WO2014046174A1 - 組換え細胞、並びに、β－フェランドレンの生産方法 - Google Patents

組換え細胞、並びに、β－フェランドレンの生産方法 Download PDF

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WO2014046174A1
WO2014046174A1 PCT/JP2013/075282 JP2013075282W WO2014046174A1 WO 2014046174 A1 WO2014046174 A1 WO 2014046174A1 JP 2013075282 W JP2013075282 W JP 2013075282W WO 2014046174 A1 WO2014046174 A1 WO 2014046174A1
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ferrandrene
synthase
nucleic acid
amino acid
acid sequence
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French (fr)
Japanese (ja)
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古谷　昌弘
章太 上西
航一郎 岩佐
郡　悌之
高橋　征司
武文 下山
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Tohoku University NUC
Sekisui Chemical Co Ltd
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Tohoku University NUC
Sekisui Chemical Co Ltd
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Priority to EP13838825.1A priority Critical patent/EP2899263A4/en
Priority to US14/429,510 priority patent/US9617563B2/en
Priority to KR1020157008333A priority patent/KR20150059649A/ko
Priority to CN201380060882.4A priority patent/CN104797704A/zh
Publication of WO2014046174A1 publication Critical patent/WO2014046174A1/ja
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
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    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01001—Dimethylallyltranstransferase (2.5.1.1)
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    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01028—Dimethylallylcistransferase (2.5.1.28)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03051—Beta-phellandrene synthase (neryl-diphosphate-cyclizing) (4.2.3.51)

Definitions

  • the present invention relates to a recombinant cell having the ability to produce ⁇ -ferrandrene, and a method for producing ⁇ -ferrandrene using the recombinant cell.
  • Monoterpene is a compound that follows the isoprene rule with 10 carbons, which is a biosynthetic precursor of geranyl diphosphate (GPP) in which dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) are condensed. It is a generic name. Currently, over 900 types of monoterpenes are known.
  • Monoterpenes have a fragrance like roses and citrus fruits and are often used for perfumes.
  • limonene is an aroma component contained in citrus fruits such as lemon, and is also used as a solvent and an adhesive raw material.
  • Menthol has a refreshing fragrance and is used as a refreshing agent in confectionery and pharmaceuticals.
  • ⁇ -pinene, ⁇ -pinene, limonene, ⁇ -ferrandolene and the like are studied as monomer raw materials for adhesives and transparent resins (Non-patent Document 1).
  • ⁇ -Phellandrene a kind of monoterpene, is expected to be used as a new polymer material.
  • ⁇ -Ferlandolene may give a higher molecular weight polymer than ⁇ -Ferlandolene.
  • ⁇ -ferrandolene is obtained by a synthetic chemical method, formation of ⁇ -ferrandolene, which is an isomer, is unavoidable, and separation of these isomers is extremely difficult (Non-patent Document 2). ). For this reason, it is difficult to obtain high-purity ⁇ -ferrandolene, which makes it difficult to investigate the polymer physical properties of ⁇ -ferrandolene.
  • the biosynthetic pathway of ⁇ -ferrandrene includes geranyl diphosphate (IPP) to geranyl diphosphate (IPP) by the action of geranyl diphosphate (GPP) synthase or neryl diphosphate (NPP) synthase. GPP) or neryl diphosphate (NPP) is biosynthesized.
  • GPP geranyl diphosphate
  • NPP neryl diphosphate
  • ⁇ -ferrandolene is biosynthesized from GPP or NPP by the action of ⁇ -ferrandolene synthase.
  • ⁇ -ferrandrene synthase has been found in tomato and lavender (Non-patent Documents 3 and 4).
  • Patent Document 1 describes a method for producing a monoterpene using a transformant of a C1 metabolic host cell into which a nucleic acid encoding a cyclic terpene synthase (cyclic terpene synthase) has been introduced.
  • a nucleic acid encoding a cyclic terpene synthase cyclic terpene synthase
  • limonene was produced using a transformant of the genus Methylomonas
  • ⁇ -ferrandrene no examples are given.
  • There are also references to ⁇ -ferrandolene synthase but specific examples and methods for obtaining ⁇ -ferrandolene synthase and its genes, as well as specific configurations and construction methods for transformants capable of producing ⁇ -ferrandolene are shown. Not.
  • an object of the present invention is to provide a series of techniques for obtaining ⁇ -ferrandrene in high purity and in large quantities.
  • At least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl diphosphate synthase and a nucleic acid encoding neryl diphosphate synthase, It is a recombinant cell having the ability to produce ⁇ -ferrandrene, wherein a nucleic acid encoding ⁇ -ferrandrene synthase is introduced into a host cell, and these nucleic acids are expressed in the host cell.
  • the present invention relates to a recombinant cell having the ability to produce ⁇ -ferrandrene.
  • the recombinant cell of the present invention is “at least one nucleic acid selected from the group consisting of a nucleic acid encoding a geranyl diphosphate (GPP) synthase and a nucleic acid encoding a neryl diphosphate (NPP) synthase”, “Nucleic acid encoding ⁇ -ferrandrene synthase” is introduced into a host cell, and these nucleic acids are expressed in the host cell.
  • GPP geranyl diphosphate
  • NPP neryl diphosphate
  • GPP is synthesized from isopentenyl diphosphate (IPP) by the action of GPP synthase expressed in the cell, and / or by the action of NPP synthase expressed in the cell.
  • IPP isopentenyl diphosphate
  • NPP is synthesized from IPP.
  • ⁇ -ferrandolene is synthesized from GPP and / or NPP by the action of ⁇ -ferrandolene synthase expressed in cells.
  • the host cell does not have methane monooxygenase.
  • the host cell is E. coli or yeast.
  • ⁇ -ferrandol Preferably, 10 mg or more of ⁇ -ferrandol can be produced per 1 g of wet cells of the recombinant cells.
  • the nucleic acid encoding geranyl diphosphate synthase encodes the following protein (a), (b) or (c).
  • C a protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase.
  • the nucleic acid encoding neryl diphosphate synthase encodes the following protein (d), (e) or (f).
  • F A protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase.
  • the nucleic acid encoding ⁇ -ferrandrene synthase encodes the following protein (g), (h) or (i).
  • a nucleic acid encoding at least one enzyme acting in the isopentenyl diphosphate synthesis pathway is further introduced, and the nucleic acid is expressed in the host cell.
  • IPP serving as a substrate for GPP synthase or NPP synthase is efficiently supplied.
  • the synthesis route of isopentenyl diphosphate is the mevalonate route.
  • the mevalonate pathway is a mevalonate pathway of yeast or actinomycetes.
  • Another aspect of the present invention is a method for producing ⁇ -ferrandrene, wherein the recombinant cells are cultured to produce ⁇ -ferrandrene.
  • the present invention relates to a method for producing ⁇ -ferrandrene.
  • ⁇ -ferrandrene is produced in the recombinant cells by culturing the above-described recombinant cells.
  • ⁇ -ferrandrene can be produced in high purity and in large quantities.
  • ⁇ -ferrandrene is produced per 1 g of wet cells of the recombinant cells.
  • ⁇ -ferrandolene released outside the recombinant cells is recovered.
  • ⁇ -ferrandrene is recovered from the gas phase of the recombinant cell culture system.
  • ⁇ -ferrandrene can be produced in high purity and in large quantities.
  • FIG. 5 is a total ion chromatogram showing the results of GC-MS analysis of the gas phase fraction of the culture performed in Example 5, wherein (a) is a control recombinant, and (b) is ⁇ -ferrandrene-producing ability. This is the case of recombinants having the same.
  • FIG. 1B is a mass spectrum showing the result of identifying each peak in FIG. 1B by GC-MS, where FIG. 1A is peak A ( ⁇ -ferrandrene), FIG. 1B is peak B (limonene), and FIG. This is the case for peak C (Myrcene).
  • the recombinant cell of the present invention comprises at least one nucleic acid selected from the group consisting of a nucleic acid encoding geranyl diphosphate (GPP) synthase and a nucleic acid encoding neryl diphosphate (NPP) synthase, and ⁇ - It is a recombinant cell in which a nucleic acid encoding ferrandolene synthase is introduced into a host cell, and these nucleic acids are expressed in the host cell, and has the ability to synthesize ⁇ -ferrandrene.
  • GPP geranyl diphosphate
  • NPP neryl diphosphate
  • the GPP synthase is not particularly limited as long as it can exhibit the enzyme activity in a recombinant cell.
  • GPP synthase examples include those derived from Arabidopsis thaliana (GenBank Accession No .: Y17376 / At2g34630; Bouvier, F., et al., Plant J ,. 2000, 24, 241-52.), Those derived from Mycobacterium tuberculosis; (GenBank Accession No .: NP — 215504; Mann, FM, et al., FEBS Lett., 2011, 585, 549-54.), And the like.
  • SEQ ID NO: 1 shows the amino acid sequence corresponding to the nucleotide sequence of the nucleic acid (DNA) encoding the Arabidopsis thaliana-derived GPP synthase
  • SEQ ID NO: 2 shows only the amino acid sequence.
  • DNA having the base sequence represented by SEQ ID NO: 1 is an example of a nucleic acid encoding GPP synthase.
  • the nucleic acid encoding the GPP synthase includes at least a nucleic acid encoding the following protein (a), (b), or (c).
  • C a protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having the activity of geranyl diphosphate synthase.
  • the homology of the amino acid sequence in (c) is more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.
  • NPP synthase examples include those derived from tomato (Solanum lycopersicum) (GenBank Accession No .: FJ797956).
  • SEQ ID NO: 3 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the tomato-derived NPP synthetase, and SEQ ID NO: 4 shows only the amino acid sequence.
  • the DNA having the base sequence represented by SEQ ID NO: 3 is an example of a nucleic acid encoding NPP synthase.
  • the nucleic acid encoding NPP synthase includes at least a nucleic acid encoding the following protein (d), (e), or (f).
  • (D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4
  • (E) a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 and having the activity of neryl diphosphate synthase
  • the amino acid sequence homology in (f) is more preferably 80% or more, still more preferably 90% or more, and particularly preferably 95% or more.
  • nucleic acid encoding geranyl diphosphate (GPP) synthase and “nucleic acid encoding neryl diphosphate (NPP) synthase”. May have been introduced, or both nucleic acids may be introduced.
  • ⁇ -ferrandrene synthase and nucleic acid encoding the same include those derived from tomato (Solanum lycopersicum) (GenBank Accession No .: FJ797957; Schilmiller, AL, et al., Proc Natl Acad Sci US A., 2009, 106, 10865-70.), Lavender (Lavandula angustifolia) (GenBank Accession No .: HQ404305; Demissie, ZA, et al., Planta, 2011 ,. 233, 685-96), etc. .
  • SEQ ID NO: 5 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the tomato-derived ⁇ -ferrandrene synthase
  • SEQ ID NO: 6 shows only the amino acid sequence
  • SEQ ID NO: 7 shows the amino acid sequence corresponding to the base sequence of the nucleic acid (DNA) encoding the lavender-derived ⁇ -ferrandrene synthase
  • SEQ ID NO: 8 shows only the amino acid sequence.
  • the DNA having the base sequence represented by SEQ ID NO: 5 or 7 is an example of a nucleic acid encoding ⁇ -ferrandrene synthase.
  • the nucleic acid encoding ⁇ -ferrandrene synthase includes at least a nucleic acid encoding the following protein (g), (h) or (i).
  • (G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or 8,
  • (H) a protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 or 8, and having the activity of ⁇ -ferrandrene synthase
  • the homology of the amino acid sequence in (i) is more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.
  • the host cell in the recombinant cell of the present invention is not particularly limited, and may be either a prokaryotic cell or a eukaryotic cell.
  • prokaryotic cells include bacteria and actinomycetes.
  • bacteria include Escherichia bacteria such as E. coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria, cyanobacteria (Cyanobacteria), Clostridium bacteria, Examples include bacteria belonging to the genus Corynebacterium and bacteria belonging to the genus Ralstonia. Among these, Escherichia coli which can be easily cultured in large quantities is particularly preferable.
  • eukaryotic cells include yeast, filamentous fungi, eukaryotic microalgae, plant cells, and animal cells.
  • yeasts examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces uvarum, Saccharomyces staticacvar sacchartices Candida tropicalis, Candida maltosa, Candida parapsilosis, Pichia pastoris, Pichia farinosa, Pichia pinus (Pichia pinus) ⁇ Pichia varijii, Pichia fermentans, Pichia guilliermondii, Pichia stipitis, Saccharomyces Telluris (Saccharomyces elluris), Candida utilis, Candida guilliermondii, Hansenula henricii, Hansenula sensensen, ula polymorph (Hansenula capsulata), Hansenula capsulata Hansenula saturnus (Hansenula saturnus), Lipomyces kononenkoae, Kluyveromysces marxianus, Candida lipolytica (
  • the host cell is preferably one that does not have methane monooxygenase (EC 1.14.13.25).
  • the method for introducing each nucleic acid encoding GPP synthase, NPP synthase, and ⁇ -ferrandolene synthase into the host cell is not particularly limited, and may be appropriately selected depending on the type of the host cell.
  • a vector that can be introduced into a host cell and can express an integrated nucleic acid can be used.
  • the vector is a promoter that can replicate autonomously in the host cell or can be integrated into a chromosome and can transcribe the inserted nucleic acid (DNA). What is contained can be used.
  • a vector that can be used when the host cell is Escherichia coli is preferably a so-called multicopy-type vector, such as a plasmid having a replication origin derived from ColE1, such as a pUC-based plasmid, a pBR322-based plasmid, or a derivative thereof.
  • pUC19 [Gene, 33, 103 (1985)]
  • pUC18, pBR322, pHelix1 manufactured by Roche Diagnostics
  • pKK233-2 manufactured by Amersham Pharmacia Biotech
  • pSE280 Invitrogen
  • pGEMEX-1 Promega
  • pQE-8 Qiagen
  • pET-3 Novagen
  • pBluescript II KS (+) (Stratagene)
  • pSTV28 manufactured by Takara Bio Inc.
  • pUC118 manufactured by Takara Bio Inc.
  • the like can be used.
  • the promoter on the vector may be any as long as it can operate in a host cell such as E. coli.
  • a host cell such as E. coli.
  • Trp promoter Trp promoter
  • lac lac promoter
  • PL promoter PL promoter
  • PR promoter PR promoter
  • promoters derived from E. coli such as T7 promoter, phages, etc.
  • tac promoter lacT7 promoter.
  • Other promoters can also be used.
  • the vector is composed of a replication system for maintaining it stably in the host yeast, a promoter capable of transcribing the inserted nucleic acid (DNA), and a terminator sequence. It is preferable to have.
  • the vector may be a plasmid that can replicate in the host yeast or be integrated into the host chromosome.
  • the vector may also be capable of encoding the expression of repetitive copies of the desired DNA sequence, each separated by a selective cleavage site.
  • the type of promoter operable in yeast is not particularly limited.
  • the promoter of isocitrate lyase gene AOX1 promoter, GAPDH promoter, PHO5 promoter, glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter, ADHI promoter, MF ⁇ 1 A promoter, a GAL10 promoter, and the like can be used.
  • each nucleic acid When introducing each nucleic acid into a host cell using a vector, each nucleic acid may be incorporated into one vector or may be incorporated into separate vectors. Furthermore, when incorporating a plurality of nucleic acids into one vector, each nucleic acid may be expressed under a common promoter or may be expressed under separate promoters.
  • the host cell has ⁇ -ferrandolene synthase
  • this operation is usually not necessary at least when bacteria or yeast are used as host cells.
  • nucleic acids may be introduced in addition to the nucleic acids encoding GPP synthase, NPP synthase, and ⁇ -ferrandrene synthase.
  • a nucleic acid encoding at least one enzyme that acts in the synthetic pathway of isopentenyl diphosphate (IPP) is further introduced and the nucleic acid is expressed in the host cell.
  • the nucleic acid to be introduced may be only one kind or two or more kinds.
  • the synthesis route of IPP is broadly divided into two types: a mevalonate pathway (MVA pathway) and a non-mevalonate pathway (MEP pathway).
  • the mevalonate pathway is provided by eukaryotes and starts from acetyl CoA.
  • enzymes that act in the mevalonate pathway include, in order from the upstream, acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, and diphosphomevalonate decarboxylase.
  • the non-mevalonic acid pathway is provided by prokaryotes, chloroplasts, and plastids, and starts with glyceraldehyde 3-phosphate (GAP) and pyruvic acid.
  • Enzymes acting in the non-mevalonate pathway include, in order from the upstream, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphoditidyl-2-C-methyl-D- Examples include erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, and HMB-PP reductase.
  • the “enzyme that acts in the IPP synthesis pathway” encoded by the nucleic acid further introduced in this embodiment is preferably an enzyme that acts in the mevalonate pathway.
  • Enzymes that act in the mevalonate pathway include acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase .
  • an enzyme group consisting of HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase is expressed in the host cell.
  • the nucleic acid to be introduced may be selected.
  • Non-eukaryotes that have a mevalonate pathway include Streptomyces sp. Strain CL190 (Takagi M. et al., J. Bacteriol. 2000, 182 (15), 4153-7), Streptomyces griseolosporeus MF730 -N6 (Hamano Y. et al., Biosci. Biotechnol. Biochem. 2001, 65 (7), 1627-35).
  • Examples of bacteria include Lactobacillus helvecticus (Smeds A et al., DNA seq.
  • Aeropyrum genus In the archaea, Aeropyrum genus, Sulfolobus genus, Desulfurococcus genus, Thermoproteus genus, Halobacterium genus, Methanococcus genus, Thermococcus genus, Pyrococcus genus, Methanopyrus genus, Thermoplasma genus, etc. (Lombard J. et al., Mol. Biol. Evol. 2010, 28 (1), 87-99).
  • the origin of the enzyme group acting in the mevalonate pathway is not particularly limited, but an enzyme group acting in the mevalonate pathway of yeast or actinomycetes is particularly preferably employed.
  • enzymes may be naturally occurring or modified enzymes of each enzyme.
  • it may be an amino acid substitution mutant of each enzyme or a polypeptide that is a partial fragment of each enzyme and has the same enzyme activity.
  • the method for culturing the recombinant cell of the present invention is not particularly limited, and can be appropriately performed according to the type of the host cell.
  • the recombinant cell is aerobic or obligately anaerobic, for example, aeration and agitation culture using a liquid medium can be performed. If the recombinant cell is absolutely anaerobic, for example, replace the gas phase with a high-purity or deoxygenated gas such as nitrogen, and add an appropriate amount of a reducing agent such as sodium sulfide or cysteine to the culture solution. Thus, culture can be performed.
  • the medium is not particularly limited as long as it is a medium in which recombinant cells grow.
  • an organic carbon source such as a saccharide or a protein degradation product.
  • the saccharide include monosaccharides (such as glucose), disaccharides (such as maltose), oligosaccharides, polysaccharides (such as starch), and sugar alcohols.
  • the protein degradation product include peptone, tryptone, casamino acid and the like.
  • ⁇ -ferrandrene By culturing the recombinant cells of the present invention, a large amount of ⁇ -ferrandrene can be produced. As the production capacity of ⁇ -ferrandrene, a production amount of 10 mg or more of ⁇ -ferrandrene per 1 g of wet cells can be realized.
  • the produced ⁇ -ferrandrene is released out of the recombinant cells or accumulated in the cells.
  • any ⁇ -ferrandrene may be recovered. Specifically, it can be recovered from a microbial cell disruption product, a culture solution (culture supernatant), a gas phase of a culture system, or the like.
  • ⁇ -ferrandrene released to the outside of the cell is recovered, and specifically, recovered from the culture solution (culture supernatant) or the gas phase of the culture system.
  • a culture solution (culture supernatant) is extracted with an appropriate solvent such as pentane, and further subjected to reverse phase chromatography or gas chromatography. Etc., and can be purified to a high purity by chromatography. Since ⁇ -ferrandrene released to the outside of the cell also evaporates into the gas phase, it can be liquefied and recovered with a cold trap or the like.
  • recombinant Escherichia coli introduced with a tomato-derived NPP synthase gene and a tomato-derived ⁇ -ferrandrene synthase gene was prepared, and the recombinant Escherichia coli was cultured to produce ⁇ -ferrandrene.
  • Tomato-derived ⁇ -ferrandrene synthase gene Tomato-derived ⁇ -ferrandrene was synthesized by RT-PCR using tomato-derived total RNA as a template and primers represented by SEQ ID NO: 11 and SEQ ID NO: 12.
  • a nucleic acid encoding a synthase (tomato-derived ⁇ -ferrandrene synthase gene, SEQ ID NO: 5, GenBank Accession No .: FJ797957) was amplified.
  • the obtained nucleic acid was cleaved with NcoI and BamHI and then introduced into a pACYCDuet-1 vector (Novagen).
  • the base sequence of the nucleic acid introduced into the vector was confirmed, and it was confirmed that there was no difference.
  • a tomato-derived ⁇ -ferrandrene synthase expression vector pACTPD was prepared.
  • the cells were disrupted, and the supernatant of the disrupted solution was extracted with pentane.
  • this extracted fraction was analyzed by gas chromatography, ⁇ -ferrandrene was detected. Further, when the gas phase fraction was analyzed, ⁇ -ferrandrene was also detected in the gas phase.
  • the culture supernatant was also extracted with pentane. When this extracted fraction was analyzed by gas chromatography, ⁇ -ferrandrene was detected.
  • the control recombinant Escherichia coli into which only the pET23a vector or the pACYCDuet-1 vector was introduced ⁇ -ferrandrene was not detected from any of the cells, the culture supernatant, and the gas phase.
  • ⁇ -ferrandolene can be produced by culturing recombinant Escherichia coli introduced with a tomato-derived NPP synthase gene and a tomato-derived ⁇ -ferrandrene synthase gene.
  • the produced ⁇ -ferrandrene was recoverable from the cells, the culture supernatant, and the gas phase.
  • E. coli BL21BPD2 was cultured in 2 ⁇ YT medium containing ampicillin 100 ⁇ g / mL and chloramphenicol 34 ⁇ g / mL at 30 ° C. and 110 rpm (rotation) for 30 hours. At this time, the system was closed 16 hours after the start of the culture. After completion of the culture, the cells and the culture supernatant were obtained by centrifugation. The cells were disrupted, and the supernatant of the disrupted solution was extracted with pentane. When this extracted fraction was analyzed by gas chromatography, ⁇ -ferrandrene was detected. The culture supernatant was also extracted with pentane.
  • ⁇ -ferrandrene can be produced by culturing recombinant Escherichia coli into which an Arabidopsis thaliana-derived GPP synthase gene and a lavender-derived ⁇ -ferrandrene synthase gene were introduced.
  • the produced ⁇ -ferrandrene was recoverable from the cells, the culture supernatant, and the gas phase.
  • a recombinant yeast into which a tomato-derived NPP synthase gene and a tomato-derived ⁇ -ferrandrene synthase gene were introduced was prepared, and the recombinant yeast was cultured to produce ⁇ -ferrandolene.
  • Tomato-derived ⁇ -ferrandrene synthase gene Tomato-derived ⁇ -ferrandrene was synthesized by RT-PCR using tomato-derived total RNA as a template and primers represented by SEQ ID NO: 19 and SEQ ID NO: 20.
  • a nucleic acid encoding a synthase (tomato-derived ⁇ -ferrandrene synthase gene, SEQ ID NO: 5, GenBank Accession No: FJ797957) was amplified.
  • the obtained nucleic acid was cleaved with BamHI and then introduced into a pPIC3.5K vector (Invitrogen). The base sequence of the nucleic acid introduced into the vector was confirmed, and it was confirmed that there was no difference.
  • pPIC3.5K vector Invitrogen
  • a recombinant yeast GSNP-1 having a plurality of copies of the tomato-derived NPP synthase gene and the tomato-derived ⁇ -ferrandrene synthase gene was obtained.
  • a geneticin (Invitrogen) resistant strain having a concentration of 1.5 mg / mL into which only the pPIC3.5K vector into which no nucleic acid had been inserted was introduced was obtained (recombinant yeast GS115).
  • MGY medium 1.34% (w / v) YNB, 1% (w / v) glycerol, 4 X 10 -5 % (w / v) biotin; YMB: 13.4% (w / v) yeast nitrogen base, 10% (w / v) ammonium sulfate) (Invitrogen manual No. 25-0043), the cells were collected, and the collected cells were collected in a Fermentation basal salts medium.
  • the cells and the culture supernatant were collected by centrifugation.
  • the cells were disrupted by the glass bead method (Invitrogen manual No. 25-0043) to obtain a supernatant.
  • the pentane extract of the supernatant was analyzed by gas chromatography, ⁇ -ferrandrene was detected.
  • the culture supernatant was also extracted with pentane.
  • ⁇ -ferrandrene was detected.
  • the gas phase fraction was analyzed, ⁇ -ferrandrene was also detected in the gas phase.
  • ⁇ -ferrandrene was not detected from any of the cells, the culture supernatant, and the gas phase. From the above, it was shown that ⁇ -ferrandrene can be produced by culturing recombinant yeast introduced with a tomato-derived NPP synthase gene and a tomato-derived ⁇ -ferrandrene synthase gene. The produced ⁇ -ferrandrene was recoverable from the cells, the culture supernatant, and the gas phase.
  • a recombinant yeast into which an Arabidopsis thaliana-derived GPP synthase gene and a lavender-derived ⁇ -ferrandrene synthase gene were introduced was prepared, and the recombinant yeast was cultured to produce ⁇ -ferrandolene.
  • ⁇ -ferrandrene can be produced by culturing a recombinant yeast introduced with an Arabidopsis thaliana-derived GPP synthase gene and a lavender-derived ⁇ -ferrandrene synthase gene.
  • the produced ⁇ -ferrandrene was recoverable from the cells, the culture supernatant, and the gas phase.
  • a recombinant Escherichia coli introduced with a tomato-derived NPP synthase gene and a lavender-derived ⁇ -ferrandrene synthase gene was prepared, and the recombinant Escherichia coli was cultured to confirm the production of ferrandolene and Identification was performed.
  • the pACLPD prepared in Example 2 was cleaved with NCOI and EcoRI, and the lavender-derived ⁇ -ferrandrene synthase gene was excised and introduced into the NcoI and EcoRI cleavage sites of pCOLADuet-1 (Novagen) to construct pCODLFS.
  • PCR was performed using pT21TNPP produced in Example 1 as a template and the primers of SEQ ID NOs: 25 and 26 to amplify the tomato-derived NPP synthase gene.
  • the amplified fragment was cleaved with NdeI and KpnI, and introduced into the NdeI and KpnI cleavage sites of the above pCODLFS to construct a co-expression vector pCOLDFSSNS.
  • the above-described pCOLDFSSNS was introduced into Rosetta 2 (DE3) to obtain a recombinant E. coli ROFSSNS that co-expressed lavender-derived ⁇ -ferrandrene synthase and tomato-derived NPP synthase.
  • a control E. coli a recombinant E. coli ROCOLA having only pCOLA-Duet-1 was also prepared.
  • a nucleic acid (SEQ ID NO: 29) encoding S. griseolosporeus mevalonate pathway enzyme is amplified by PCR using the genomic DNA of Streptomyces griseolosporeus (Kitasatospora griseola) and the primers represented by SEQ ID NO: 27 and SEQ ID NO: 28 did.
  • This nucleic acid includes gene clusters encoding Mevalonate kinase, Mevalonate diphosphate decarboxylase, Phosphomevalonate kinase, IPP isomerase, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (HMGR), and HMG-CoA synthase. ing.
  • the obtained amplified DNA fragment was cloned into the pT7-Blue T vector to construct pT7SMV.

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