WO2013125666A1 - Micro-organisme produisant de l'inositol avec haut rendement, et procédé pour la fabrication d'inositol en utilisant lesdits micro-organismes - Google Patents

Micro-organisme produisant de l'inositol avec haut rendement, et procédé pour la fabrication d'inositol en utilisant lesdits micro-organismes Download PDF

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WO2013125666A1
WO2013125666A1 PCT/JP2013/054455 JP2013054455W WO2013125666A1 WO 2013125666 A1 WO2013125666 A1 WO 2013125666A1 JP 2013054455 W JP2013054455 W JP 2013054455W WO 2013125666 A1 WO2013125666 A1 WO 2013125666A1
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inositol
gene
enzyme
phosphate
activity
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PCT/JP2013/054455
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English (en)
Japanese (ja)
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仙波　尚
勇介 宮▲崎▼
里栄 寺坂
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株式会社日本触媒
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01009—Glucose-6-phosphate isomerase (5.3.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y505/00—Intramolecular lyases (5.5)
    • C12Y505/01—Intramolecular lyases (5.5.1)
    • C12Y505/01004—Inositol-3-phosphate synthase (5.5.1.4)

Definitions

  • the present invention relates to a high inositol-producing microorganism and a method for producing inositol using the same.
  • Inositol is an important substance as a vitamin for higher animals, and plays an important role in the metabolism of fat and cholesterol, and is therefore effective for hypercholesterolemia. For this reason, it is used for nutritional foods, feed additives, pharmaceuticals, and the like.
  • the method of extracting from rice bran, corn steep liquor, etc. has a low yield of inositol and contains many impurities other than inositol, so that purification is difficult and there is a problem in terms of production efficiency.
  • the method for culturing and producing baker's yeast has low productivity, is still economically problematic, and has no industrial achievement.
  • the method of culturing and producing microorganisms belonging to the genus Candida has not been satisfactory in terms of industrial yield and yield.
  • the Opi gene that negatively regulates the expression of inositol-1-phosphate synthase (hereinafter referred to as “INO1”) gene in microorganisms belonging to the genus Saccharomyces has been destroyed.
  • INO1 inositol-1-phosphate synthase
  • the INO1 gene is increased by replacing the promoter of the INO1 gene with the promoter of the glyceraldehyde-3-phosphate (GAP) gene.
  • GAP glyceraldehyde-3-phosphate
  • Various mutant strains including mutant strains that have been expressed have been prepared and used for inositol production (Patent Documents 1-3).
  • An object of the present invention is to provide a microorganism capable of producing inositol in high yield and high yield.
  • Another object of the present invention is to provide a method for producing inositol capable of producing inositol in high yield and high yield.
  • the present inventors have reduced or deleted the expression level or activity of glycolytic enzymes and highly expressed inositol synthesis genes compared to the wild type.
  • the present inventors have found that the mutated microorganism can produce inositol in high yield and high yield, and have completed the present invention.
  • the present invention includes the following.
  • Glycolytic enzymes are phosphoglucose isomerase, phosphofructokinase, fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase and pyruvate
  • the mutant microorganism of [1] which is one or more enzymes selected from the group consisting of kinases.
  • the inositol synthesis gene is one or more genes encoding an enzyme selected from inositol-1-phosphate synthase and inositol-1-phosphate phosphatase [1] to [3] Any of the mutant microorganisms.
  • [8] A method for producing inositol, comprising culturing the mutant microorganism of any one of [1] to [7].
  • Inositol is a type of cyclitol having a structure (1,2,3,4,5,6-cyclohexanehexaol) in which one hydrogen atom on each carbon of cyclohexane is replaced with a hydroxy group.
  • Inositol in the present invention includes any of these isomers, but the present invention is particularly suitable for the production of myo-inositol.
  • the mutant microorganism of the present invention can be obtained by adding a mutation that reduces or eliminates the expression level or activity of a glycolytic enzyme and highly expresses the inositol synthesis gene to the host microorganism.
  • the “host microorganism” is not particularly limited as long as it is a microorganism having a glycolytic system, more specifically, a microorganism that can biosynthesize glucose-6-phosphate from glucose, and various microorganisms can be used. .
  • the host microorganism may be isolated from the natural environment, or may be produced by mutation treatment or gene recombination. Further, the host microorganism itself may or may not have inositol-producing ability.
  • the host microorganism can be either prokaryotic or eukaryotic, and for example, Escherichia coli or yeast can be used, and yeast is preferred.
  • yeast include Saccharomyces, Candida, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Hansenula, Kluyveromyces, Debaryomyces, Geotrichum, Wickerhamia, Poroboloces, Preferably, those belonging to the genus Saccharomyces or Zygosaccharomyces, more preferably Saccharomyces cerevisiae.
  • the “glycolytic enzyme” means an enzyme that catalyzes various glycolytic reactions.
  • the “glycolytic enzyme” is one that can suppress or inhibit glucose-6-phosphate metabolism by reducing or eliminating the expression level or activity of the enzyme.
  • Examples of such an enzyme include an enzyme having an activity of converting glucose-6-phosphate to fructose-6-phosphate, an enzyme having an activity of converting fructose-6-phosphate to fructose 1,6-bisphosphate, An enzyme having an activity to convert fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, an enzyme having an activity to convert glyceraldehyde 3-phosphate into 3-phosphoglycerophosphate, Enzyme with activity to convert seraldehyde 3-phosphate to 1,3-bisphosphoglycerate, enzyme with activity to convert 3-phosphoglycerate to 1,3-diphosphoglycerate, 3-phosphoglycerin An enzyme having an activity to convert acid to 2-phosphoglycerate, an enzyme having an activity to convert 2-phosphoglycerate to phosphoenolpyruvate, Such as an enzyme having an activity of converting an bins acid to pyruvic acid, but are not limited to.
  • phosphoglucose isomerase (hereinafter referred to as “PGI”) (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), fructose bisphosphate aldolase (EC 4.1.2.13), triose phosphate isomerase (EC 5.3.1.1), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglyceromutase (EC 5.4.2.1), enolase (EC 4.2.1.11) ) And enzymes such as pyruvate kinase (EC 2.7.1.40).
  • PGI phosphoglucose isomerase
  • “glycolytic enzyme” includes one or more of the above enzymes. That is, in the mutant microorganism of the present invention, the expression level or activity of one or more of the above enzymes (2, 3, 4, 5, 6, 7, 8, or 9 combinations) is reduced or deleted.
  • amino acid sequence and base sequence of the published databases are Accession No. M37267,03M26943,01M26944, X15003, J01366, J01324, J01342, X06408, J01322, X14400 Examples include registered ones.
  • PGI derived from Saccharomyces cerevisiae comprising the amino acid sequence of SEQ ID NO: 19 can be mentioned.
  • the “glycolytic enzyme” has a deletion, substitution, addition or insertion of 1 to several amino acids in the above published database and the amino acid sequence shown in SEQ ID NO: 19, and is still an enzyme Proteins that retain activity are also included.
  • the term “several” means 1 to 20, preferably 1 to 10.
  • the “glycolytic enzyme” is calculated using the above published database, the amino acid sequence shown in SEQ ID NO: 19, and BLASTP, TBLASTN, BLASTX, etc. (for example, default or default parameters). Occasionally, proteins comprising amino acid sequences having identity of 80% or more, more preferably 90% or more, most preferably 95% or more, and still retain enzymatic activity are also included.
  • Reduction or deletion of the expression level or activity of a glycolytic enzyme means that the expression level and / or activity of the glycolytic enzyme in the host microorganism is reduced or deleted compared to that in the wild type. More specifically, reducing or eliminating the expression level or activity of a glycolytic enzyme suppresses or inhibits glucose-6-phosphate metabolism in the glycolysis system of the host microorganism compared to that in the wild type. Means that.
  • the method for reducing or eliminating the expression level or activity of glycolytic enzymes is not particularly limited, and various techniques can be used.
  • a negative regulator that negatively regulates the expression of glycolytic enzymes For example, a negative regulator that negatively regulates the expression of glycolytic enzymes. , Introduction of a negative regulator that negatively regulates the activity of glycolytic enzymes, and the introduction of mutations that reduce or eliminate the expression level or activity of glycolytic enzymes (but are not limited thereto) ).
  • glycolytic genes transcription inhibitors of genes encoding glycolytic enzymes
  • a glycolytic gene transcription inhibitor can be obtained by a general screening method known to those skilled in the art. For example, a DNA construct in which a reporter gene (for example, luciferase gene, ⁇ -glucuronidase gene, green fluorescent protein (GFP) gene, etc.) is functionally linked downstream of the DNA including the expression control region of the glycolytic gene is prepared. In a situation where the reporter gene can be transcribed from the DNA construct, the DNA construct is brought into contact with a candidate factor for a transcription inhibitor in the presence and absence of a test substance.
  • a reporter gene for example, luciferase gene, ⁇ -glucuronidase gene, green fluorescent protein (GFP) gene, etc.
  • the glycolytic gene expression control region is, for example, genomic DNA using a primer or probe complementary to a desired gene sequence designed based on the base sequence of the glycolytic gene disclosed in the above published database. It can be obtained by techniques known to those skilled in the art, such as library screening, inverse PCR, and genome walking.
  • Antisense RNA consists of an antisense strand consisting of a sequence complementary to a part of the glycolytic gene mRNA (target mRNA portion) and a sense strand complementary to the base sequence of the antisense strand.
  • target mRNA portion a part of the glycolytic gene mRNA
  • sense strand complementary to the base sequence of the antisense strand.
  • the antisense strand hybridizes with the target mRNA portion to promote degradation of the mRNA (RNA interference) and significantly reduce glycolytic gene expression. Or can be deleted.
  • the antisense strand and the sense strand have a length of about 19 to 35 bases.
  • the sequence and length of the antisense strand and the sense strand can be selected by a known method based on the base sequence encoding the glycolytic enzyme shown in the above-mentioned database (for example, S. M. Elbashir et al. (2001) Nature 411: 494-498; S. M. Elbashir et al. (2001) Genes & Dev. 15: 188-200; S. M. Elbashir et al. (2001). EMBO J. 20: 6877-6888; Q. Ge et al. (2003) Proc. Natl. Acad. Sci. U. S. A.100: 2718-23; J. Harborth et al. (2001). J.
  • the antisense strand and the sense strand may be linked via a linker molecule, and may have a folded structure that forms a loop structure at the linker portion.
  • Examples of negative regulators that negatively regulate the activity of glycolytic enzymes include enzyme inhibitors.
  • Such an enzyme inhibitor can be obtained by a general screening method known to those skilled in the art. For example, the activity of glycolytic enzymes is compared in the presence and absence of the test substance. When the enzyme activity in the presence of the test substance is reduced or deleted compared to the enzyme activity in the absence of the test substance, the test substance can be identified as an enzyme inhibitor.
  • Examples of such enzyme inhibitors include, but are not limited to, 2-deoxyglucose, sodium fluoride (NaF), BZL101, sesquiterpene lactone, lithium iodoacetic acid, and the like.
  • a mutation introduction method known to those skilled in the art (for example, a homologous recombination method or a mutation induction method) is used. can do.
  • a DNA fragment for mutagenesis comprising a base sequence having a substitution, deletion, insertion and / or addition of one or more nucleotides in the base sequence of a glycolytic gene or the expression control region of the gene
  • the DNA fragment for mutagenesis is deleted so that a part or all of the gene or the expression control region of the gene is reduced or deleted to the extent that the expression of the gene or the activity of the protein encoded by the gene is reduced or deleted.
  • DNA fragment for gene disruption containing a base sequence obtained by inserting or replacing another base sequence or another base sequence (for example, a base sequence encoding a selection marker gene) is preferable.
  • selectable markers include formaldehyde resistance markers, drug resistance markers such as kanamycin, ampicillin, tetracycline, chloramphenicol, and auxotrophic markers such as leucine, histidine, lysine, methionine, arginine, tryptophan, and uracil. It is not limited to this.
  • the DNA fragment for mutagenesis is obtained by the electric pulse method (Y. Kurusu et al., Agric. Biol. Chem. 54: 443-447 (1990)), the method using calcium ions (Proc. Natl. Acad. Sci. USA) , 69, 2110 (1972)), protoplast method (JP 63-2483942), electroporation method (Nucleic Acids Res., 16, 6127 (1988)), etc. can do.
  • the introduced DNA fragment is introduced into the host chromosome through homologous recombination sites contained at both ends.
  • Mutagenesis is performed by irradiating a parent strain (for example, a wild strain or a mutant strain in which the following inositol synthesis gene is highly expressed) with ultraviolet rays, or a mutagenic agent (for example, N-methyl-N′-nitro-N-nitrosoguanidine, After treatment with ethyl methanesulfonic acid, etc., the expression level or activity of the above glycolytic enzyme is reduced or deleted compared to the parent strain, more preferably glucose-6-phosphate metabolism is suppressed. Select stocks.
  • a parent strain for example, a wild strain or a mutant strain in which the following inositol synthesis gene is highly expressed
  • a mutagenic agent for example, N-methyl-N′-nitro-N-nitrosoguanidine
  • the “inositol synthesis gene” includes a gene encoding an enzyme that catalyzes any reaction in the inositol biosynthesis reaction.
  • the “inositol synthesis gene” is a gene that can increase the synthesis amount of inositol by increasing the expression level and / or activity of the enzyme encoded by the gene.
  • an enzyme examples include, but are not limited to, an enzyme having an activity to synthesize myo-inositol-1-phosphate, an enzyme having an activity to dephosphorylate myo-inositol-1-phosphate, and the like. More specifically, inositol-1-phosphate synthase (EC 5.5.1.4) (hereinafter referred to as “INO1”), inositol-1-phosphate dephosphorylating enzyme (hereinafter referred to as “INM”) Enzymes such as
  • the “inositol synthesis gene” includes one or more genes encoding the enzyme. That is, in the mutant microorganism of the present invention, one or a plurality of genes (a combination of two) encoding the enzyme are highly expressed.
  • the inositol synthesis gene a gene derived from an animal including humans, a gene derived from a plant, or a gene derived from a microorganism can be used, but a gene derived from the same type of microorganism as the host microorganism is preferably used. That is, when the host microorganism is yeast, genes derived from yeast, Saccharomyces spp., Candida sp., Torulopsis sp., Zygosaccharomyces sp., Schizosaccharomyces sp., Pichia sp., Yarrowia sp., Hansenula sp., Kluyveromyces sp. Inositol synthesis genes derived from yeasts belonging to the genus, Wickerhamia genus, Fellomyces genus, Sporobolomyces genus and the like can be suitably used.
  • inositol synthesis genes include those registered in public databases (GenBank, EMBL, DDBJ) as Accession No. ⁇ J04453, AL396312, L22737.
  • the “inositol synthetic gene” has a deletion, substitution, addition or insertion of 1 to several bases in the above-mentioned database and the nucleotide sequences shown in SEQ ID NOs: 20 and 21, and still A base sequence encoding a protein that retains enzyme activity is also included.
  • the term “several” means 1 to 20, preferably 1 to 10.
  • the “inositol synthetic gene” includes a DNA that can hybridize under stringent conditions with the database disclosed above or a DNA comprising a base sequence complementary to the base sequences shown in SEQ ID NOs: 20 and 21. And a base sequence encoding a protein that still retains enzyme activity.
  • Stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • 2 to 6 ⁇ SSC composition of 1 ⁇ SSC: 0.15M NaCl, 0.015M Hybridize at 42-55 ° C in a solution containing sodium citrate, pH 7.0) and 0.1-0.5% SDS, and 55-65 ° C in a solution containing 0.1-0.2xSSC and 0.1-0.5% SDS Refers to the conditions for washing.
  • the “inositol synthetic gene” is calculated using the database disclosed above, the base sequences shown in SEQ ID NOS: 20 and 21, BLAST and the like (for example, default or default parameters)
  • a base sequence consisting of a base sequence having an identity of 80% or more, more preferably 90% or more, most preferably 95% or more, and still encoding an enzyme activity is also included.
  • High expression of the inositol synthesis gene means that the expression level and / or activity of the enzyme encoded by the inositol synthesis gene in the host microorganism is increased compared to that in the wild type. More specifically, high expression of the inositol synthesis gene means that the inositol synthesis ability in the host microorganism is increased as compared to that in the wild type, or that the inositol synthesis ability in the host microorganism is established.
  • the method for highly expressing the inositol synthesis gene is not particularly limited, and various methods can be used. For example, the introduction of a mutation that highly expresses the inositol synthesis gene or the transcription of the gene is negatively affected. Examples include (but are not limited to) inactivation of negative regulators to be regulated.
  • Methods for introducing a mutation that highly expresses an inositol synthesis gene include a method for introducing an inositol synthesis gene, a method for exchanging the promoter of the inositol synthesis gene with a promoter that causes the gene to be highly expressed, and a method by mutation induction Etc.
  • Inositol synthesis gene is introduced by linking the gene or a part thereof to an appropriate vector and introducing one or more of the obtained recombinant vectors into a host microorganism so that the target gene can be expressed, Alternatively, it can be carried out by inserting the target gene or a part thereof at an arbitrary position on the chromosome by homologous recombination. “Part” refers to a part of each gene capable of expressing the protein encoded by each gene when introduced into a host.
  • the inositol synthesis gene can be prepared by a general cloning method known to those skilled in the art. That is, a genomic library is screened by using a probe complementary to a desired gene sequence designed and synthesized based on the database disclosed above and the nucleotide sequence information shown in SEQ ID NOs: 20 and 21, and the sequence is isolated. Once this is done, the DNA can be amplified using standard amplification methods such as polymerase chain reaction (PCR) to obtain an amount of DNA suitable for transformation (gene transfer).
  • PCR polymerase chain reaction
  • An inositol synthetic gene with an unknown sequence can be obtained by a hybridization method or a PCR method using a synthetic DNA primer appropriately designed based on the known gene sequence as a template.
  • the vector is not particularly limited as long as it can be replicated in a host cell, and examples thereof include plasmids, phages and cosmids.
  • the chromosomally integrated vector may be any vector as long as it can replicate in E. coli.
  • Examples of the plasmid include E.
  • coli plasmids for example, pET21a (+), pET32a (+), pET39b (+), pET40b (+), pET43.1a (+), pET44a (+), pKK223-3, pGEX4T, pUC118, pUC119, pUC18, pUC19, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) pPICZ ⁇ , pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPIC9K, pPIC6, pGAPZ, pPIC9K, pPIC3.5K, pAO815, pFLD, pJL-IX, pJL-SX, etc.).
  • a commercially available cloning vector such as
  • an appropriate promoter is linked upstream of the inserted inositol synthesis gene to ensure that the inserted inositol synthesis gene is expressed.
  • the promoter to be used is not limited as long as it is a promoter capable of expressing a linked inositol synthesis gene in culture under the following conditions, and can be appropriately selected by those skilled in the art depending on the host. Examples include, but are not limited to, promoters such as glyceraldehyde-3-phosphate dehydrogenase gene, RNA polymerase gene, DNA gyrase gene, 5s rRNA gene, 16s rRNA gene, and 23s rRNA gene, and PGK promoter. Not.
  • a cis element such as an enhancer, a splicing signal, a poly
  • a addition signal such as an enhancer, a splicing signal, a poly
  • a selection marker such as a ribosome binding sequence (SD sequence) and the like may be linked to the vector, if desired.
  • the selection marker include those described above.
  • the constructed gene introduction vector can be introduced into a host microorganism by the gene introduction method such as the electric pulse method.
  • the method of inserting an inositol synthesis gene into an arbitrary position on a chromosome by homologous recombination is to insert an inositol synthesis gene linked to an appropriate promoter into a sequence homologous to the sequence on the genome, and this DNA fragment is electropulsed.
  • a strain in which homologous recombination has occurred can be easily selected by using a DNA fragment ligated with a selection marker gene.
  • a gene linked to a drug resistance gene and a gene that becomes lethal under specific conditions is inserted into the chromosome by homologous recombination using the above method, and then becomes lethal under specific conditions with the drug resistance gene.
  • the target gene can also be introduced using homologous recombination in the form of replacing the gene.
  • the method for exchanging the promoter can be carried out, for example, by exchanging the inositol synthetic gene promoter on the genome with the target promoter (for example, the above-mentioned one) by homologous recombination according to the above method.
  • the method of mutagenesis is carried out by irradiating a parent strain (for example, a wild strain or a mutant strain in which the expression level or activity of the glycolytic enzyme is reduced or deleted) with a UV ray, or a mutagenic agent (for example, N-methyl-N After treatment with '-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid, etc.), a strain that highly expresses inositol synthesis gene can be selected.
  • a parent strain for example, a wild strain or a mutant strain in which the expression level or activity of the glycolytic enzyme is reduced or deleted
  • a mutagenic agent for example, N-methyl-N After treatment with '-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid, etc.
  • the method of inactivating a negative regulator that negatively regulates the transcription of the inositol synthesis gene can be performed using the same method as the method of reducing or deleting the expression level or activity of the glycolytic enzyme. That is, introduction of a mutation that reduces or deletes the activity of the negative regulator or the expression level of the gene encoding the negative regulator.
  • negative regulators that negatively regulate transcription of inositol synthesis genes include, but are not limited to, the Opi gene.
  • the method for selecting mutant microorganisms of the present invention is not particularly limited, and can be selected by culturing under the following conditions, measuring the amount of inositol in the medium by HPLC or the like, and comparing.
  • the method for producing inositol of the present invention includes culturing the mutant microorganism of the present invention using a medium for producing inositol.
  • the medium may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like and can cultivate mutant yeast efficiently.
  • a carbon source for example, sugars such as glucose, sucrose, lactose, maltose, trehalose, cellobiose, polyols such as glycerin, alcohols such as ethanol, or organic acids such as pyruvic acid, succinic acid or citric acid are used. be able to.
  • a nitrogen source for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean koji alkali extract, alkylamines such as methylamine, or ammonia or a salt thereof can be used.
  • salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, a specific amino acid, a specific vitamin, an antifoaming agent, and the like may be used as necessary.
  • Cultivation is usually carried out under aerobic conditions such as shaking culture or aeration and agitation culture, preferably at 0 to 40 ° C., more preferably at 10 to 37 ° C., particularly preferably at 15 to 37 ° C.
  • the pH of the medium can be changed as appropriate as long as mutant microorganisms can grow and the activity of the inositol synthesis gene is not impaired, but is preferably in the range of about pH 3-9.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
  • inositol When inositol is produced in the microbial cells after culturing, generally known methods such as mechanical methods, enzymatic methods using lysozyme, or chemical treatment using a surfactant, etc. Inositol can be extracted by destroying by. When inositol is produced outside the cells, the culture solution is used as it is, or the cells are removed by centrifugation or the like.
  • the method for collecting and isolating inositol from the culture is not particularly limited. That is, it can be carried out by combining conventionally known ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods and other methods.
  • inositol crystals can be obtained by removing cells by centrifugation, removing ionic substances with a cation and anion exchange resin, and concentrating. Inositol accumulated in the culture may be used as it is without being isolated.
  • glucose-6-phosphate is rapidly metabolized to gain energy due to the action of glycolysis, and the amount of inositol produced is very small.
  • the expression level or activity of the glycolytic enzyme is reduced or deleted, and therefore glucose-6-phosphate metabolism is suppressed or inhibited, and inositol synthesis is performed. It is considered that inositol is efficiently produced from glucose-6-phosphate because inositol synthesis ability is increased due to high gene expression.
  • inositol is obtained in a higher yield than glucose, that is, 17% or more, 18% or more, 19% or more, 20% or more, 25% or more, 30% or more, 31% or more, 32% or more, 33%
  • it can be obtained in a yield of 35% or more, 40% or more, or more.
  • PCR Polymerase chain reaction
  • the enzyme for PCR was KOD-plus- (manufactured by TOYOBO), and the reaction solution composition and reaction conditions were determined according to the protocol attached to the product.
  • purification was performed using Wizard SV gel and PCR Clean-up System manufactured by Promega.
  • the purified DNA fragment was cleaved with restriction enzymes SacI and PstI and purified again.
  • the purified DNA fragment was inserted between the SacI and PstI sites of pUC18 according to a conventional method to construct pUC18-pgi.
  • PCR was carried out to amplify a DNA fragment containing the kanamycin resistance gene (KanMX).
  • KanMX kanamycin resistance gene
  • the DNA fragment obtained by inverse PCR and the DNA fragment containing KanMX were purified and then cleaved with the restriction enzyme XbaI, and both were combined according to a conventional method to obtain pUC18- ⁇ pgi :: KanMX.
  • 1-2 Construction of a phosphoglucose isomerase gene disruption strain Saccharomyces cerevisiae KK4 was transformed with a ⁇ pgi :: KanMX cassette, and YPFDG agar medium (Yeast Extract 10 g / L, Bacto Peptone 20 g / L, Fructose 20 g / L, Glucose 0.5 g / L, Agar 20 g / L, Geneticin 200 ⁇ g / L). Transformation was performed according to the method described in the yeast gene experiment manual (translated by Maruzen Co., Ltd., Junichi Ohya, p111-112). The agar medium coated with the bacterial solution was statically cultured in a 30 ° C.
  • PCR was performed using a plurality of colonies generated on an agar medium as a template and the primers shown in Table 1.
  • the PCR product was subjected to agarose gel electrophoresis, and only a DNA fragment of about 2.5 kbp could be confirmed as a phosphoglucose isomerase gene disrupted strain.
  • the PCR enzyme was KOD-plus- manufactured by TOYOBO, and the reaction solution composition and reaction conditions were determined according to the protocol attached to the product.
  • the amplified DNA was purified using the Wizard SV gel and PCR Clean-up System manufactured by Promega. The purified DNA was cleaved with restriction enzymes MunI and EcoRI, and inserted into the EcoRI site of pUC18 according to a conventional method to obtain pUC18-PGAP.
  • PCR was carried out using, and a DNA fragment containing the upstream and downstream sequences of the glyceraldehyde-3-phosphate dehydrogenase gene was amplified.
  • the amplified DNA was purified, cleaved with the restriction enzyme EcoRV, and cloned into pCR4-TOPO according to a conventional method to obtain pCR4-PGAP-TGAP.
  • PCR was performed to amplify the open reading frame (ORF) portion of the inositol-1-phosphate synthase gene.
  • the amplified DNA was purified, cleaved with restriction enzyme SalI, and cloned into the SalI site of pCR4-PGAP-TGAP according to a conventional method to obtain pCR4-PGAP-INO1-TGAP.
  • PCR was performed, and the upstream region of the glyceraldehyde-3-phosphate dehydrogenase gene, the ORF region of the inositol-1-phosphate synthase gene, and the downstream region of the glyceraldehyde-3-phosphate dehydrogenase gene A DNA fragment containing was amplified.
  • the amplified DNA was purified, cut with the restriction enzyme XbaI, and cloned into the XbaI site of pAUR101 according to a conventional method to obtain pAUR101-PGAP-INO1-TGAP.
  • Inositol-1-phosphate synthase gene high expression of phosphoglucose isomerase gene-disrupted strain pAUR101-PGAP-INO1-TGAP was digested with restriction enzyme BsiWI and purified and constructed as described above
  • a phosphoglucose isomerase gene-disrupted strain of Saccharomyces cerevisiae KK4 was transformed.
  • the transformation solution was applied to YPFDA agar medium (Yeast Extract 10g / L, Bacto Peptone 20g / L, Fructose 20g / L, Glucose 0.5g / L, Agar 20g / L, Aureobasidin 0.5 ⁇ g / L).
  • the inositol-1-phosphate synthase gene high expression strain was designated.
  • Inositol productivity evaluation test ⁇ Method A breeding strain of Saccharomyces cerevisiae KK4 constructed as described above (a strain in which the phosphoglucose isomerase gene was disrupted and the inositol-1-phosphate synthase gene was highly expressed) was cultured in an YPFD medium (Yeast Extract 10g / L, Bacto Peptone 20g / L, Fructose 20g / L, Glucose 0.5g / L) was inoculated into 10 test tubes into which 5mL was dispensed. The inoculated test tube was cultured for 24 hours at a rotation speed of 300 rpm using a reciprocating shake culture apparatus set at 30 ° C.
  • YPFD medium Yeast Extract 10g / L, Bacto Peptone 20g / L, Fructose 20g / L, Glucose 0.5g / L
  • the culture medium (Yeast Extract 20g / L, Bacto Peptone 40g / L, Fructose 100g / L, Glucose 4g / L) is placed in a 2L jar fermenter (Biott) containing 1.2L. The whole amount was inoculated. The temperature of the culture solution was controlled to 30 ° C., and the pH was controlled to be 5.5 with 25% aqueous ammonia. The air flow rate was 1.2 L / min, and the rotation speed was 680 rpm. After the start of culture, the culture solution was sampled over time and analyzed by liquid chromatography to monitor the concentration of various components in the culture solution. The glucose decreased with the cultivation was supplemented by adding a 50% (w / v) aqueous glucose solution, and the cultivation was carried out for 31.5 hours.
  • the phosphoglucose isomerase gene disruption strain of Saccharomyces cerevisiae KK4 constructed in 1-2 of Example 1 was inoculated into a medium test tube into which 5 mL of YPFD medium was dispensed. After inoculation, using a reciprocating shake culture apparatus (TXY-16R-3F manufactured by Takasaki Kagaku Co., Ltd.) set at 30 ° C., culture was performed for 24 hours at a rotation speed of 300 rpm.
  • TXY-16R-3F manufactured by Takasaki Kagaku Co., Ltd.
  • a main culture medium Yeast Extract 10 g / L, Bacto Peptone 20 g / L, Fructose 20 g / L, Glucose 3 g / L.
  • a reciprocating shake culture apparatus set at 30 ° C. under the condition of 120 rpm.
  • the culture solution was sampled over time and analyzed by liquid chromatography to monitor the concentration of various components in the culture solution.
  • the glucose decreased with the culture was supplemented by adding a 50% (w / v) aqueous glucose solution. The culture was stopped after 64 hours from the start of the culture.
  • the transformation solution was applied to YPDA agar medium (Yeast Extract 10g / L, Bacto Peptone 20g / L, Glucose 20g / L, Agar 20g / L, Aureobasidin 0.5 ⁇ g / L), and the resulting colonies were inositol-1-phosphate A synthase gene high expression strain was designated.
  • YPDA agar medium Yeast Extract 10g / L, Bacto Peptone 20g / L, Glucose 20g / L, Agar 20g / L, Aureobasidin 0.5 ⁇ g / L
  • culturing was started using a reciprocating shake culture apparatus set at 30 ° C. under the condition of 120 rpm. After the start of culture, the culture solution was sampled over time and analyzed by liquid chromatography to monitor the concentration of various components in the culture solution. The glucose decreased with the culture was supplemented by adding a 50% (w / v) aqueous glucose solution. The culture was stopped after 47 hours from the start of the culture.
  • Inositol is a highly useful substance used in various fields such as nutritional foods, feed additives, and pharmaceuticals. According to the present invention, inositol can be produced in a significantly higher yield and yield than the prior art. The present invention is expected to make a significant contribution in the field of inositol production.

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Abstract

L'invention concerne: un micro-organisme mutant capable de produire de l'inositol avec un haut rendement ; et un procédé pour fabriquer de l'inositol en utilisant le micro-organisme mutant. L'invention concerne un micro-organisme mutant capable de produire de l'inositol, dans lequel, comparé aux micro-organismes de type sauvage, la quantité d'expression ou l'activité des enzymes glycolytiques est réduite ou délétée et le gène de synthèse de l'inositol est exprimé à un niveau élevé. L'invention concerne aussi un procédé de fabrication d'inositol par l'utilisation dudit micro-organisme mutant.
PCT/JP2013/054455 2012-02-23 2013-02-22 Micro-organisme produisant de l'inositol avec haut rendement, et procédé pour la fabrication d'inositol en utilisant lesdits micro-organismes WO2013125666A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018511343A (ja) * 2015-04-17 2018-04-26 チェンドゥ ユアンホン バイオロジカル テクノロジー カンパニー リミテッド イノシトールの調製方法
CN113913451A (zh) * 2020-07-08 2022-01-11 山东福洋生物科技股份有限公司 一株产肌醇的巴斯德毕赤酵母工程菌的构建方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09220093A (ja) * 1996-02-16 1997-08-26 Toray Ind Inc イノシトール−１−リン酸合成酵素をコードするｄｎａ、それを含む組換えｄｎａ、形質転換体およびそれを用いたイノシトールの製造方法
JP2005507108A (ja) * 2001-10-26 2005-03-10 ザ・レジェンツ・オブ・ザ・ユニバーシティ・オブ・カリフォルニア 酵母代謝をモデリングするための組成物および方法
WO2006109479A1 (fr) * 2005-03-30 2006-10-19 Niigata Bio-Research Park, Inc. Cassette d’expression de gene et transformant, et procede pour la production de 2-desoxy-scyllo-inosose et procede pour la purification de 2-desoxy-scyllo-inosose en utilisant le transformant
JP2011055722A (ja) * 2009-09-07 2011-03-24 Nippon Shokubai Co Ltd イノシトールの製造方法
WO2011063304A1 (fr) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Méthodes, systèmes et compositions de bio-production microbienne de biomolécules employant au titre de produits de départ des composants de gaz de synthèse ou des sucres
WO2011063363A2 (fr) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Production d'un acide organique, et/ou produits chimiques connexes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09220093A (ja) * 1996-02-16 1997-08-26 Toray Ind Inc イノシトール−１−リン酸合成酵素をコードするｄｎａ、それを含む組換えｄｎａ、形質転換体およびそれを用いたイノシトールの製造方法
JP2005507108A (ja) * 2001-10-26 2005-03-10 ザ・レジェンツ・オブ・ザ・ユニバーシティ・オブ・カリフォルニア 酵母代謝をモデリングするための組成物および方法
WO2006109479A1 (fr) * 2005-03-30 2006-10-19 Niigata Bio-Research Park, Inc. Cassette d’expression de gene et transformant, et procede pour la production de 2-desoxy-scyllo-inosose et procede pour la purification de 2-desoxy-scyllo-inosose en utilisant le transformant
JP2011055722A (ja) * 2009-09-07 2011-03-24 Nippon Shokubai Co Ltd イノシトールの製造方法
WO2011063304A1 (fr) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Méthodes, systèmes et compositions de bio-production microbienne de biomolécules employant au titre de produits de départ des composants de gaz de synthèse ou des sucres
WO2011063363A2 (fr) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Production d'un acide organique, et/ou produits chimiques connexes
US20110125118A1 (en) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Production of an Organic Acid and/or Related Chemicals

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHI, YIHUI. ET AL.: "Genetic Perturbation of Glycolysis Results in Inhibition of de Novo Inositol Biosynthesis", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 51, 23 December 2005 (2005-12-23), pages 41805 - 41810, XP055082108 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018511343A (ja) * 2015-04-17 2018-04-26 チェンドゥ ユアンホン バイオロジカル テクノロジー カンパニー リミテッド イノシトールの調製方法
US10597682B2 (en) 2015-04-17 2020-03-24 Chengdu Yuanhong Biological Technology Co., Ltd. Inositol preparation method
US11028414B2 (en) 2015-04-17 2021-06-08 Bonumose, Inc. Inositol preparation method
CN113913451A (zh) * 2020-07-08 2022-01-11 山东福洋生物科技股份有限公司 一株产肌醇的巴斯德毕赤酵母工程菌的构建方法
CN113913451B (zh) * 2020-07-08 2023-05-26 山东福洋生物科技股份有限公司 一株产肌醇的巴斯德毕赤酵母工程菌的构建方法

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