PCR method using TTTTATGTCCTCAGAGAGACAAACTGTTAGCCCTTAGACTTGATA (SEQ ID NO: 13) and CAATTTCTTGTTTTAGCATCCATGTTATAGGAATAGTTTGATTGT (SEQ ID NO: 77). Using the genomic DNA of S.
  • ATCCGTCCGACCGGAATTTCTTATGATTTTATGA SEQ ID NO: 15
  • TATCAAGTCTAGGGCTACAGTTGTTCTCTGGATCATAGAA SEQ ID NO: 16
  • SEQ ID NO: 76 Amplified by the PCR method, the obtained DNA fragment was digested with restriction enzymes Sal and BamHI, and an about 1.4 kbp codon-modified Escherichia coli containing DNA sequences encoding ADH1 terminator and ADH1 promoter Coli CoA transferase fragment was obtained (codon-modified atoA).
  • the genomic DNA of the Saccharomyces cerevisiae X2180 strain was used as a template, and CGGGATCCCAGGGCCCTTTTCCTTTGT (SEQ ID NO: 56) and GCTCTTAGAGATGAGAGTGTATAACTGCGAA (SEQ ID NO: 6) were amplified by the PCR method.
  • the obtained DNA fragment was digested with restriction enzymes BamHI and XbaI to obtain a DNA sequence encoding a CYC1 terminator.
  • the genomic DNA of Saccharomyces cerevisiae X2180 strain was used as a template, and ACAATCAACTATCTCATATACAATGTTCCATGAACTTTACCATTG (SEQ ID NO: 78) and CGGGATCCCTCATATCTTTTCATGACAADAG PCR
  • the genome DNA of Saccharomyces cerevisiae X2180 strain was used as a template, and GCTCTGAACTCGTAGCCCTAGACTTG (SEQ ID NO: 3) and CAATGTATAACGTTCTGGACATTGTATATGAGATAGTTGATTGT (SEQ ID NO: 8)
  • the two DNA fragments obtained were amplified by the PCR method using GCTCTAGAACTGTTAGCCCTAGACTTG (SEQ ID NO: 3) and CGGGATCCCTCATATTTTTCAATGACAATAG (SEQ ID NO: 79), and the resulting DNA fragment
  • the above ADH1 promoter, thiolase fragment, CYC1 terminator, and the fragment obtained by digesting the plasmid pYEplacKAN with the restriction enzyme XbaI were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd.) DNA-903) was transformed to obtain transformants that grew on LB Broth, Miller culture medium agar plates containing 100 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 100 ⁇ g / mL of ampicillin, and the plasmid pERG10-2 was recovered from the obtained cells.
  • the above codon-modified acetoacetate decarboxylase fragment and the fragment obtained by digesting plasmid pERG10-2 with restriction enzymes EcoRI and KpnI were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd. DNA-903) was transformed to obtain transformants that grew on LB Broth, Miller agar plates containing ampicillin 100 ⁇ g / mL. The obtained colonies were cultured overnight at 37 ° C. in LB Broth, Miller liquid medium containing 100 ⁇ g / mL of ampicillin, and plasmid pADCc-ERG10 was recovered from the obtained cells.
  • the above codon-modified isopropyl alcohol dehydrogenase fragment and the fragment obtained by digesting the plasmid pYEplacNAT with the restriction enzyme EcoRI were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd., DNA- 903) to obtain transformants that grow on LB Broth, Miller culture medium agar plates containing 100 ⁇ g / mL of ampicillin. The obtained colonies were cultured overnight at 37 ° C. in an LB Broth, Miller liquid medium containing 100 ⁇ g / mL of ampicillin, and the plasmid pIPAc2 was recovered from the resulting cells.
  • a fragment obtained by digesting the plasmid pATODAc with the restriction enzymes PstI and XbaI and a fragment obtained by digesting the plasmid IPAc2 with the restriction enzymes PstI and XbaI are mixed, ligated using ligase, and then Escherichia coli DH5 ⁇ strain Transformants were grown into competent cells (Toyobo Co., Ltd., DNA-903) and grown on LB Broth, Miller agar plates containing ampicillin 100 ⁇ g / mL. The obtained colonies were cultured overnight at 37 ° C. in LB Broth, Miller liquid medium containing 100 ⁇ g / mL of ampicillin, and the plasmid pIPAc2-ATODAc was recovered from the obtained cells.
  • a fragment obtained by digesting plasmid pctfABm with restriction enzymes PstI and XbaI and a fragment obtained by digesting plasmid pIPAc2 with restriction enzymes PstI and XbaI are mixed, ligated with ligase, and then Escherichia coli DH5 ⁇ strain Transformants were transformed into competent cells to obtain transformants that grew on LB Broth, Miller agar plates containing ampicillin 100 ⁇ g / mL. The obtained colonies were cultured overnight at 37 ° C. in LB Broth, Miller liquid medium containing 100 ⁇ g / mL of ampicillin, and the plasmid pIPAc2-CTFABc was recovered from the obtained cells.
  • Codon-modified isopropyl alcohol dehydrogenase gene pIPAc2-CTFABc containing codon-modified Clostridial CoA transferase gene, codon-modified isopropyl alcohol dehydrogenase gene, pIPAc2-ATODAc containing CoA transferase gene from Escherichia coli, codon-modified acetoacetate de PADCc-ERG10 containing a carboxylase gene and a thiolase gene derived from Saccharomyces cerevisiae was transformed into Saccharomyces cerevisiae strain X2180, and YPD Broth agar plate containing Geneticin 200 ⁇ g / mL and Noaceosricin 200 ⁇ g / mL at 30 ° C.
  • Isolation of isopropyl alcohol gene by culturing overnight Group expression vector transformants [pIPAc2-CTFABc pADCc-ERG10 / X2180 strain] and [pIPAc2-ATODA pADCc-ERG10 / X2180 strain] were obtained.
  • the AAAAAATGTGAAAAAAAAAGCTGGGTGTTACAATATGGGACTTCC SEQ ID NO: 84
  • CAATGTAAACGTTCTGAGAATTTGTAATGAGATAGAGTTGTATTGT SEQ ID NO: 80
  • ATACAGATTTAGACGTACTCAAAGTTTAATTTACCTTTTTTTCGGAGGGC SEQ ID NO: 85
  • GGAAGTCCATATTGTTACACCCAGCTTTTTCTTTCTCATTTTTTTTTTTT SEQ ID NO: 86.
  • the obtained four DNA fragments were amplified by PCR using TTCTGTGAGATGATTATTTTCG (SEQ ID NO: 82) and CTTTTCTGCGGTTACACCC (SEQ ID NO: 81).
  • the plasmid was introduced on a plasmid, transformed into competent cells of Escherichia coli DH5 ⁇ strain, and transformants that grew on LB Broth, Miller agar plates containing 100 ⁇ g / mL of ampicillin were obtained. The obtained colonies are cultured overnight at 37 ° C.
  • the plasmid pGEM-T easy-pERG10-URA3-pADH1-ERG10 containing the 2.9 kbp thiolase fragment containing the DNA sequence was recovered.
  • Saccharomyces cerevisiae W303-1A strain (ATCC 201238) can be obtained from the American Type Culture Collection, a cell / microorganism / gene bank.
  • a fragment obtained by cleaving the above plasmid pGEM-T easy-pERG10-URA3-pADH1-ERG10 with the restriction enzyme NotI was transformed into Saccharomyces cerevisiae uracil-sensitive X2180 strain, and SD culture solution from which uracil was removed (0.67% YRG Nitrogen base w / o Amino Acids Difco 239210, 2% glucose) was cultured on an agar plate at 30 ° C. overnight to obtain ERG10 genome-enhanced X2180 strain.
  • the plasmids pIPAc2-ATODAAc and pADCc prepared in Example 5 were transformed into the Saccharomyces cerevisiae ERG10 genome-enhanced X2180 strain, and overnight on a YPD Broth agar plate containing Geneticin 200 ⁇ g / mL and Noaceosricin 200 ⁇ g / mL at 30 ° C. By culturing, an isopropyl alcohol synthase gene group expression vector transformant [pIPAc2-ATODAc and pADCc / ERG10 genome-enhanced X2180 strain] was obtained.
  • Example 7 ⁇ Saccharomyces cerevisiae [pIPAc2-CTFABc and pADCc-ERG10 / X2180 strain], [pIPAc2-ATODAAc and pADCc-ERG10 / X2180 strain], Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc / ERG10 genome enhanced X2180 strain] Production of isopropyl alcohol> As a pre-culture, 5 mL of YPD Broth liquid medium containing geneticin 200 ⁇ g / mL and noaceosricin 200 ⁇ g / mL was placed in a 14 mL plastic tube (FALCON 2057).
  • Saccharomyces cerevisiae [pIPAc2-ATODAAc and pADCc-ERG10 / X2180 strain] Saccharomyces cerevisiae obtained in Example 6 [pIPAc2-CTFABc and pADCc / ERG10 genome-enhanced X2180] Strain] was inoculated and cultured overnight with shaking at 30 ° C. and 120 rpm. Further, Saccharomyces cerevisiae [pYEplacNAT and YEplacKAN / X2180 strain] was cultured with shaking under the same medium composition and culture conditions.
  • the total amount of each preculture was transferred to a 100 mL Erlenmeyer flask containing 40 mL of YPD Broth liquid medium containing Geneticin 200 ⁇ g / mL and Noaceosricin 200 ⁇ g / mL.
  • the culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • the cell culture solution was sampled, the cells were removed by centrifugation, and the amount of isopropyl alcohol accumulated in the obtained culture supernatant was measured by HPLC according to a standard method. The results are shown in Table 8.
  • Example 8 ⁇ Isopropyl alcohol production by Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc-ERG10 / X2180 strain], Escherichia coli pGAP-Iaaa / B strain] and cell viability after acid treatment>
  • 5 mL of YPD Broth liquid medium containing 200 ⁇ g / mL geneticin and 200 ⁇ g / mL noaceosricin was placed in a 14 mL plastic tube (FALCON 2057).
  • Saccharomyces cerevisiae obtained in Example 6 [pIPAc-ATODAc And pADCc-ERG10 / X2180 strain] were inoculated and cultured overnight with shaking at 30 ° C. and 120 rpm.
  • Cells corresponding to the preculture liquid OD600 Abs4 (4 ⁇ 10 7 cells) were transferred to a 100 mL Erlenmeyer flask containing 40 mL of YPD Broth liquid medium containing 200 ⁇ g / mL geneticin and 200 ⁇ g / mL noaceosricin.
  • the culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • Escherichia coli pIaaa / B strain described in WO2009 / 008377 was inoculated into 5 mL of LB Broth, Miller culture solution containing 100 ⁇ g / mL ampicillin in a plastic tube (FALCON 2057) as a preculture overnight. Then, shaking culture was performed at a culture temperature of 30 ° C. and 120 rpm. The cells of the preculture solution OD660 Abs0.4 (4 ⁇ 10 7 cells) were transferred to a 100 mL Erlenmeyer flask containing 40 mL of LB Broth, Miller culture solution containing ampicillin 100 ⁇ g / mL and glucose 2 g / L. It was. The culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • a part of the cell culture solution 96 hours after the start of the culture was removed by centrifugation, and then the obtained Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc-ERG10 / X2180 strain] in the culture supernatant.
  • the amount of isopropyl alcohol accumulated was measured by HPLC according to a conventional method.
  • a part of the Escherichia coli pIaaa / B strain cell culture solution 72 hours after the start of culture was removed by centrifugation, and the amount of isopropyl alcohol accumulated was measured by HPLC according to a conventional method.
  • the concentration of isopropyl alcohol in the culture solution of Saccharomyces cerevisiae is 32 mg / L
  • the concentration of isopropyl alcohol in the culture solution of Escherichia coli pIAaa / B strain is 2.65 g / L was shown.
  • Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc-ERG10 / X2180 strain] culture medium containing cells corresponding to OD600 Abs10 (1 ⁇ 10 8 cells), or 72 hours after the start of the culture, A culture solution of Escherichia coli pIaaa / B strain containing cells corresponding to OD660 Abs1 (1 ⁇ 10 8 cells) is sampled, and after removing the culture solution by centrifugation, each of the citrate-phosphate buffer at pH 7 and pH 5.4 is obtained.
  • citrate-phosphate buffer pH 3.8 citrate-phosphate buffer, pH 3.4 citrate-phosphate buffer, pH 3 citrate-phosphate buffer, pH 2.6 citrate-phosphate buffer
  • citrate-phosphate buffer with the same pH as used after washing 4mL 25 °C in and allowed to 24 hours quietly reaction.
  • the cells were cultured overnight at 30 ° C., the number of colonies was counted, and the cell viability was measured.
  • a part of the sample reacted with Escherichia coli pIaaa / B was diluted with LB Broth, Miller culture medium, and inoculated on an LB Broth, Miller culture medium agar plate containing 100 ⁇ g of ampicillin.
  • the cells were cultured overnight at 30 ° C., the number of colonies was counted, and the cell viability was measured.
  • the results are shown in Table 9. Thus, it was found that by treatment with an acid having a pH of 3 or less, Saccharomyces cerevisiae can survive and Escherichia coli can be killed.
  • Example 9 ⁇ Recycling study after acid treatment in Saccharomyces cerevisiae [pIPAc2-CTFABc and pADCc-ERG10 / X2180 strain], Escherichia coli pIaaa / B strain> The remainder of the Saccharomyces cerevisiae [pIPAc2-ATODAAc and pADCc-ERG10 / X2180 strain] sample reacted with the citrate-phosphate buffer in Example 8 was washed with a YPD culture solution, geneticin 200 ⁇ g / mL, noaceosricin The suspension was suspended in 20 mL of YPD Broth liquid medium containing 200 ⁇ g / mL, transferred to a 100 mL Erlenmeyer flask, and cultured.
  • the remainder of the Escherichia coli pIaaa / B sample that had been reacted with the citrate-phosphate buffer was washed with LB Broth, Miller culture solution and suspended in 20 mL of LB Broth, Miller culture solution containing 100 ⁇ g of ampicillin. It was transferred to a 100 mL Erlenmeyer flask and cultured.
  • Saccharomyces cerevisiae can produce isopropyl alcohol even after treatment with an acid having a pH of 2.6 or higher. It was also found that the pH is desirably 3 or more. It was found that Escherichia coli can produce isopropyl alcohol even after acid treatment at pH 3.8 and 5.4.
  • Example 10 ⁇ Isopropyl alcohol production by Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc / ERG10 genome-enhanced X2180 strain] and cell viability after acid treatment>
  • the Saccharomyces cerevisiae [pIPAc-ATODAAc and pADCc / ERG10 genome-enhanced X2180 strain] obtained in Example 7 was subjected to the same culture operation as in Example 8, and the amount of isopropyl alcohol accumulated in the culture supernatant was determined by HPLC.
  • Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc / ERG10 genome-enhanced X2180] containing cells corresponding to OD600 Abs10 (1 ⁇ 10 8 cells) was used in the same manner as in Example 8.
  • the cells were treated with an acid-phosphate buffer, the number of colonies was counted, and the cell viability was measured. The results are shown in Table 11. Thus, it was found that Saccharomyces cerevisiae can survive by treatment with an acid having a pH of 3 or less.
  • Example 11 ⁇ Recycling study after acid treatment in Saccharomyces cerevisiae [pIPAc2-CTFABc and pADCc / ERG10 genome-enhanced X2180 strain]> The remainder of the Saccharomyces cerevisiae [pIPAc2-ATODAAc and pADCc / ERG10 genome-enhanced X2180 strain] sample reacted with the citrate-phosphate buffer in Example 10 was washed with a YPD culture solution, geneticin 200 ⁇ g / mL, The suspension was suspended in 20 mL of YPD Broth liquid medium containing 200 ⁇ g / mL of noaceosricin, transferred to a 100 mL Erlenmeyer flask, and cultured.
  • Saccharomyces cerevisiae [pIPAc2-ATODAc and pADCc / ERG10 genome-enhanced X2180 strain] in the culture supernatant
  • the amount of isopropyl alcohol accumulated was measured by HPLC according to a conventional method. The results are shown in Table 12. As a result, it was found that Saccharomyces cerevisiae was treated with an acid having a pH of 5.4 or less, and the productivity after acid treatment was higher than that treated with pH 7.
  • Example 12 ⁇ Construction of Codon-Modified Clostridium Bacteria Isopropyl Alcohol Dehydrogenase Gene Expression Vector and the Expression Vector Transformant>
  • the DNA fragment of SEQ ID NO: 52 was used as a template, and was amplified by PCR using CCCCGCTAGCATGAAAGGTTTTGCAATGTT (SEQ ID NO: 89) and GGGGGGATCTCTATATAGATACTACTGCGCTT (SEQ ID NO: 90).
  • the fragment was digested with restriction enzymes NheI and BamHI to obtain an approximately 1.1 kbp codon-modified isopropyl alcohol dehydrogenase fragment.
  • the obtained DNA fragment and the fragment obtained by digesting the previously prepared plasmid pGK426 with restriction enzymes NheI and BglII were mixed and ligated using ligase, and then Escherichia coli DH5 ⁇ strain competent cell (Toyobo Co., Ltd.) Company DNA-903) was transformed to obtain transformants that grew on LB agar plates containing 50 ⁇ g / mL of ampicillin.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pIPAc3 was recovered from the obtained cells.
  • the genomic DNA of Clostridium begerinki NRRL B-593 was used as a template, CCCCGCTAGTAGGAGAAGGTTTGCAATGCT (SEQ ID NO: 91), and GGGGGGATCTCTATAATAACTACTGCTT (SEQ ID NO: 92) by PCR method Then, the obtained DNA fragment was digested with restriction enzymes NheI and BamHI to obtain an isopropyl alcohol dehydrogenase fragment of about 1.1 kbp.
  • the obtained DNA fragment and the previously prepared plasmid pGK426 were mixed with the fragments obtained by digesting with restriction enzymes NheI and BglII, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell.
  • a transformant that grows on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight at 37 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pIPA2 was recovered from the obtained cells.
  • PTHI2 containing the thiolase gene derived from Clostridium bacteria prepared in Example 3 and codon-unmodified pATOD and pATOA containing the CoA transferase gene derived from Escherichia coli prepared in Example 3 and acetoacetic acid dehydrated prepared in Example 3
  • a pADC containing a carboxylase gene and a pIPAc3 containing a codon-modified isopropyl alcohol dehydrogenase gene, or a codon-unmodified pIPA are transformed into a Saccharomyces cerevisiae YPH499 strain, and does not contain adenine, histidine, tryptophan, leucine, or uracil.
  • an isopropyl alcohol synthase gene group expression vector transformant [pIPA2, pADC, pATOA, pATOD, THI2 / YPH499 share] to give the [pIPAc3 pADC pATOAc2 pATODc2 pTHI2 / YPH499 shares].
  • the culture was performed at a stirring speed of 150 rpm and a culture temperature of 30 ° C. After 24 hours from the start of the culture, the cell culture solution was sampled, and after removing the cells by centrifugation, the amount of isopropyl alcohol accumulated in the obtained culture supernatant was measured by GC-MS according to a conventional method. The results are shown in Table 13. Thus, it has been clarified that isopropyl alcohol can be produced in yeast by using at least codon-modified IPAdh.
  • the total amount of each preculture was transferred to a 100 mL Erlenmeyer flask containing 40 mL of YPD Broth liquid medium containing Geneticin 200 ⁇ g / mL and Noaceosricin 200 ⁇ g / mL.
  • the culture was performed at a stirring speed of 120 rpm and a culture temperature of 30 ° C.
  • the cell culture solution was sampled, the cells were removed by centrifugation, and the amount of isopropyl alcohol accumulated in the obtained culture supernatant was measured by HPLC according to a standard method.
  • Saccharomyces cerevisiae [pIPAc2-atoDAc and pADCc / X2180 strain] and Saccharomyces cerevisiae [pYElacNAT and YElacKAN / X2180 strain], there was no accumulation of isopropyl alcohol.
  • the isopropyl alcohol-producing yeast according to the present invention can produce isopropyl alcohol only by optimizing the codons in the base sequence of the gene encoding isopropyl dehydrogenase. Is possible. Furthermore, from the results of Examples 5, 6, and 7, the isopropyl alcohol-producing yeast according to the present invention can obtain higher productivity by inserting and enhancing genes on the genome such as enhancing the promoter sequence of the genome. is there. Furthermore, CoA transferase, acetoacetate decarboxylase and isopropyl alcohol dehydrogenase used in the present invention are expected to show high productivity when inserted into the genome.
  • the isopropyl alcohol-producing yeast according to the present invention can be used repeatedly by washing with an acid. Furthermore, from the results of Example 8, Escherichia coli cannot survive in a citrate-phosphate buffer at pH 3. On the other hand, from the results of Examples 8 and 9, Saccharomyces cerevisiae can produce isopropyl alcohol even after treatment with citrate-phosphate buffer at pH 3 and pH 2.6, and Saccharomyces cerevisiae is a strong acid. It is thought that sterilization by can be performed. From these facts, it can be said that the isopropyl alcohol-producing yeast according to the present invention is remarkably superior to the prior art in the practical application of plant-derived isopropyl alcohol production.
  • the isopropyl alcohol-producing yeast according to the present invention can be improved in productivity by being washed with an acid having a pH of 5.4 or lower than when being washed with an acid having a pH of 7; This is considered to be because not only sterilization but also productivity can be improved by sterilization with a strong acid.
  • the yeast obtained by the present invention is not only a CoA transferase activity, an acetoacetate decarboxylase activity and an isopropyl alcohol dehydrogenase activity, but also a thiolase originally present in the yeast genome. It can be seen that isopropyl alcohol is not produced unless the activity of the enzyme encoded by the gene is also imparted.
  • the present invention by performing an acid treatment, it can be reused and isopropyl alcohol can be continuously produced. That is, according to the present invention, it is possible to provide an isopropyl alcohol-producing yeast that produces isopropyl alcohol that can be used repeatedly, and a method for producing isopropyl alcohol using the yeast.

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Abstract

La levure productrice d'alcool isopropylique d'acide urique selon l'invention possède les activités des enzymes productrices d'alcool isopropylique comprenant l'activité alcool isopropylique déshydrogénase, acétoacétate décarboxylase, CoA transférase et thiolase et alcool isopropylique déshydrogénase, lesdites activités ayant été conférées ou améliorées par gène transféré dans ladite levure sous la forme d'un gène modifié. Un procédé de production d'alcool isopropylique à l'aide de ladite levure productrice d'alcool isopropylique est également décrit.
PCT/JP2010/067596 2009-10-09 2010-10-06 Levure productrice d'alcool isopropylique et procédé de production d'alcool isopropylique Ceased WO2011043401A1 (fr)

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