WO2023286776A1 - Mel-producing recombinant microorganism - Google Patents

Mel-producing recombinant microorganism Download PDF

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WO2023286776A1
WO2023286776A1 PCT/JP2022/027444 JP2022027444W WO2023286776A1 WO 2023286776 A1 WO2023286776 A1 WO 2023286776A1 JP 2022027444 W JP2022027444 W JP 2022027444W WO 2023286776 A1 WO2023286776 A1 WO 2023286776A1
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mel
gene
strain
pseudozyma
pep carboxykinase
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French (fr)
Japanese (ja)
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あずさ 雜賀
友岳 森田
圭介 和田
達也 藤井
周平 山本
知宏 菅原
顕生 川原
淳 川井
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国立研究開発法人産業技術総合研究所
東洋紡株式会社
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

Definitions

  • a technique for producing mannosylerythritol lipids using microorganisms is disclosed.
  • Mannosylerythritol lipid is a substance having a structure (Fig. 1) in which fatty acid is ester-bonded to mannosylerythritol (also referred to as "ME”) in which erythritol is glycoside-bonded to mannose. is.
  • MEL has attracted attention as a substance (biosurfactant) produced by microorganisms and having a function as a surfactant.
  • MEL has various structures with different positions and numbers of fatty acid residues and acetyl groups that bind to mannose.
  • the structure in which R1 and R2 are fatty acid residues and R3 and R4 are acetyl groups is called MEL-A.
  • a structure in which R1 and R2 are fatty acid residues , R3 is a hydrogen atom, and R4 is an acetyl group is called MEL-B.
  • a structure in which R1 and R2 are fatty acid residues, R3 is an acetyl group and R4 is a hydrogen atom is called MEL-C.
  • a structure in which R 1 and R 2 are fatty acid residues and R 3 and R 4 are hydrogen atoms is called MEL-D.
  • FIG. 2(a) shows MEL having a sugar skeleton of 4-O- ⁇ -D-mannopyranosyl-erythritol, which is referred to as 4-O- ⁇ -D-MEL.
  • the former taxonomic name is produced by Pseudozyma antarctica).
  • MEL having 1-O- ⁇ -D-mannopyranosyl-erythritol as a sugar skeleton, and is called 1-O- ⁇ -D-MEL. Produces seed MEL. Compared to 4-O- ⁇ -MEL, 1-O- ⁇ -MEL has improved hydration and high vesicle-forming ability, making it a promising biomaterial for skin care agents.
  • Patent Document 1 Various attempts have been made to improve the production efficiency of MEL by microorganisms (for example, Patent Document 1).
  • One challenge is to provide a means of producing MEL more efficiently.
  • Item 1 A mutant mannosylerythritol lipid-producing microorganism in which a PEP carboxykinase gene and/or a malic enzyme gene are strongly expressed.
  • Item 2 Item 1. The microorganism according to Item 1, which belongs to the genus Pseudozyma or Moesziomyces.
  • Item 3 Item 3. A method for producing MEL using the microorganism according to Item 1 or 2.
  • R 1 to R 5 each represent a hydrogen atom, an acetyl group, or a fatty acid residue having 3 to 18 carbon atoms.
  • the structure of 4-O- ⁇ -D-MEL (a) and the structure of 1-O- ⁇ -D-MEL (b) are shown.
  • the culture profile of M. antarcticus is shown. (A) is the case of using glucose as the carbon source, and (B) is the case of using olive oil as the carbon source. Squares indicate the carbon source, circles indicate the amount of bacterial cells, and diamonds indicate the amount of MEL produced. Metabolome analysis results of M. antarcticus are shown.
  • G6P is glucose-6-phosphate
  • 6PG 6-phosphogluconate
  • F6P fructose-6-phosphate
  • E4P is erythrose-4-phosphate
  • GAP is glyceraldehyde-3-phosphate
  • S7P is sedoheptulose-7-phosphate
  • R5P is ribose-5-phosphate
  • X5P is xylulose-5-phosphate
  • RU5P is ribulose-5-phosphate
  • FBP is fructose-1,6-bisphosphate
  • DHAP dihydroxyacetone Phosphate
  • 1,3BPG for 1,3-bisphosphoglycerate
  • 3PG for 3-phosphoglycerene phosphate
  • PEP for phosphoenolpyruvate
  • PYR for pyruvate
  • AcCoA for acetyl-CoA
  • OAA for oxaloacetate
  • CIT citric acid Acid
  • ACO cis
  • G indicates the result of culturing using carbon source
  • O indicates the result of culturing using olive oil as carbon source.
  • the structure of the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK is shown.
  • the culture profile of the P. tsukubaensis 1E5 strain introduced with the PEP carboxykinase gene expression vector is shown.
  • “hyg” indicates strains introduced with the expression vector pHSG2_hyg
  • PEPCK indicates strains introduced with the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK.
  • PaLIPA+hyg indicates a lipase-introduced strain into which the expression vector pHSG2_hyg was introduced
  • PaLIPA+PEPCK shows a lipase-introduced strain into which the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK was introduced.
  • (a) is the dry cell weight
  • (b) is the residual amount of oil
  • (c) is the MEL production amount. The results of jar culture of a P.
  • tsukubaensis 1E5/pUCT_neo::PaLIPA (E5Ptef) strain lipase-introduced strain introduced with a PEP carboxykinase gene expression vector are shown.
  • PaLIPA+hyg indicates a lipase-introduced strain into which the expression vector pHSG2_hyg was introduced
  • PaLIPA+PEPCK shows a lipase-introduced strain into which the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK was introduced.
  • (a) is the dry cell weight
  • (b) is the residual amount of oil
  • (c) is the MEL production amount.
  • the PEP carboxykinase gene and/or the malic enzyme gene are strongly expressed in the mannosylerythritol lipid-producing microorganism.
  • the PEP carboxykinase gene and the malic enzyme gene are strongly expressed in the mannosylerythritol lipid-producing microorganism.
  • the PEP carboxykinase gene is preferably strongly expressed in the mannosylerythritol lipid-producing microorganism.
  • MEL-producing microorganisms are cultured in a medium containing vegetable oil (for example, olive oil), fumaric acid and malic acid are relatively low in the metabolic system involved in the production of MEL in microorganisms, as shown by the results of metabolome analysis in Examples. accumulate a lot. Therefore, by strongly expressing the PEP carboxykinase gene and the malic enzyme gene, these substances are converted to phosphoenolpyruvate and pyruvate, and by promoting the flow to MEL biosynthesis, the production efficiency of MEL is improved. can be raised.
  • vegetable oil for example, olive oil
  • the PEP carboxykinase gene is a gene that encodes PEP carboxykinase.
  • a malic enzyme gene is a gene that encodes malic enzyme.
  • PEP carboxykinase is an enzyme that produces phosphoenolpyruvate and carbon dioxide from oxaloacetate and ATP.
  • Malic enzyme is an enzyme that decarboxylates malic acid to produce pyruvate.
  • the origin of the PEP carboxykinase gene is not particularly limited.
  • the PEP carboxykinase gene may be microbial, plant or animal.
  • the preferred PEP carboxykinase is of microbial origin.
  • preferred microorganisms from which PEP carboxykinase is derived are the genera Pseudozyma, Moesziomyces, Ustilago, Sporisorium, Melanopsichium, and Kurtzmanomyces.
  • Preferred Pseudozyma microorganisms are Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma hubeiensis, and Pseudozyma tsukubaensis.
  • Preferred microorganisms of the genus Moesziomyces are Moesziomyces antarcticus, Moesziomyces aphidis (old taxonomy Pseudozyma aphidis), Moesziomyces partarcticus (old taxonomy Pseudozyma partarctica), and Moesziomyces rugulosus (old taxonomy Pseudozyma rugulosa).
  • Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis.
  • Preferred Sporisorium microorganisms are Sporisorium graminicola (formerly known as Pseudozyma graminicola), Sporisorium reilianum and Sporisorium scitamineum.
  • a preferred Melanopsichium microorganism is Melanopsichium pennsylvanicum.
  • a preferred Kurtzmanomyces microorganism is Kurtzmanomyces sp. I-11.
  • the origin of the malic enzyme gene is not particularly limited.
  • the malic enzyme gene may be any of microorganisms, plants and animals.
  • the preferred malic enzyme is of microbial origin.
  • the preferred microorganisms from which the malic enzyme is derived are the genera Pseudozyma, Moesziomyces, Ustilago, Sporisorium, Melanopsichium, and Kurtzmanomyces.
  • Preferred Pseudozyma microorganisms are Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma hubeiensis, and Pseudozyma tsukubaensis.
  • Preferred Moesziomyces microorganisms are Moesziomyces antarcticus, Moesziomyces aphidis, Moesziomyces paratarcticus, and Moesziomyces rugulosus.
  • Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis.
  • Preferred Sporisorium microorganisms are Sporisorium graminicola, Sporisorium reilianum and Sporisorium scitamineum.
  • a preferred Melanopsichium microorganism is Melanopsichium pennsylvanicum.
  • a preferred Kurtzmanomyces microorganism is Kurtzmanomyces sp. I-11.
  • the gene is strongly expressed means that the expression level of the gene is increased compared to before modification (mutation).
  • the means for overexpressing the gene is arbitrary and not particularly limited, and known techniques can be selected as appropriate. For example, by constructing an expression vector in which a gene is placed under the control of a control element (e.g., promoter) capable of overexpressing the gene and introducing it into a microorganism, the gene can be overexpressed.
  • a control element e.g., promoter
  • Any promoter can be used, but for example, the native promoter of the gene, the glyceraldehyde triphosphate dehydrogenase gene promoter (Pgap), the elongation factor EF-1 promoter (Ptef), or the ubiquitin gene promoter (Pubq) etc. can be mentioned.
  • genes under the control of specific promoters can be integrated into the genome of microorganisms by genome editing.
  • the construction of the expression vector is arbitrary as long as it has a gene encoding PEP carboxykinase or malic enzyme under the control of a specific promoter as described above.
  • the expression vector may have multiple genes encoding PEP carboxykinase and/or genes encoding malic enzyme under the control of a specific promoter.
  • multiple genes encoding PEP carboxykinase may be of the same type or of different types.
  • multiple genes encoding malic enzymes may be of the same type or of different types.
  • the expression vector may contain multiple cassettes containing a gene encoding PEP carboxykinase and/or a gene encoding malic enzyme under the control of a specific promoter.
  • the multiple genes encoding PEP carboxykinase contained in the expression vector may be of the same species or of different species.
  • multiple genes encoding malic enzymes may be homologous or heterologous to each other.
  • specific promoters contained in each cassette may be of the same or different type.
  • the type and structure of the expression vector are not particularly limited as long as they are capable of replication and expression within the host cell (microorganism).
  • the type of vector can be appropriately selected according to the type of host cell.
  • Specific examples of vectors include plasmid vectors, cosmid vectors, phage vectors, virus vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, etc.).
  • a preferred vector in one embodiment is a plasmid vector.
  • preferred vectors are pUXV1-neo, pPAX1-neo, pPAA1-neo, pUC_neo, pUCT_neo, pHSG_hyg, and pHSG2_hyg.
  • An expression vector containing a selectable marker can also be used as the expression vector. Insertion of selection marker genes into expression vectors, insertion of promoters, etc. can be performed using standard recombinant DNA techniques (see, for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). .
  • the method of introducing the vector into the host cell is arbitrary and can be selected as appropriate according to the type of host cell and vector.
  • the vector introduction method can be carried out, for example, by electroporation, calcium phosphate coprecipitation method, lipofection, microinjection, lithium acetate method and the like.
  • the vector is preferably introduced into the host cell after the plasmid vector is integrated by restriction enzyme treatment. This enables stable transformation by integrating the introduced gene into the genomic gene.
  • a MEL-producing microorganism is a microorganism that has the ability to produce MEL.
  • the type of MEL-producing microorganism in which the gene is strongly expressed is not particularly limited. Examples thereof include MEL-producing microorganisms belonging to the genus Pseudozyma, Moesziomyces or Sporisorium.
  • preferred MEL-producing microorganisms are microorganisms belonging to Moesziomyces antarcticus, Pseudozyma tsukubaensis, Moesziomyces rugulosus, Moesziomyces aphidis, Moesziomyces partarcticus, Pseudozyma hubeiensis and Sporisorium graminicola.
  • a preferred MEL-producing microorganism in one embodiment is Pseudozyma tsukubaensis.
  • MEL-producing microorganisms may be microorganisms modified by genes other than the PEP carboxykinase gene and the malic enzyme gene.
  • the MEL-producing microorganism may be a microorganism modified to overexpress the lipase gene. Examples of such microorganisms include those described in Patent Document 1.
  • Any method can be used to produce MEL using mutant microorganisms. For example, it can be carried out by culturing the microorganism in a medium suitable for producing MEL.
  • the type of fatty acid is not particularly limited, and can be appropriately selected according to the type of MEL desired. Examples include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, behenic acid, and nervonic acid.
  • a preferred fatty acid in one embodiment is oleic acid.
  • the type of vegetable oil is not particularly limited, and examples thereof include soybean oil, olive oil, rapeseed oil, safflower oil, sesame oil, palm oil, sunflower oil, coconut oil, cocoa butter, and castor oil.
  • the conditions for culturing microorganisms are not particularly limited.
  • the microorganism belongs to the genus Moesziomyces or Pseudozyma, it can be cultured for 3 to 14 days under conditions of pH 3-8, preferably pH 4-7, temperature 20-35°C, preferably 22-28°C.
  • MEL can be recovered from the culture medium according to a standard method.
  • MELs are 1-O- ⁇ -MEL and 4-O- ⁇ -MEL.
  • 1-O- ⁇ -MEL and 4-O- ⁇ -MEL may be any of MEL-A, MEL-B, MEL-C and MEL-D.
  • Metabolism Enzymes involved in the synthesis of MEL include the enzyme that synthesizes the sugar skeleton (EMT1), the enzyme that binds acyl groups to mannose (MAC1, MAC2), and the enzyme that binds acetyl groups to mannose (MAT1). ing.
  • EMT1 sugar skeleton
  • MAC1, MAC2 mannose
  • MAT1 mannose
  • fatty acids produced when the vegetable oil is degraded by extracellular lipase are taken into the cell and converted to the acyl-CoA type, followed by the ⁇ -oxidation pathway. It is known that medium-chain fatty acids with shortened are used as fatty acids constituting MEL.
  • MEL biosynthesis involves not only the direct MEL synthesis pathway but also complex metabolic pathways such as the TCA cycle, glycolysis/gluconeogenesis, and the pentose phosphate pathway involved in the synthesis of erythritol.
  • Metabolome analysis of M. antarcticus is known as an effective means for analyzing intracellular metabolism in more detail. Unlike the transcriptome, which evaluates the expression level of genes, this method analyzes the amount of metabolites in cells. Allows forecasting from quantity.
  • FIG. 3 shows the culture profile when M. antarcticus was cultured using glucose or olive oil as a carbon source for metabolomic analysis.
  • FIG. 3A shows the results of culturing using glucose as a carbon source
  • FIG. 3B shows the results of culturing using olive oil as a carbon source. In both cases, the production of MEL (diamonds in the figure) and dry cell weight (circles in the figure) increased linearly.
  • Figure 4 shows the results of collecting the cells after culturing for 72 hours, extracting intracellular metabolites, detecting them by GC-MS and LC-MS, and conducting metabolome analysis. It can be seen that fatty acids and glycerol are olive oil>glucose and glucose is olive oil ⁇ glucose. This result indicates that when olive oil is used as the raw material, the intracellular amounts of fatty acids and glycerol, which are degradation products of olive oil, are high, and when glucose is used as the raw material, the intracellular amount of glucose is large. After fatty acids are taken up into cells, they are converted to acyl-CoA and finally degraded to acetyl-CoA. I understand.
  • Enhancing the expression of PEP carboxykinase and malic enzyme genes As a means of enhancing the reactions that direct surplus FUM and MAL toward PYR and PEP, it is necessary to enhance the expression of PEP carboxykinase and malic enzyme genes. Conceivable. Therefore, the sequence information of the gene is obtained from the genome information of M. antarcticus, P. tsukubaensis, etc., and the gene expression strain is constructed according to the standard method. As a result, it becomes clear that the amount of MEL production is increased by enhancing the expression of the gene.
  • enhancement of the expression of the PEP carboxykinase gene of P. tsukubaensis will be specifically described.
  • Pseudozyma tsukubaensis strain 1E5 (deposit number JCM16987) Pseudozyma tsukubaensis 1E5/pUCT_neo::PaLIPA (E5Ptef) strain (lipase-introduced strain; prepared by introducing the lipase gene of M. antarcticus T-34 strain into P.
  • tsukubaensis according to the method described in the example of Patent Document 1) ⁇ Genomic DNA Pseudozyma tsukubaensis strain 1E5, plasmid expression vector pHSG2_hyg ⁇ Culture medium YM medium with glycerol: prepared by dissolving 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and 50 g of glycerol in 1 L of deionized water.
  • MEL Production Medium Prepared by dissolving 5 g of yeast extract, 3 g of sodium nitrate, 0.3 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate heptahydrate, and 20 g of glycerol in 1 L of deionized water.
  • SEQ ID NO: 1 is the nucleotide sequence (including upstream 1 kb and downstream 0.5 kb of the gene) encoding PEP carboxykinase of Pseudozyma tsukubaensis 1E5 strain.
  • a forward primer SEQ ID NO: 2 with a BamHI site added upstream of the sequence
  • a reverse primer SEQ ID NO: 3 with an XbaI site added downstream of the sequence were prepared.
  • the genomic DNA of Pseudozyma tsukubaensis 1E5 strain was used as a template for gene amplification.
  • the amplified gene was added to the expression vector pHSG2_hyg (containing the origin of replication (UARS) derived from filamentous fungi (Ustilago maydis) and the hygromycin resistance gene (hyg)) cleaved at the BamHI site and the XbaI site.
  • Ligation high Ver.2 ( Toyobo) to construct the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK.
  • the structure of the expression vector is shown in FIG.
  • Fwd (for PEP carboxykinase amplification, SEQ ID NO: 2) CGGGGATCCGCCGGATCACGGCTTTGCTG
  • Rvs (for PEP carboxykinase amplification, SEQ ID NO: 3) GACTCTAGAGATAGATGCACCAACAGAGC
  • HPLC condition detector Evaporative Light Scattering Detector 1260 Infinity II (manufactured by Agilent)
  • A:B 100:0
  • Flow rate 1mL/min
  • hyg indicates the strain introduced with the expression vector pHSG2_hyg (control)
  • PEPCK indicates the strain introduced with the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK.
  • PaLIPA+hyg indicates the expression vector pHSG2_hyg-introduced strain (control)
  • PaLIPA+PEPCK indicates the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK-introduced strain.
  • PaLIPA+hyg indicates the expression vector pHSG2_hyg-introduced strain (control)
  • PaLIPA+PEPCK indicates the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK-introduced strain.
  • FIG. 8(a) it was confirmed that the PaLIPA+PEPCK strain increased the amount of cell growth compared to the control.
  • FIG. 8(c) it was confirmed that the PaLIPA+PEPCK strain improved the MEL production amount more than the control.

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Abstract

The present invention provides a means for more efficiently producing MEL. This mutated-type mannosyl erythritol lipid-producing microorganism expresses, at a high level, a PEP carboxykinase gene and/or a malic enzyme gene.

Description

ＭＥＬ産生組換え微生物MEL-producing recombinant microorganism
　微生物を用いたマンノシルエリスリトールリピッドの生産に関する技術が開示される。 A technique for producing mannosylerythritol lipids using microorganisms is disclosed.
　マンノシルエリスリトールリピッド（「MEL」とも称される。）は、マンノースにエリスリトールがグリコシド結合したマンノシルエリスリトール（「ME」とも称される。）に、更に脂肪酸がエステル結合した構造（図１）を有する物質である。MELは、微生物によって生産される界面活性剤として機能を有する物質（バイオサーファクタント）として近年注目されている。 Mannosylerythritol lipid (also referred to as "MEL") is a substance having a structure (Fig. 1) in which fatty acid is ester-bonded to mannosylerythritol (also referred to as "ME") in which erythritol is glycoside-bonded to mannose. is. In recent years, MEL has attracted attention as a substance (biosurfactant) produced by microorganisms and having a function as a surfactant.
　MELは、マンノースに結合する脂肪酸残基及びアセチル基の位置及び数等が異なる種々の構造が存在する。図１に示す構造において、Ｒ１及びＲ２が脂肪酸残基であり、Ｒ３及びＲ４がアセチル基である構造物はMEL-Aと呼ばれる。Ｒ１及びＲ２が脂肪酸残基であり、Ｒ３が水素原子であり、Ｒ４がアセチル基である構造物はMEL-Bと呼ばれる。Ｒ１及びＲ２が脂肪酸残基であり、Ｒ３がアセチル基でＲ４が水素原子である構造物はMEL-Cと呼ばれる。Ｒ１及びＲ２が脂肪酸残基であり、Ｒ３及びＲ４が水素原子である構造物はMEL-Dと呼ばれる。 MEL has various structures with different positions and numbers of fatty acid residues and acetyl groups that bind to mannose. In the structure shown in Figure 1, the structure in which R1 and R2 are fatty acid residues and R3 and R4 are acetyl groups is called MEL-A. A structure in which R1 and R2 are fatty acid residues , R3 is a hydrogen atom, and R4 is an acetyl group is called MEL-B. A structure in which R1 and R2 are fatty acid residues, R3 is an acetyl group and R4 is a hydrogen atom is called MEL-C. A structure in which R 1 and R 2 are fatty acid residues and R 3 and R 4 are hydrogen atoms is called MEL-D.
　また、マンノースと結合するエリスリトールのヒドロキシメチル基が１位の炭素に由来するか、４位の炭素に由来するかによって、MELの構造は図２の（ａ）と（ｂ）に示す２種類が存在する。図２の（ａ）に示されるのは、4－O－β－D－mannopyranosyl－erythritolを糖骨格とするMELであり、4－O－β－D－MELと称され、例えば、Moesziomyces antarcticus(旧分類名はPseudozyma antarctica)によって生産される。図２（ｂ）に示されるのは、1－O－β－D－mannopyranosyl－erythritolを糖骨格とするMELであり、1－O－β－D－MELと称され、例えば、Pseudozyma tsukubaensisがこの種のMELを生産する。1－O－β－MELは、4－O－β－MELと比べて水和性が向上し、ベシクル形成能も高いという特徴を持ち、スキンケア剤などとして有望なバイオ素材である。 In addition, depending on whether the hydroxymethyl group of erythritol that binds to mannose is derived from the 1-position carbon or from the 4-position carbon, there are two types of MEL structures shown in Figure 2 (a) and (b). exist. FIG. 2(a) shows MEL having a sugar skeleton of 4-O-β-D-mannopyranosyl-erythritol, which is referred to as 4-O-β-D-MEL. The former taxonomic name is produced by Pseudozyma antarctica). FIG. 2(b) shows MEL having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton, and is called 1-O-β-D-MEL. Produces seed MEL. Compared to 4-O-β-MEL, 1-O-β-MEL has improved hydration and high vesicle-forming ability, making it a promising biomaterial for skin care agents.
　これまでに微生物によるMELの生産効率を改善するため種々の試みがなされている（例えば、特許文献１）。 Various attempts have been made to improve the production efficiency of MEL by microorganisms (for example, Patent Document 1).
WO2021/010264WO2021/010264
　MELをより効率的に生産する手段を提供することが１つの課題である。 One challenge is to provide a means of producing MEL more efficiently.
　下記に代表される発明が提供される。
項１
PEPカルボキシキナーゼ遺伝子及び／又はリンゴ酸酵素遺伝子が強発現されている、変異型マンノシルエリスリトールリピッド産生微生物。
項２
Pseudozyma属またはMoesziomyces属に属する、項１に記載の微生物。
項３
項１又は２に記載の微生物を用いてMELを生産する方法。 The inventions represented below are provided.
Item 1
A mutant mannosylerythritol lipid-producing microorganism in which a PEP carboxykinase gene and/or a malic enzyme gene are strongly expressed.
Item 2
Item 1. The microorganism according to Item 1, which belongs to the genus Pseudozyma or Moesziomyces.
Item 3
Item 3. A method for producing MEL using the microorganism according to Item 1 or 2.
　効率的なMELの生産が可能となる。 Efficient MEL production becomes possible.
MELの基本構造を示す。Ｒ１～Ｒ５は、水素原子、アセチル基、または炭素数３～１８の脂肪酸残基を示す。The basic structure of MEL is shown. R 1 to R 5 each represent a hydrogen atom, an acetyl group, or a fatty acid residue having 3 to 18 carbon atoms. 4－O－β－D－MELの構造（ａ）、及び1－O－β－D－MELの構造（ｂ）を示す。The structure of 4-O-β-D-MEL (a) and the structure of 1-O-β-D-MEL (b) are shown. M. antarcticusの培養プロファイルを示す。（Ａ）は、炭素源としてグルコースを用いた場合であり、（Ｂ）は炭素源としてオリーブ油を用いた場合である。四角は炭素源、丸は菌体量、菱形はMELの生産量を示す。The culture profile of M. antarcticus is shown. (A) is the case of using glucose as the carbon source, and (B) is the case of using olive oil as the carbon source. Squares indicate the carbon source, circles indicate the amount of bacterial cells, and diamonds indicate the amount of MEL produced. M. antarcticusのメタボローム解析結果を示す。図中、G6Pはグルコース-6-リン酸、6PGは6-ホスホグルコン酸、F6Pはフルクトース-6-リン酸、E4Pはエリトロース-4-リン酸、GAPはグリセルアルデヒド-3-リン酸、S7Pはセドヘプツロース-7-リン酸、R5Pはリボース-5-リン酸、X5Pはキシルロース-5-リン酸、RU5Pはリブロース-5-リン酸、FBPはフルクトース-1,6-ビスリン酸、DHAPはジヒドロキシアセトンリン酸、1,3BPGは1,3-ビスホスホグリセリン酸、3PGは3-ホスホグリセンリン酸、PEPはホスホエノールピルビン酸、PYRはピルビン酸、AcCoAはアセチルCoA、OAAはオキサロ酢酸、CITはクエン酸、ACOはcis-アコニット酸、ICITはイソクエン酸、αKGはα-ケトグルタル酸、SUCCoAはスクシニルCoA、SUCはコハク酸、FUMはフマル酸、MALはリンゴ酸、ATPはアデノシン三リン酸、ADPはアデノシン二リン酸、AMPはアデノシン一リン酸を指す。各グラフにおいて「G」は炭素源として培養した結果を示し、「O」は、オリーブ油を炭素源として培養した結果を示す。Metabolome analysis results of M. antarcticus are shown. In the figure, G6P is glucose-6-phosphate, 6PG is 6-phosphogluconate, F6P is fructose-6-phosphate, E4P is erythrose-4-phosphate, GAP is glyceraldehyde-3-phosphate, S7P is sedoheptulose-7-phosphate, R5P is ribose-5-phosphate, X5P is xylulose-5-phosphate, RU5P is ribulose-5-phosphate, FBP is fructose-1,6-bisphosphate, DHAP is dihydroxyacetone Phosphate, 1,3BPG for 1,3-bisphosphoglycerate, 3PG for 3-phosphoglycerene phosphate, PEP for phosphoenolpyruvate, PYR for pyruvate, AcCoA for acetyl-CoA, OAA for oxaloacetate, CIT for citric acid Acid, ACO for cis-aconitate, ICIT for isocitrate, αKG for α-ketoglutarate, SUCCoA for succinyl-CoA, SUC for succinate, FUM for fumarate, MAL for malate, ATP for adenosine triphosphate, ADP for Adenosine diphosphate, AMP refers to adenosine monophosphate. In each graph, "G" indicates the result of culturing using carbon source, and "O" indicates the result of culturing using olive oil as carbon source. PEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCKの構造を示す。The structure of the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK is shown. P. tsukubaensis 1E5株にPEPカルボキシキナーゼ遺伝子発現ベクターを導入した株の培養プロファイルを示す。各グラフにおいて「hyg」は発現ベクターpHSG2_hygを導入した株を示し、「PEPCK」はPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCKを導入した株を示す。（a）は乾燥菌体重量、（b）はMEL生産量、（c）はオレイン酸の残存量を示す。The culture profile of the P. tsukubaensis 1E5 strain introduced with the PEP carboxykinase gene expression vector is shown. In each graph, "hyg" indicates strains introduced with the expression vector pHSG2_hyg, and "PEPCK" indicates strains introduced with the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK. (a) is the dry cell weight, (b) is the MEL production amount, and (c) is the residual amount of oleic acid. P. tsukubaensis 1E5/pUCT_neo::PaLIPA（E5Ptef）株(リパーゼ導入株)にPEPカルボキシキナーゼ遺伝子発現ベクターを導入した株のフラスコ培養の結果を示す。各グラフにおいて「PaLIPA+hyg」はリパーゼ導入株に発現ベクターpHSG2_hygを導入した株を示し、「PaLIPA+PEPCK」はリパーゼ導入株にPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCKを導入した株を示す。（a）は乾燥菌体重量、（b）は油脂の残存量、（c）はMEL生産量を示す。The results of flask culture of a P. tsukubaensis 1E5/pUCT_neo::PaLIPA (E5Ptef) strain (lipase-introduced strain) introduced with a PEP carboxykinase gene expression vector are shown. In each graph, "PaLIPA+hyg" indicates a lipase-introduced strain into which the expression vector pHSG2_hyg was introduced, and "PaLIPA+PEPCK" shows a lipase-introduced strain into which the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK was introduced. (a) is the dry cell weight, (b) is the residual amount of oil, and (c) is the MEL production amount. P. tsukubaensis 1E5/pUCT_neo::PaLIPA（E5Ptef）株(リパーゼ導入株)にPEPカルボキシキナーゼ遺伝子発現ベクターを導入した株のジャー培養の結果を示す。各グラフにおいて「PaLIPA+hyg」はリパーゼ導入株に発現ベクターpHSG2_hygを導入した株を示し、「PaLIPA+PEPCK」はリパーゼ導入株にPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCKを導入した株を示す。（a）は乾燥菌体重量、（b）は油脂の残存量、（c）はMEL生産量を示す。The results of jar culture of a P. tsukubaensis 1E5/pUCT_neo::PaLIPA (E5Ptef) strain (lipase-introduced strain) introduced with a PEP carboxykinase gene expression vector are shown. In each graph, "PaLIPA+hyg" indicates a lipase-introduced strain into which the expression vector pHSG2_hyg was introduced, and "PaLIPA+PEPCK" shows a lipase-introduced strain into which the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK was introduced. (a) is the dry cell weight, (b) is the residual amount of oil, and (c) is the MEL production amount.
　マンノシルエリスリトールリピッド産生微生物は、PEPカルボキシキナーゼ遺伝子及び／又はリンゴ酸酵素遺伝子が強発現されていることが好ましい。マンノシルエリスリトールリピッド産生微生物は、PEPカルボキシキナーゼ遺伝子及びリンゴ酸酵素遺伝子が強発現されていることが好ましい。一実施形態において、マンノシルエリスリトールリピッド産生微生物は、PEPカルボキシキナーゼ遺伝子が強発現されていることが好ましい。 It is preferable that the PEP carboxykinase gene and/or the malic enzyme gene are strongly expressed in the mannosylerythritol lipid-producing microorganism. Preferably, the PEP carboxykinase gene and the malic enzyme gene are strongly expressed in the mannosylerythritol lipid-producing microorganism. In one embodiment, the PEP carboxykinase gene is preferably strongly expressed in the mannosylerythritol lipid-producing microorganism.
　植物油（例えば、オリーブ油）を含む培地でMEL産生微生物を培養した場合、実施例のメタボローム解析結果が示すように、微生物内のMELの生産に関与する代謝系において、フマル酸やリンゴ酸が比較的多く蓄積する。よって、PEPカルボキシキナーゼ遺伝子及びリンゴ酸酵素遺伝子を強発現させることにより、これらの物質をホスホエノールピルビン酸やピルビン酸に変換し、MEL生合成への流れを促進することで、MELの生産効率を上げることができる。 When MEL-producing microorganisms are cultured in a medium containing vegetable oil (for example, olive oil), fumaric acid and malic acid are relatively low in the metabolic system involved in the production of MEL in microorganisms, as shown by the results of metabolome analysis in Examples. accumulate a lot. Therefore, by strongly expressing the PEP carboxykinase gene and the malic enzyme gene, these substances are converted to phosphoenolpyruvate and pyruvate, and by promoting the flow to MEL biosynthesis, the production efficiency of MEL is improved. can be raised.
　PEPカルボキシキナーゼ遺伝子とは、PEPカルボキシキナーゼをコードする遺伝子である。リンゴ酸酵素遺伝子とは、リンゴ酸酵素をコードする遺伝子である。PEPカルボキシキナーゼは、オキサロ酢酸とＡＴＰからホスホエノールピルビン酸と二酸化炭素を生成する酵素である。リンゴ酸酵素は、リンゴ酸を脱炭酸しピルビン酸を生成する酵素である。 The PEP carboxykinase gene is a gene that encodes PEP carboxykinase. A malic enzyme gene is a gene that encodes malic enzyme. PEP carboxykinase is an enzyme that produces phosphoenolpyruvate and carbon dioxide from oxaloacetate and ATP. Malic enzyme is an enzyme that decarboxylates malic acid to produce pyruvate.
　PEPカルボキシキナーゼ遺伝子の由来は特に制限されない。例えば、PEPカルボキシキナーゼ遺伝子は、微生物、植物及び動物のいずれでもよい。一実施形態において好ましいPEPカルボキシキナーゼは微生物由来である。一実施形態において、PEPカルボキシキナーゼの由来として好ましい微生物は、Pseudozyma属、Moesziomyces属、Ustilago属、Sporisorium属、Melanopsichium属、及びKurtzmanomyces属である。好ましいPseudozyma属微生物は、Pseudozyma siamensis、Pseudozyma shanxiensis、Pseudozyma crassa、Pseudozyma churashimaensis、Pseudozyma hubeiensis、及びPseudozyma tsukubaensisである。好ましいMoesziomyces属微生物はMoesziomyces antarcticus、Moesziomyces aphidis（旧分類名Pseudozyma aphidis）、Moesziomyces parantarcticus（旧分類名Pseudozyma parantarctica）、Moesziomyces rugulosus（旧分類名Pseudozyma rugulosa）である。好ましいUstilago属微生物は、Ustilago hordei及びUstilago maydisである。好ましいSporisorium属微生物は、Sporisorium graminicola（旧分類名Pseudozyma graminicola）、Sporisorium reilianum及びSporisorium scitamineumである。好ましいMelanopsichium属微生物は、Melanopsichium pennsylvanicumである。好ましいKurtzmanomyces属微生物は、Kurtzmanomyces sp. I-11である。 The origin of the PEP carboxykinase gene is not particularly limited. For example, the PEP carboxykinase gene may be microbial, plant or animal. In one embodiment the preferred PEP carboxykinase is of microbial origin. In one embodiment, preferred microorganisms from which PEP carboxykinase is derived are the genera Pseudozyma, Moesziomyces, Ustilago, Sporisorium, Melanopsichium, and Kurtzmanomyces. Preferred Pseudozyma microorganisms are Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma hubeiensis, and Pseudozyma tsukubaensis. Preferred microorganisms of the genus Moesziomyces are Moesziomyces antarcticus, Moesziomyces aphidis (old taxonomy Pseudozyma aphidis), Moesziomyces partarcticus (old taxonomy Pseudozyma partarctica), and Moesziomyces rugulosus (old taxonomy Pseudozyma rugulosa). Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis. Preferred Sporisorium microorganisms are Sporisorium graminicola (formerly known as Pseudozyma graminicola), Sporisorium reilianum and Sporisorium scitamineum. A preferred Melanopsichium microorganism is Melanopsichium pennsylvanicum. A preferred Kurtzmanomyces microorganism is Kurtzmanomyces sp. I-11.
　リンゴ酸酵素遺伝子の由来は特に制限されない。例えば、リンゴ酸酵素遺伝子は、微生物、植物及び動物のいずれでもよい。一実施形態において好ましいリンゴ酸酵素は微生物由来である。一実施形態において、リンゴ酸酵素の由来として好ましい微生物は、Pseudozyma属、Moesziomyces属、Ustilago属、Sporisorium属、Melanopsichium属、及びKurtzmanomyces属である。好ましいPseudozyma属微生物は、Pseudozyma siamensis、Pseudozyma shanxiensis、Pseudozyma crassa、Pseudozyma churashimaensis、Pseudozyma hubeiensis、及びPseudozyma tsukubaensisである。好ましいMoesziomyces属微生物はMoesziomyces antarcticus、Moesziomyces aphidis、Moesziomyces parantarcticus、Moesziomyces rugulosusである。好ましいUstilago属微生物は、Ustilago hordei及びUstilago maydisである。好ましいSporisorium属微生物は、Sporisorium graminicola 、Sporisorium reilianum及びSporisorium scitamineumである。好ましいMelanopsichium属微生物は、Melanopsichium pennsylvanicumである。好ましいKurtzmanomyces属微生物は、Kurtzmanomyces sp. I-11である。 The origin of the malic enzyme gene is not particularly limited. For example, the malic enzyme gene may be any of microorganisms, plants and animals. In one embodiment, the preferred malic enzyme is of microbial origin. In one embodiment, the preferred microorganisms from which the malic enzyme is derived are the genera Pseudozyma, Moesziomyces, Ustilago, Sporisorium, Melanopsichium, and Kurtzmanomyces. Preferred Pseudozyma microorganisms are Pseudozyma siamensis, Pseudozyma shanxiensis, Pseudozyma crassa, Pseudozyma churashimaensis, Pseudozyma hubeiensis, and Pseudozyma tsukubaensis. Preferred Moesziomyces microorganisms are Moesziomyces antarcticus, Moesziomyces aphidis, Moesziomyces paratarcticus, and Moesziomyces rugulosus. Preferred Ustilago microorganisms are Ustilago hordei and Ustilago maydis. Preferred Sporisorium microorganisms are Sporisorium graminicola, Sporisorium reilianum and Sporisorium scitamineum. A preferred Melanopsichium microorganism is Melanopsichium pennsylvanicum. A preferred Kurtzmanomyces microorganism is Kurtzmanomyces sp. I-11.
　「遺伝子が強発現されている」とは、遺伝子の発現レベルが改変（変異）前と比較して高められていることを意味する。遺伝子を強発現させる手段は任意であり、特に制限されず、公知の手法を適宜選択できる。例えば、遺伝子を強発現させることが可能な制御因子（例えば、プロモーター）の制御下に遺伝子を配置した発現ベクターを構築し、これで微生物に導入することで、当該遺伝子を強発現させることができる。使用できるプロモーターは任意であるが、例えば、当該遺伝子のネイティブプロモーター、グリセルアルデヒド三リン酸デヒドロゲナーゼ遺伝子のプロモーター（Pgap）、伸長因子EF-1のプロモーター（Ptef）、又はユビキチン遺伝子のプロモーター（Pubq）等を挙げることができる。その他、ゲノム編集により微生物のゲノム上に特定のプロモーターの制御下にある遺伝子を組み込むことができる。 "The gene is strongly expressed" means that the expression level of the gene is increased compared to before modification (mutation). The means for overexpressing the gene is arbitrary and not particularly limited, and known techniques can be selected as appropriate. For example, by constructing an expression vector in which a gene is placed under the control of a control element (e.g., promoter) capable of overexpressing the gene and introducing it into a microorganism, the gene can be overexpressed. . Any promoter can be used, but for example, the native promoter of the gene, the glyceraldehyde triphosphate dehydrogenase gene promoter (Pgap), the elongation factor EF-1 promoter (Ptef), or the ubiquitin gene promoter (Pubq) etc. can be mentioned. In addition, genes under the control of specific promoters can be integrated into the genome of microorganisms by genome editing.
　発現ベクターは、上述のとおり特定のプロモーターの制御下にあるPEPカルボキシキナーゼ、またはリンゴ酸酵素をコードする遺伝子を有する限り、その構成は任意である。例えば、発現ベクターは、特定のプロモーターの制御下にPEPカルボキシキナーゼをコードする遺伝子及び／又はリンゴ酸酵素をコードする遺伝子を複数有していてもよい。この場合、PEPカルボキシキナーゼをコードする複数の遺伝子は互いに同種であってもよく、異種であってもよい。同様に、リンゴ酸酵素をコードする複数の遺伝子は互いに同種であってもよく、異種であってもよい。発現ベクターは、特定のプロモーターの制御下にあるPEPカルボキシキナーゼをコードする遺伝子及び／又はリンゴ酸酵素をコードする遺伝子を含むカセットを複数含んでいてもよい。この場合も発現ベクターに含まれるPEPカルボキシキナーゼをコードする複数の遺伝子は互いに同種であってもよく、異種であってもよい。同様に、リンゴ酸酵素をコードする複数の遺伝子は互いに同種でも異種でもよい。また、各カセットに含まれる特定のプロモーターも互いに同種でも異種でもよい。 The construction of the expression vector is arbitrary as long as it has a gene encoding PEP carboxykinase or malic enzyme under the control of a specific promoter as described above. For example, the expression vector may have multiple genes encoding PEP carboxykinase and/or genes encoding malic enzyme under the control of a specific promoter. In this case, multiple genes encoding PEP carboxykinase may be of the same type or of different types. Similarly, multiple genes encoding malic enzymes may be of the same type or of different types. The expression vector may contain multiple cassettes containing a gene encoding PEP carboxykinase and/or a gene encoding malic enzyme under the control of a specific promoter. Also in this case, the multiple genes encoding PEP carboxykinase contained in the expression vector may be of the same species or of different species. Similarly, multiple genes encoding malic enzymes may be homologous or heterologous to each other. In addition, specific promoters contained in each cassette may be of the same or different type.
　発現ベクターの種類は、宿主細胞（微生物）内で複製及び発現が可能である限り、その種類や構造は特に限定されない。ベクターの種類は、宿主細胞の種類に応じて適宜選択できる。ベクターの具体例としては、プラスミドベクター、コスミドベクター、ファージベクター、ウイルスベクター（アデノウイルスベクター、アデノ随伴ウイルスベクター、レトロウイルスベクター、ヘルペスウイルスベクター等）等を挙げることができる。一実施形態において好ましいベクターは、プラスミドベクターである。 The type and structure of the expression vector are not particularly limited as long as they are capable of replication and expression within the host cell (microorganism). The type of vector can be appropriately selected according to the type of host cell. Specific examples of vectors include plasmid vectors, cosmid vectors, phage vectors, virus vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, etc.). A preferred vector in one embodiment is a plasmid vector.
　プラスミドベクターとしては、例えば、pUXV1　ATCC　77463、pUXV2　ATCC　77464、pUXV5　ATCC　77468、pUXV6　ATCC　77469、pUXV7　ATCC　77470、pUXV8　ATCC　77471、pUXV3　ATCC　77465、pU2X1　ATCC　77466、pU2X2　ATCC　77467、pUXV1-neo、pPAX1-neo、pPAA1-neo、pUC_neo、pUCT_neo、pHSG_hyg、及びpHSG2_hyg等を例示することができる。一実施形態において、好ましいベクターは、pUXV1-neo、pPAX1-neo、pPAA1-neo、pUC_neo、pUCT_neo、pHSG_hyg、及びpHSG2_hygである。 プラスミドベクターとしては、例えば、pUXV1　ATCC　77463、pUXV2　ATCC　77464、pUXV5　ATCC　77468、pUXV6　ATCC　77469、pUXV7　ATCC　77470、pUXV8　ATCC　77471、pUXV3　ATCC　77465、pU2X1　ATCC　77466、pU2X2　ATCC　77467、pUXV1-neo、pPAX1-neo , pPAA1-neo, pUC_neo, pUCT_neo, pHSG_hyg, and pHSG2_hyg. In one embodiment, preferred vectors are pUXV1-neo, pPAX1-neo, pPAA1-neo, pUC_neo, pUCT_neo, pHSG_hyg, and pHSG2_hyg.
　発現ベクターとして、選択マーカーを含む発現ベクターを使用することもできる。発現ベクターへの選択マーカー遺伝子の挿入、プロモーターの挿入等は標準的な組換えDNA技術（例えば、Molecular　Cloning，　Third　Edition，　1.84，　Cold　Spring　Harbor　Laboratory　Press，　New　York参照）を用いて行うことができる。 An expression vector containing a selectable marker can also be used as the expression vector. Insertion of selection marker genes into expression vectors, insertion of promoters, etc. can be performed using standard recombinant DNA techniques (see, for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). .
　宿主細胞へのベクターの導入方法は任意であり、宿主細胞及びベクターの種類等に応じて適宜選択できる。ベクターの導入方法は、例えば、エレクトロポレーション、リン酸カルシウム共沈降法、リポフェクション、マイクロインジェクション、及び酢酸リチウム法等によって実施することができる。一実施形態において、宿主細胞へのベクターの導入方法は、プラスミドベクターを制限酵素処理によって一本化してから導入することが好ましい。これにより、導入した遺伝子をゲノム遺伝子に組み込むことで安定的な形質転換ができる。 The method of introducing the vector into the host cell is arbitrary and can be selected as appropriate according to the type of host cell and vector. The vector introduction method can be carried out, for example, by electroporation, calcium phosphate coprecipitation method, lipofection, microinjection, lithium acetate method and the like. In one embodiment, the vector is preferably introduced into the host cell after the plasmid vector is integrated by restriction enzyme treatment. This enables stable transformation by integrating the introduced gene into the genomic gene.
　MEL産生微生物とは、MELを産生する能力を有する微生物である。遺伝子が強発現される、MEL産生微生物の種類は特に制限されない。例えば、Pseudozyma属、Moesziomyces属又はSporisorium属に属するMEL産生微生物が挙げられる。一実施形態において、好ましいMEL生産微生物は、Moesziomyces antarcticus、Pseudozyma tsukubaensis、Moesziomyces rugulosus、Moesziomyces aphidis、Moesziomyces parantarcticus、Pseudozyma hubeiensis及びSporisorium graminicolaに属する微生物である。一実施形態において好ましいMEL生産微生物は、Pseudozyma tsukubaensisである。 A MEL-producing microorganism is a microorganism that has the ability to produce MEL. The type of MEL-producing microorganism in which the gene is strongly expressed is not particularly limited. Examples thereof include MEL-producing microorganisms belonging to the genus Pseudozyma, Moesziomyces or Sporisorium. In one embodiment, preferred MEL-producing microorganisms are microorganisms belonging to Moesziomyces antarcticus, Pseudozyma tsukubaensis, Moesziomyces rugulosus, Moesziomyces aphidis, Moesziomyces partarcticus, Pseudozyma hubeiensis and Sporisorium graminicola. A preferred MEL-producing microorganism in one embodiment is Pseudozyma tsukubaensis.
　MEL産生微生物は、PEPカルボキシキナーゼ遺伝子及びリンゴ酸酵素遺伝子以外の遺伝子によって改変された微生物であってもよい。例えば、MEL産生微生物は、リパーゼ遺伝子を強発現するように改変された微生物であってもよい。そのような微生物としては、例えば、特許文献１に記載されるものを挙げることができる。 　MEL-producing microorganisms may be microorganisms modified by genes other than the PEP carboxykinase gene and the malic enzyme gene. For example, the MEL-producing microorganism may be a microorganism modified to overexpress the lipase gene. Examples of such microorganisms include those described in Patent Document 1.
　変異型微生物を用いたMELの生産は任意の方法で行うことができる。例えば、MELの生産に適した培地で微生物を培養することによって実施できる。一実施形態において、微生物を用いてMELを生産する場合、培地に脂肪酸又はそのエステル類、或いは植物油脂を添加することが好ましい。脂肪酸の種類は特に制限されず、目的とするMELの種類等に応じて適宜選択することができる。例えば、カプリル酸、カプリン酸、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレン酸、アラキドン酸、ベヘン酸、及びネルボン酸等を上げることができる。一実施形態において好ましい脂肪酸はオレイン酸である。植物油脂の種類は特に制限されず、例えば、大豆油、オリーブ油、ナタネ油、紅花油、ゴマ油、パームオイル、ひまわり油、ココナッツ油、カカオバター、及びひまし油等を挙げることができる。 Any method can be used to produce MEL using mutant microorganisms. For example, it can be carried out by culturing the microorganism in a medium suitable for producing MEL. In one embodiment, when producing MEL using microorganisms, it is preferable to add fatty acids or esters thereof, or vegetable oils and fats to the medium. The type of fatty acid is not particularly limited, and can be appropriately selected according to the type of MEL desired. Examples include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, behenic acid, and nervonic acid. A preferred fatty acid in one embodiment is oleic acid. The type of vegetable oil is not particularly limited, and examples thereof include soybean oil, olive oil, rapeseed oil, safflower oil, sesame oil, palm oil, sunflower oil, coconut oil, cocoa butter, and castor oil.
　微生物の培養条件は特に制限されない。例えば、微生物が、Moesziomyces属又はPseudozyma属の場合には、pH3～8、好ましくはpH4～7、温度20～35℃、好ましくは22～28℃の条件で3～14日間培養することができる。MELは、定法にしたがって培養液中から回収することができる。 The conditions for culturing microorganisms are not particularly limited. For example, when the microorganism belongs to the genus Moesziomyces or Pseudozyma, it can be cultured for 3 to 14 days under conditions of pH 3-8, preferably pH 4-7, temperature 20-35°C, preferably 22-28°C. MEL can be recovered from the culture medium according to a standard method.
　微生物が産生するマンノシルエリスリトールリピッドの種類は特に制限されず目的に応じて適宜選択できる。一実施形態において、好ましいMELは、1－O－β－MEL及び4－O－β－MELである。また、1－O－β－MEL及び4－O－β－MELは、MEL－A、MEL－B、MEL－C、及びMEL－Dのいずれであってもよい。 The type of mannosylerythritol lipid produced by microorganisms is not particularly limited and can be appropriately selected according to the purpose. In one embodiment, preferred MELs are 1-O-β-MEL and 4-O-β-MEL. 1-O-β-MEL and 4-O-β-MEL may be any of MEL-A, MEL-B, MEL-C and MEL-D.
　以下、実施例により本発明についてさらに詳細に説明するが、本発明はこれらに制限されるものではない。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.
１．代謝
　MELを合成するに関与する酵素には、糖骨格を合成する酵素（EMT1）、アシル基をマンノースに結合する酵素（MAC1, MAC2）、アセチル基をマンノースに結合する酵素（MAT1）が知られている。また、MEL生産効率の高い原料である植物油を使用した場合に、植物油が細胞外リパーゼで分解されて生じる脂肪酸が細胞内に取り込まれ、アシルCoA型に変換された後、β酸化経路で鎖長が短くなった中鎖脂肪酸が、MELを構成する脂肪酸として利用されていることが知られている。一方、これらMEL生合成に直接関わる反応以外に、一次代謝経路を中心とする様々な反応が、MEL生産量や生産効率に関連すると考えられる。脂肪酸はβ酸化で分解され、一部はMELを構成する脂肪酸として使用されるとしても、最終的にアセチルCoAにまで分解された後、TCA回路に流れていく。もう一つの油脂分解物であるグリセロールも細胞内に取り込まれた後、解糖系／糖新生に流れていく。グルコースを原料として培養した場合もやはり、取り込まれたグルコースは解糖系／糖新生を経て様々な代謝物に変換されていく。すなわち、MEL生合成には、直接的なMEL合成経路だけでなく、TCA回路や解糖系／糖新生、さらにエリスリトールの合成に関わるペントースリン酸経路といった複雑な代謝経路が関連している。 1. Metabolism Enzymes involved in the synthesis of MEL include the enzyme that synthesizes the sugar skeleton (EMT1), the enzyme that binds acyl groups to mannose (MAC1, MAC2), and the enzyme that binds acetyl groups to mannose (MAT1). ing. In addition, when vegetable oil, which is a raw material with high MEL production efficiency, is used, the fatty acids produced when the vegetable oil is degraded by extracellular lipase are taken into the cell and converted to the acyl-CoA type, followed by the β-oxidation pathway. It is known that medium-chain fatty acids with shortened are used as fatty acids constituting MEL. On the other hand, in addition to these reactions directly involved in MEL biosynthesis, various reactions mainly in the primary metabolic pathway are thought to be related to MEL production and production efficiency. Fatty acids are degraded by β-oxidation, and even if some of them are used as fatty acids that make up MEL, they are finally degraded to acetyl-CoA and flow into the TCA cycle. Glycerol, which is another oil-decomposed product, is also taken into cells and then flows into glycolysis/gluconeogenesis. When cultured using glucose as a raw material, the taken-up glucose is converted into various metabolites through glycolysis/gluconeogenesis. In other words, MEL biosynthesis involves not only the direct MEL synthesis pathway but also complex metabolic pathways such as the TCA cycle, glycolysis/gluconeogenesis, and the pentose phosphate pathway involved in the synthesis of erythritol.
２．M. antarcticusのメタボローム解析
　細胞内の代謝をより詳しく解析する有効な手段としてメタボローム解析が知られている。これは、遺伝子の発現量を評価するトランスクリプトームとは異なり、細胞内の代謝物量を分析する手法であり、細胞内で代謝の流れがどのようになっているかを、実際に存在する化合物の量から予測することを可能にする。メタボローム解析を行うために、M. antarcticusを、炭素源としてグルコースあるいはオリーブ油を使用して培養した際の培養プロファイルを図３に示す。図３の（Ａ）は、グルコースを炭素源として培養した結果であり、（Ｂ）はオリーブ油を炭素源として培養した結果である。いずれの場合も、MELの生産量（図中、菱形）及び乾燥菌体重量（図中、丸）は直線的に上昇した。炭素源（図中、四角）として使用したグルコースあるいはオリーブ油も、培養時間の経過に伴って減少した。これらの結果は、細胞の生育やMEL生合成が適切に進行していることを示す。また、矢印で示した培養72時間後の状態は、細胞増殖とMEL生合成の両方が適度に進行しており、メタボローム解析を行うための条件を兼ね備えていると判断できた。 2. Metabolome analysis of M. antarcticus Metabolome analysis is known as an effective means for analyzing intracellular metabolism in more detail. Unlike the transcriptome, which evaluates the expression level of genes, this method analyzes the amount of metabolites in cells. Allows forecasting from quantity. FIG. 3 shows the culture profile when M. antarcticus was cultured using glucose or olive oil as a carbon source for metabolomic analysis. FIG. 3A shows the results of culturing using glucose as a carbon source, and FIG. 3B shows the results of culturing using olive oil as a carbon source. In both cases, the production of MEL (diamonds in the figure) and dry cell weight (circles in the figure) increased linearly. Glucose or olive oil used as carbon sources (squares in the figure) also decreased with the passage of culture time. These results indicate that cell growth and MEL biosynthesis proceed appropriately. In addition, in the state indicated by the arrow after 72 hours of culture, both cell proliferation and MEL biosynthesis are progressing moderately, and it could be judged that the conditions for performing metabolome analysis are combined.
　72時間培養後の細胞を回収し、細胞内代謝物を抽出し、GC-MSおよびLC-MSで検出し、メタボローム解析を行った結果を図４に示す。脂肪酸およびグリセロールは、オリーブ油＞グルコースであり、グルコースはオリーブ油＜グルコースであることが分かる。この結果は、オリーブ油を原料とした場合はオリーブ油の分解物である脂肪酸とグリセロールの細胞内量が多く、グルコースを原料とした場合はグルコースの細胞内量が多いことを示す。脂肪酸は、細胞内に取り込まれた後、アシルCoAに変換され、最終的にアセチルCoAまで分解されるが、図４に示すとおり、オリーブ油で培養した場合にはアセチルCoAの量が極端に低下することが分かる。 Figure 4 shows the results of collecting the cells after culturing for 72 hours, extracting intracellular metabolites, detecting them by GC-MS and LC-MS, and conducting metabolome analysis. It can be seen that fatty acids and glycerol are olive oil>glucose and glucose is olive oil<glucose. This result indicates that when olive oil is used as the raw material, the intracellular amounts of fatty acids and glycerol, which are degradation products of olive oil, are high, and when glucose is used as the raw material, the intracellular amount of glucose is large. After fatty acids are taken up into cells, they are converted to acyl-CoA and finally degraded to acetyl-CoA. I understand.
　図４の下側に記載するTCA回路に着目すると、オリーブ油では、CIT, αKG, SUCがやや減少しているのに対してFUM, MALはグルコース培養と同程度である。すなわち、オリーブ油培養でCITからMALへの反応が働いているために、代謝物のバランスが変化しているといえる。FUMやMALの量が多くなっていることは今回のメタボローム解析で初めて明らかになった。さらに今回のメタボローム解析で、PYRの量がオリーブ油培養で顕著に少ないことも分かった。すなわち、メタボローム解析の結果から、余剰気味になっているFUMやMALを、PYRやPEPに向かわせる反応を強化すれば、MELの糖骨格を構成するマンノースやエリスリトールの合成量が増加し、結果的にMEL生産量が向上することが示唆された。 Focusing on the TCA cycle shown in the lower part of Fig. 4, in olive oil, CIT, αKG, and SUC are slightly reduced, while FUM and MAL are comparable to those in glucose culture. In other words, it can be said that the balance of metabolites is changed due to the reaction from CIT to MAL in the olive oil culture. This metabolome analysis revealed for the first time that the amounts of FUM and MAL were increased. Furthermore, the present metabolomic analysis also revealed that the amount of PYR was significantly lower in the olive oil culture. In other words, from the results of metabolomic analysis, if the reaction that directs the surplus FUM and MAL to PYR and PEP is strengthened, the amount of synthesis of mannose and erythritol that constitute the sugar skeleton of MEL will increase, resulting in It was suggested that MEL production was improved in
３． PEPカルボキシキナーゼ及びリンゴ酸酵素遺伝子の発現強化
　余剰気味になっているFUMやMALを、PYRやPEPに向かわせる反応を強化するための手段としては、PEPカルボキシキナーゼとリンゴ酸酵素遺伝子の発現強化が考えられる。そこで、当該遺伝子の配列情報をM. antarcticus 及びP.tsukubaensis等のゲノム情報から取得し、定法に従って、遺伝子発現株の構築を行う。その結果、当該遺伝子の発現強化によってMEL生産量が増加することが明らかとなる。以下、P. tsukubaensisのPEPカルボキシキナーゼ遺伝子の発現強化について具体的に示す。 3. Enhancing the expression of PEP carboxykinase and malic enzyme genes As a means of enhancing the reactions that direct surplus FUM and MAL toward PYR and PEP, it is necessary to enhance the expression of PEP carboxykinase and malic enzyme genes. Conceivable. Therefore, the sequence information of the gene is obtained from the genome information of M. antarcticus, P. tsukubaensis, etc., and the gene expression strain is constructed according to the standard method. As a result, it becomes clear that the amount of MEL production is increased by enhancing the expression of the gene. Hereinafter, enhancement of the expression of the PEP carboxykinase gene of P. tsukubaensis will be specifically described.
３－１．材料
・使用菌体
Pseudozyma tsukubaensis 1E5株（寄託番号JCM16987）
Pseudozyma tsukubaensis 1E5/pUCT_neo::PaLIPA（E5Ptef）株（リパーゼ導入株；特許文献１の実施例に記載の方法に従って、M. antarcticus T-34株のリパーゼ遺伝子をP. tsukubaensisに導入して作成した）
・ゲノムDNA
Pseudozyma tsukubaensis 1E5株
・プラスミド
発現ベクターpHSG2_hyg
・培地　
グリセロール添加YM培地：脱イオン水1Lに、酵母エキス3g、麦芽エキス3g、ペプトン5g、グルコース10g、及びグリセロール50gを溶かして調製した。
MEL生産培地：脱イオン水1Lに、酵母エキス5g、硝酸ナトリウム3g、リン酸二水素カリウム0.3g、硫酸マグネシウム・七水和物0.3g、及びグリセロール20gを溶かして調製した。 3-1. Materials/Bacteria used
Pseudozyma tsukubaensis strain 1E5 (deposit number JCM16987)
Pseudozyma tsukubaensis 1E5/pUCT_neo::PaLIPA (E5Ptef) strain (lipase-introduced strain; prepared by introducing the lipase gene of M. antarcticus T-34 strain into P. tsukubaensis according to the method described in the example of Patent Document 1)
・Genomic DNA
Pseudozyma tsukubaensis strain 1E5, plasmid expression vector pHSG2_hyg
·Culture medium
YM medium with glycerol: prepared by dissolving 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and 50 g of glycerol in 1 L of deionized water.
MEL Production Medium: Prepared by dissolving 5 g of yeast extract, 3 g of sodium nitrate, 0.3 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate heptahydrate, and 20 g of glycerol in 1 L of deionized water.
３－２．発現ベクター構築
　配列番号１に示す遺伝子を発現する発現ベクターを次の手順で構築した。配列番号１は、Pseudozyma tsukubaensis 1E5株のPEPカルボキシキナーゼをコードする塩基配列（遺伝子の上流1kbおよび下流0.5kbを含む）の配列である。まず、配列番号１を参照して、配列の上流にBamHIサイトを付加したフォワードプライマー（配列番号２）、および配列の下流にXbaIサイトを付加したリバースプライマー（配列番号３）を調製した。これらを用いて、Pseudozyma tsukubaensis 1E5株のゲノムDNAをテンプレートに遺伝子の増幅を行った。増幅した遺伝子を、BamHIサイトおよびXbaIサイトで切断した発現ベクターpHSG2_hyg（糸状菌（Ustilago maydis）由来の複製開始点（UARS）、ハイグロマイシン耐性遺伝子（hyg）を含む）に、Ligation high Ver.2（Toyobo）を用いて連結し、PEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCKを構築した。発現ベクターの構造を図５に示す。
Fwd:（PEPカルボキシキナーゼ増幅用、配列番号２）CGGGGATCCGCCGGATCACGGCTTTGCTG
Rvs:（PEPカルボキシキナーゼ増幅用、配列番号３）GACTCTAGAGATAGATGCACCAACAGAGC 3-2. Construction of Expression Vector An expression vector expressing the gene shown in SEQ ID NO: 1 was constructed by the following procedure. SEQ ID NO: 1 is the nucleotide sequence (including upstream 1 kb and downstream 0.5 kb of the gene) encoding PEP carboxykinase of Pseudozyma tsukubaensis 1E5 strain. First, referring to SEQ ID NO: 1, a forward primer (SEQ ID NO: 2) with a BamHI site added upstream of the sequence and a reverse primer (SEQ ID NO: 3) with an XbaI site added downstream of the sequence were prepared. Using these, the genomic DNA of Pseudozyma tsukubaensis 1E5 strain was used as a template for gene amplification. The amplified gene was added to the expression vector pHSG2_hyg (containing the origin of replication (UARS) derived from filamentous fungi (Ustilago maydis) and the hygromycin resistance gene (hyg)) cleaved at the BamHI site and the XbaI site. Ligation high Ver.2 ( Toyobo) to construct the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK. The structure of the expression vector is shown in FIG.
Fwd: (for PEP carboxykinase amplification, SEQ ID NO: 2) CGGGGATCCGCCGGATCACGGCTTTGCTG
Rvs: (for PEP carboxykinase amplification, SEQ ID NO: 3) GACTCTAGAGATAGATGCACCAACAGAGC
３－３．形質転換体の調製
　上記３－２．で得られたPEPカルボキシキナーゼ遺伝子発現ベクターを制限酵素SspIで処理したものを用いて直線化した後、エレクトロポレーション法にてPseudozyma tsukubaensis 1E5株およびリパーゼ導入株を形質転換した。また、コントロールとしてインサートを含まないベクターpHSG2_hygも同様に、制限酵素SspI処理で直線化した後、エレクトロポレーション法にてPseudozyma tsukubaensis 1E5株およびリパーゼ導入株に導入した。形質転換体の選別には、ハイグロマイシンを使用した。 3-3. Preparation of transformant 3-2 above. The PEP carboxykinase gene expression vector obtained in 1. was treated with the restriction enzyme SspI and linearized, and then the Pseudozymatus tsukubaensis 1E5 strain and the lipase-introduced strain were transformed by electroporation. As a control, the vector pHSG2_hyg containing no insert was similarly linearized by restriction enzyme SspI treatment, and then introduced into the Pseudozymatus tsukubaensis 1E5 strain and the lipase-introduced strain by electroporation. Hygromycin was used for selection of transformants.
３－４．1E5株形質転換体のMEL生産能および原料消費能力の評価
　1E5株の各形質転換体をグリセロール添加YM培地2mLで25℃、250rpmで2日間振とう培養し、前培養液を得た。次いで、前培養液1mLをMEL培地に6%オレイン酸を添加した培地20mLに接種し、25℃、250rpmで6日間振とう培養した。培養3日目及び5日目に6%オレイン酸を追加した（添加油脂量合計18%）。得られた菌体培養液に等量の酢酸エチルを添加し、十分撹拌した後、酢酸エチル層を分取した。残った水層にメタノールを加えた後遠心し、沈殿した菌体を回収して乾燥させた後に重量を測定した（図６（a））。酢酸エチル層に含まれるMELは高速液体クロマトグラフィー（HPLC）を用いて定量した（図６（b））。また、残存オレイン酸は薄層クロマトグラフィー（TLC）で評価した（図６（c））。各分析条件は下記の通りである。 3-4. Evaluation of MEL production ability and material consumption ability of 1E5 strain transformant Each transformant of 1E5 strain was cultured in 2 mL of glycerol-added YM medium with shaking at 25°C and 250 rpm for 2 days to obtain a preculture solution. rice field. Then, 1 mL of the preculture solution was inoculated into 20 mL of MEL medium supplemented with 6% oleic acid, and cultured with shaking at 25° C. and 250 rpm for 6 days. 6% oleic acid was added on the 3rd and 5th day of culture (total amount of added oil and fat was 18%). An equal amount of ethyl acetate was added to the obtained cell culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate layer was separated. Methanol was added to the remaining aqueous layer, followed by centrifugation, and the precipitated cells were collected, dried, and weighed (Fig. 6(a)). MEL contained in the ethyl acetate layer was quantified using high performance liquid chromatography (HPLC) (Fig. 6(b)). In addition, residual oleic acid was evaluated by thin layer chromatography (TLC) (Fig. 6(c)). Each analysis condition is as follows.
HPLC条件
検出器：蒸発光散乱検出器 1260 Infinity II（Agilent社製）
カラム：Inertsil SIL-100A（GL Science社製）
　移動相A：クロロホルム 
　移動相B：メタノール
　グラジエント：分析開始時A:B=100:0、分析15分A:B=0:100、分析15分から25分までA:B=0:100、分析25.1分から分析終了時（35分）までA:B = 100:0
　流速：1mL/min
　カラム温度：室温
　サンプルインジェクション量：10μL HPLC condition detector: Evaporative Light Scattering Detector 1260 Infinity II (manufactured by Agilent)
Column: Inertsil SIL-100A (manufactured by GL Science)
Mobile phase A: chloroform
Mobile phase B: Methanol Gradient: A:B = 100:0 at the start of analysis, 15 minutes of analysis A:B = 0:100, from 15 minutes to 25 minutes of analysis A:B = 0:100, from 25.1 minutes of analysis to end of analysis ( 35 minutes) A:B = 100:0
Flow rate: 1mL/min
Column temperature: Room temperature Sample injection volume: 10 μL
TLC条件
シリカゲルプレート：TLC Silica gel 60 F254（Merck社製）
展開相：クロロホルム：メタノール：12%アンモニア水＝55：25：2
検出試薬：2%アンスロン硫酸試薬
検出温度：95℃、5分間 TLC conditions Silica gel plate: TLC Silica gel 60 F254 (manufactured by Merck)
Development phase: chloroform: methanol: 12% aqueous ammonia = 55:25:2
Detection reagent: 2% anthrone sulfate reagent Detection temperature: 95°C for 5 minutes
　図６中、hygは発現ベクターpHSG2_hyg導入株（コントロール）を、PEPCKはPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCK導入株を示す。乾燥菌体重量により形質転換体の増殖を評価した結果、コントロールとPseudozyma tsukubaensis 1E5株由来のPEPカルボキシキナーゼ発現ベクターを導入した株（PEPCK株）で菌体増殖量に有意な差は見られなかった（図６（a））。図６（b）に示される通り、HPLC分析の結果、PEPCK株はコントロールよりもMELの生産量が向上することが確認された。また、原料のオレイン酸の残存量をTLCで評価したところ（図６（c））、PEPCK株ではコントロールよりも残存オレイン酸のスポットが小さい、すなわちオレイン酸の消費が促進されていた。 In FIG. 6, hyg indicates the strain introduced with the expression vector pHSG2_hyg (control), and PEPCK indicates the strain introduced with the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK. As a result of evaluating the growth of the transformant by dry cell weight, no significant difference was observed in the amount of cell growth between the control and the strain introduced with the PEP carboxykinase expression vector derived from the Pseudozyma tsukubaensis 1E5 strain (PEPCK strain). (Fig. 6(a)). As shown in FIG. 6(b), HPLC analysis confirmed that the PEPCK strain improved the production of MEL compared to the control. In addition, when the residual amount of oleic acid as a raw material was evaluated by TLC (Fig. 6(c)), the spot of residual oleic acid was smaller in the PEPCK strain than in the control, that is, the consumption of oleic acid was promoted.
３－５．リパーゼ導入株形質転換体のMEL生産能および原料消費能力の評価（フラスコ培養）
　リパーゼ導入株の各形質転換体をグリセロール添加YM培地2mLで25℃、2日間振とう培養し、前培養液を得た。次いで、前培養液1mLをMEL培地に6%オリーブ油を添加した培地20mLに接種し、25℃、250rpmで6日間振とう培養した。培養3日目及び5日目に8%オリーブ油を追加した（添加油脂量合計22%）。得られた菌体培養液に等量の酢酸エチルを添加し、十分撹拌した後、酢酸エチル層を分取した。残った水層にメタノールを加えた後遠心し、沈殿した菌体を回収して乾燥させた後に重量を測定した（図７（a））。酢酸エチル層に含まれる残存油脂およびMELは高速液体クロマトグラフィー（HPLC）を用いて定量した（図７（b）、（c））。HPLC分析条件は先述の通りである。 3-5. Evaluation of MEL production ability and raw material consumption ability of lipase-introduced strain transformants (flask culture)
Each transformant of the lipase-introduced strain was shake-cultured in 2 mL of glycerol-added YM medium at 25° C. for 2 days to obtain a preculture solution. Then, 1 mL of the preculture solution was inoculated into 20 mL of MEL medium supplemented with 6% olive oil, and cultured with shaking at 25° C. and 250 rpm for 6 days. On the 3rd and 5th days of culture, 8% olive oil was added (total amount of added oil and fat was 22%). An equal amount of ethyl acetate was added to the obtained cell culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate layer was separated. Methanol was added to the remaining aqueous layer, followed by centrifugation, and the precipitated cells were collected, dried, and weighed (Fig. 7(a)). Residual fats and oils contained in the ethyl acetate layer and MEL were quantified using high performance liquid chromatography (HPLC) (FIGS. 7(b) and (c)). The HPLC analysis conditions are as described above.
　図７中、PaLIPA＋hygは発現ベクターpHSG2_hyg導入株（コントロール）を示し、PaLIPA＋PEPCKはPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCK導入株を示す。乾燥菌体重量により形質転換体の増殖を評価した結果、コントロールのPaLIPA+hyg株とPseudozyma tsukubaensis 1E5株由来のPEPカルボキシキナーゼ発現ベクターを導入したPaLIPA＋PEPCK株では、PaLIPA＋PEPCK株の方で菌体増殖量が向上することが確認された（図７（a））。図７（b）に示される通り、HPLC分析の結果、PaLIPA＋PEPCK株はコントロールよりもオリーブ油の消費が促進されることが確認された。また、MEL生産量を評価したところ（図７（c））、PaLIPA＋PEPCK株ではコントロールよりもMEL生産量が向上することが確認された。 In FIG. 7, PaLIPA+hyg indicates the expression vector pHSG2_hyg-introduced strain (control), and PaLIPA+PEPCK indicates the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK-introduced strain. As a result of evaluating the growth of the transformants based on the dry cell weight, the growth rate of the PaLIPA+PEPCK strain was higher than that of the control PaLIPA+hyg strain and the PaLIPA+PEPCK strain introduced with the PEP carboxykinase expression vector derived from the Pseudozyma tsukubaensis 1E5 strain. An improvement was confirmed (Fig. 7(a)). As shown in FIG. 7(b), HPLC analysis confirmed that the PaLIPA+PEPCK strain promotes consumption of olive oil more than the control. Moreover, when the MEL production amount was evaluated (Fig. 7(c)), it was confirmed that the PaLIPA+PEPCK strain improved the MEL production amount more than the control.
３－６．リパーゼ導入株形質転換体のMEL生産能および原料消費能力の評価（ジャー培養）
　リパーゼ導入株の各形質転換体をグリセロール添加YM培地2mLで25℃、250rpmで2日間振とう培養し、前培養液を得た。次いで、前培養液15mLをMEL培地に12%オリーブ油を添加した培地300mL(培養槽容量1L)に接種し、25℃、580rpm、1.5vvm（0.45L/min）、加圧0.05MPaで7日間培養した。培養3日目に15%オリーブ油を、培養5日目に20%オリーブ油を追加した（添加油脂量合計47%）。得られた菌体培養液に等量の酢酸エチルを添加し、十分撹拌した後、酢酸エチル層を分取した。残った水層にメタノールを加えた後遠心し、沈殿した菌体を回収して乾燥させた後に重量を測定した（図８（a））。酢酸エチル層に含まれる残存油脂（図８（b））およびMEL（図８（c））は高速液体クロマトグラフィー（HPLC）を用いて定量した。HPLC分析条件は先述の通りである。 3-6. Evaluation of MEL production ability and raw material consumption ability of lipase-introduced strain transformants (jar culture)
Each transformant of the lipase-introduced strain was cultured in 2 mL of glycerol-added YM medium at 25° C. and 250 rpm with shaking for 2 days to obtain a preculture solution. Next, inoculate 15 mL of the preculture solution into 300 mL of medium (1 L of culture tank volume) in which 12% olive oil is added to MEL medium, and culture for 7 days at 25 ° C, 580 rpm, 1.5 vvm (0.45 L / min), pressurized 0.05 MPa. bottom. 15% olive oil was added on the 3rd day of culture, and 20% olive oil was added on the 5th day of culture (total amount of added oil and fat was 47%). An equal amount of ethyl acetate was added to the obtained cell culture solution, and the mixture was sufficiently stirred, and then the ethyl acetate layer was separated. Methanol was added to the remaining aqueous layer, followed by centrifugation, and the precipitated cells were collected, dried, and weighed (Fig. 8(a)). The residual oil (Fig. 8(b)) and MEL (Fig. 8(c)) contained in the ethyl acetate layer were quantified using high performance liquid chromatography (HPLC). The HPLC analysis conditions are as described above.
　図８中、PaLIPA＋hygは発現ベクターpHSG2_hyg導入株（コントロール）を示し、PaLIPA＋PEPCKはPEPカルボキシキナーゼ遺伝子発現ベクターpHSG2_hyg-PEPCK導入株を示す。図８（a）に示される通り、PaLIPA＋PEPCK株ではコントロールよりも菌体増殖量が向上することが確認された。また、MEL生産量を評価したところ（図８（c））、PaLIPA＋PEPCK株ではコントロールよりもMEL生産量が向上することが確認された。 In FIG. 8, PaLIPA+hyg indicates the expression vector pHSG2_hyg-introduced strain (control), and PaLIPA+PEPCK indicates the PEP carboxykinase gene expression vector pHSG2_hyg-PEPCK-introduced strain. As shown in FIG. 8(a), it was confirmed that the PaLIPA+PEPCK strain increased the amount of cell growth compared to the control. Moreover, when the MEL production amount was evaluated (FIG. 8(c)), it was confirmed that the PaLIPA+PEPCK strain improved the MEL production amount more than the control.

Claims (3)

  1. PEPカルボキシキナーゼ遺伝子及び／又はリンゴ酸酵素遺伝子が強発現されている、変異型マンノシルエリスリトールリピッド産生微生物。 A mutant mannosylerythritol lipid-producing microorganism in which a PEP carboxykinase gene and/or a malic enzyme gene are strongly expressed.
  2. Pseudozyma属またはMoesziomyces属に属する、請求項１に記載の微生物。 2. The microorganism according to claim 1, which belongs to the genus Pseudozyma or Moesziomyces.
  3. 請求項１又は２に記載の微生物を用いてMELを生産する方法。 A method for producing MEL using the microorganism according to claim 1 or 2.
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Title
TOMOTAKE MORITA, TOKUMA FUKUOKA, TOMOHIRO IMURA, DAI KITAMOTO: "Mannosylerythritol Lipids: Production and Applications", JOURNAL OF OLEO SCIENCE, vol. 64, no. 2, 1 January 2015 (2015-01-01), JP , pages 133 - 141, XP055280352, ISSN: 1345-8957, DOI: 10.5650/jos.ess14185 *

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