WO2019059337A1 - Procédé de fabrication de nootkatone - Google Patents

Procédé de fabrication de nootkatone Download PDF

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Publication number
WO2019059337A1
WO2019059337A1 PCT/JP2018/034996 JP2018034996W WO2019059337A1 WO 2019059337 A1 WO2019059337 A1 WO 2019059337A1 JP 2018034996 W JP2018034996 W JP 2018034996W WO 2019059337 A1 WO2019059337 A1 WO 2019059337A1
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strain
seq
microorganism
alcohol dehydrogenase
amino acid
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PCT/JP2018/034996
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English (en)
Japanese (ja)
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義教 田島
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/613—Unsaturated compounds containing a keto groups being part of a ring polycyclic
    • C07C49/617—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system
    • C07C49/623—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system having two rings
    • C07C49/637—Unsaturated compounds containing a keto groups being part of a ring polycyclic a keto group being part of a condensed ring system having two rings the condensed ring system containing ten carbon atoms

Definitions

  • the present invention relates to a method of producing nootkatone.
  • IPP isopentenyl diphosphate
  • DMAPP dimethylallyl diphosphate
  • FPP farnesyl diphosphate synthase
  • Nootkatone (4,4a, 5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2 (3II) -naphthalenone) is a sesquiterpenoid that is an important scent component in grapefruit.
  • Nootkatone is useful as a flavor and is used in products such as beverages and cosmetics.
  • Nootkatone is biosynthesized by converting valencene to noutokatole by monooxygenase (MO) such as cytochrome P450 and then converting noutokatol to noutokatone by alcohol dehydrogenase (ADH).
  • MO monooxygenase
  • ADH alcohol dehydrogenase
  • Non-Patent Document 1 describes that enhancing the expression of Pichia pastoris-derived alcohol dehydrogenase (ADH) increases the production amount of nootkatone.
  • Non-Patent Document 2 describes that a mutant of cytochrome P450cam derived from Pseudomonas putida can convert valencene into noutokatole.
  • Non-Patent Document 3 E.I. It is described that YahK, an alcohol dehydrogenase derived from E. coli, can recognize various aldehyde compounds as a substrate.
  • the object of the present invention is to provide an efficient method of producing nootkatone by biological methods.
  • the present inventors have found that a specific alcohol dehydrogenase can be converted to nootkatone using noutokatole as a substrate, and the specific alcohol dehydrogenase is a known enzyme in conversion of nootkatone from noutokatol (Pichia pastoris). It has been found that it is possible to efficiently convert nootkatol to nootkatone, since it can exhibit high activity as compared with the derived ADH (see Non-Patent Document 1), and the present invention has been completed.
  • the transformed microorganism is any one of the following (i) to (iii): (I) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding the alcohol dehydrogenase and a promoter operably linked thereto; (Ii) a microorganism comprising an expression unit comprising a polynucleotide encoding the alcohol dehydrogenase and a promoter operably linked thereto in a non-natural genomic region or a non-genomic region; or (iii) a polynucleotide encoding the alcohol dehydrogenase , A microorganism containing the expression unit at multiple copy numbers.
  • [4] The method of [2] or [3], wherein the transformed microorganism is a bacterium belonging to Enterobacteriaceae.
  • [5] The method according to any one of [2] to [4], wherein the transformed microorganism is Pantoea bacteria, Escherichia bacteria, or Corynebacterium bacteria.
  • [6] The method of [5], wherein the transformed microorganism is Pantoea ananatis, E. coli, or Corynebacterium glutamicum.
  • nootkatone can be efficiently produced by a biological method.
  • the present invention provides a method of producing nootkatone.
  • the method of the present invention comprises converting nootkatol into nootkatone in the presence of (i) a transformed microorganism whose activity of alcohol dehydrogenase is improved compared to a wild-type microorganism, or (ii) alcohol dehydrogenase.
  • the alcohol dehydrogenase used in the method of the invention corresponds to the following proteins: (A) a protein comprising the amino acid sequence of SEQ ID NO: 30 or 32; (B) A protein comprising an amino acid sequence comprising substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 30 or 32, and having alcohol dehydrogenase activity; or (C) sequence A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of Nos. 30 or 32, and having alcohol dehydrogenase activity.
  • one, several, or three amino acid residues may be modified by 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues it can. Mutations of amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions.
  • the term "one or several” refers to a number that does not significantly impair the activity of the protein.
  • the term "one or several” represents, for example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, still more preferably 1 to 20, particularly preferably 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).
  • the percent identity with the amino acid sequence of SEQ ID NO: 2 or 5 is 90% or more.
  • the identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • Compositional Adjustments can be performed using Conditional Compositional Score Matrix Adjustment.
  • the proteins (A) to (C) can convert noutokatole to nootkatone because it has alcohol dehydrogenase activity using nootkatol as a substrate.
  • B-1 a protein comprising an amino acid sequence comprising substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 30, and having alcohol dehydrogenase activity
  • C-1 A protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 30, and having alcohol dehydrogenase activity, when the activity is measured under specific measurement conditions (A-1) For example, 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably, based on the activity of a protein comprising the amino acid sequence of SEQ ID NO: 30 (preferably, a protein consisting of the amino acid sequence of SEQ ID NO: 30) Is 85% or more, particularly preferably 90% or more, 94% or more, 96% or more, 98% On, or equivalent (i.e.
  • (B-2) a protein comprising an amino acid sequence comprising substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 32, and having alcohol dehydrogenase activity
  • -2) A protein containing an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 32 and having alcohol dehydrogenase activity, each having an activity measured under specific measurement conditions (A- 2) 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 60% or more, preferably 70% or more, more preferably 60% or more, based on the activity of the protein (preferably, the protein consisting of the amino acid sequence of SEQ ID NO: 32) More preferably, it is 85% or more, particularly preferably 90% or more, 94% or more, 96% or more.
  • 8% or more, or equivalent may have more active.
  • the following conditions can be adopted as such specific measurement conditions.
  • a transformed microorganism that expresses a target protein is inoculated on LB medium and cultured overnight at 34 ° C.
  • the obtained cells are inoculated into MS-PIPES-Nootkatol medium containing about 20 mg / L nootkator (manufactured by Sundia), and shake culture is performed in a test tube at 37 ° C. for about 16 hours.
  • alcohol dehydrogenase activity can be evaluated by measuring the amount of nootkatone in the supernatant obtained by removing the cells by centrifugation (4 ° C., 15,000 rpm 10 min.) .
  • mutations may be introduced at sites in the catalytic domain and at sites other than the catalytic domain as long as the desired properties can be maintained.
  • the position of the amino acid residue to which a mutation may be introduced which can retain the property of interest, will be apparent to those skilled in the art. Specifically, one skilled in the art 1) compares the amino acid sequences of multiple proteins with similar properties, and 2) reveals relatively conserved regions and relatively non-conserved regions, Then, 3) from the relatively conserved regions and the relatively unsaved regions, it is possible to predict regions that may play an important role in functions and regions that may not play an important role in functions, respectively. Recognize the correlation between structure and function. Therefore, one skilled in the art can specify the position of an amino acid residue to which a mutation may be introduced in the amino acid sequence of the protein used in the present invention.
  • substitution of the amino acid residue may be a conservative substitution.
  • conservative substitution refers to the replacement of a given amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art.
  • amino acids having basic side chains eg, lysine, arginine, histidine
  • amino acids having acidic side chains eg, aspartic acid, glutamic acid
  • amino acids having non-charged polar side chains eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acid having nonpolar side chain eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • Amino acids eg, threonine, valine, isoleucine
  • amino acids having aromatic side chains eg, tyrosine, phenylalanine, tryptophan, histidine
  • amino acids having hydroxyl group eg, alcoholic or phenolic
  • conservative substitutions of amino acids include substitution between aspartic acid and glutamic acid, substitution between arginine and lysine and histidine, substitution between tryptophan and phenylalanine, phenylalanine and valine. , Leucine, isoleucine and alanine, and glycine and alanine.
  • the proteins used in the present invention may also be fusion proteins linked via peptide bonds with heterologous moieties.
  • a heterologous moiety for example, a peptide component that facilitates purification of a target protein (eg, a tag moiety such as histidine tag, Strep-tag II, etc .; glutathione-S-transferase, maltose binding protein, and variants thereof Etc.), peptide components that improve the solubility of the target protein (eg, Nus-tag), peptide components that act as chaperones (eg, trigger factor), and peptide components that have other functions (eg, Examples include full-length proteins or parts thereof), as well as linkers.
  • a target protein eg, a tag moiety such as histidine tag, Strep-tag II, etc .; glutathione-S-transferase, maltose binding protein, and variants thereof Etc.
  • proteins used in the present invention include proteins derived from bacteria belonging to Enterobacteriaceae (eg, Escherichia bacteria such as Escherichia coli, or Pantoea bacteria such as Pantoea ananatis), naturally
  • the homologues that occur or artificially produced mutant proteins can be mentioned.
  • a mutant protein can be obtained, for example, by introducing a mutation into a DNA encoding a target protein and using the obtained mutant DNA to produce a mutant protein.
  • the mutagenesis methods include, for example, site-directed mutagenesis and random mutagenesis (eg, treatment with mutagens and ultraviolet irradiation).
  • the method of the present invention can be performed using the above-mentioned protein itself used in the present invention.
  • Natural proteins or recombinant proteins can be used as the proteins used in the present invention.
  • the recombinant protein can be obtained, for example, using a cell-free vector or from a microorganism producing the protein used in the present invention.
  • the proteins used in the present invention can be used as unpurified, crudely purified or purified proteins. These proteins may be used as immobilized proteins immobilized on a solid phase in the reaction.
  • the protein of interest is obtained by isolating the protein used in the present invention by a known method and further purifying it if necessary.
  • a microorganism that produces a protein a transformed microorganism is preferable from the viewpoint of obtaining a large amount of protein and the like.
  • the culture medium for culturing the microorganism is known, and can be used, for example, by adding a carbon source, a nitrogen source, a vitamin source and the like to a nutrient medium such as LB medium or a minimal medium such as M9 medium.
  • the transformed microorganism is generally cultured at 16-42 ° C., preferably 25-37 ° C., for 5-168 hours, preferably 8-72 hours, depending on the host. Depending on the host cell, both shaking culture and stationary culture are possible, but agitation may be performed or aeration may be performed if necessary.
  • the culture can be performed by adding a promoter inducer to the medium.
  • the target protein produced is a method of utilizing molecular weight differences such as known salting out, precipitation such as isoelectric precipitation or solvent precipitation, dialysis, ultrafiltration or gel filtration from extracts of transformed microorganisms.
  • Methods using specific affinity such as ion exchange chromatography, methods using hydrophobicity, reverse phase chromatography, etc. using differences in hydrophobicity, other affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing Purification and isolation can be performed by electrophoresis, etc., or a combination thereof.
  • the culture supernatant obtained by culturing the transformed microorganism is subjected to centrifugation or the like to remove the cells, whereby a culture supernatant containing the target protein can be obtained.
  • the target protein can also be purified and isolated from this culture supernatant.
  • the method of the present invention can be performed in the presence of a transformed microorganism in which the activity of the above-mentioned protein is improved as compared to a wild-type microorganism.
  • transformation is intended not only for the introduction of a polynucleotide into a host cell but also for modifying the genome in the host cell.
  • the polynucleotide encoding the above-mentioned protein used in the present invention may be a polynucleotide selected from the group consisting of (a) to (d) below: (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 29 or 31; (B) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 29 or 31 and which encodes a protein having alcohol dehydrogenase activity; (C) a polynucleotide which comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 29 or 31 and which encodes a protein having alcohol dehydrogenase activity; and (d) (a) to (c) A degenerate variant of the polynucleotide selected from the group consisting
  • the polynucleotide may be DNA or RNA, but is preferably DNA.
  • the nucleotide sequence of SEQ ID NO: 29 encodes the amino acid sequence of SEQ ID NO: 30.
  • the nucleotide sequence of SEQ ID NO: 31 encodes the amino acid sequence of SEQ ID NO: 32.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
  • SSC sodium chloride / sodium citrate
  • 50 ° -65 ° C. in 0.2 ⁇ SSC 0.1% SDS And one or more washes.
  • the percent identity of the base sequence to the base sequence of SEQ ID NO: 29 or 31 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% % Or more, 97% or more, 98% or more, or 99% or more.
  • the term "degenerate variant” means that at least one codon encoding a predetermined amino acid residue in the polynucleotide before mutation is in another codon encoding the same amino acid residue. Refers to an altered polynucleotide variant. Since such degenerate variants are variants based on silent mutations, the protein encoded by the degenerate variants is identical to the protein encoded by the polynucleotide before mutation.
  • degenerate variants are polynucleotide variants in which the codons have been altered to match the codon usage of the host cell into which it is to be introduced.
  • a gene is expressed in a heterologous host cell (eg, a microorganism)
  • the difference in codon usage results in insufficient supply of the corresponding tRNA species, resulting in reduced translational efficiency and / or incorrect translation (eg, translation) ) May occur.
  • a heterologous host cell eg, a microorganism
  • the difference in codon usage results in insufficient supply of the corresponding tRNA species, resulting in reduced translational efficiency and / or incorrect translation (eg, translation) ) May occur.
  • the low frequency codons shown in Table A are known.
  • degenerate variants that match the codon usage of the host cell as described below.
  • degenerate variants have codons encoding one or more amino acid residues selected from the group consisting of arginine residues, glycine residues, isoleucine residues, leucine residues, and proline residues. It may be More specifically, degenerate variants have one or more codons selected from the group consisting of low frequency codons (eg, AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC) altered It may be one.
  • low frequency codons eg, AGG, AGA, CGG, CGA, GGA, AUA, CUA, and CCC
  • the degenerate variants may comprise one or more (e.g., one, two, three, four or five) codon modifications selected from the group consisting of: i) changing at least one codon selected from the group consisting of four codons encoding Arg (AGG, AGA, CGG, and CGA) to another codon encoding C (CGU or CGC); ii) changing one codon (GGA) encoding Gly to another codon (GGG, GGU or GGC); iii) changing one codon (AUA) encoding Ile to another codon (AUU or AUC); iv) Change of one codon (CUA) encoding Leu to another codon (UUG, UUA, CUG, CUU, or CUC); and v) one codon (CCC) encoding Pro Change to another codon (CCG, CCA, or CCU).
  • one codon modifications selected from the group consisting of: i) changing at least one codon selected from the group consisting of
  • nucleotide residue "U” should be used as described above, but if the degenerate variant is DNA, "T” will be used instead of nucleotide residue "U” It should be.
  • the number of mutations of nucleotide residues for adapting to the codon usage of the host cell is not particularly limited as long as it encodes the same protein before and after the mutation, for example, 1 to 400, 1 to 300, 1 to 200 Or 1 to 100.
  • degenerate variants may include changes to low frequency codons to non-low frequency codons (eg, high frequency codons).
  • methods for designing mutants are considered in consideration of not only low frequency codons but also factors such as adaptability of the production strain to the genomic GC content (Alan Villalobos et al., Gene Designer: a synthetic Biology tools for constructing artificial DNA segments, BMC Bioinformatics. 2006 Jun 6; 7: 285.), such methods may be used.
  • the above-mentioned mutant can be appropriately produced depending on the type of any host cell (eg, a microorganism as described later) into which it can be introduced.
  • the transformed microorganism in which the activity of the protein is improved as compared to a wild-type microorganism is preferably a microorganism comprising an expression unit comprising a polynucleotide encoding the protein and a promoter operably linked thereto.
  • the term "expression unit” refers to the transcription of a polynucleotide of interest, and thus of the protein encoded by said polynucleotide, comprising the predetermined polynucleotide to be expressed as a protein and a promoter operably linked thereto.
  • the expression unit may further comprise elements such as a terminator, a ribosome binding site, and a drug resistance gene.
  • the expression unit may be DNA or RNA, but is preferably DNA.
  • the expression unit may also be homologous (ie homologous) or heterologous (ie non-native) to the host cell.
  • the expression unit is also expressed as an expression unit comprising one polynucleotide to be expressed as a protein and a promoter operably linked thereto (ie an expression unit enabling expression of monocistronic mRNA) or as a protein
  • a promoter operably linked thereto ie an expression unit enabling expression of monocistronic mRNA
  • a plurality of polynucleotides eg, 2 or more, preferably 3 or more, more preferably 4 or more, still more preferably 5 or more, particularly preferably 10 or more polynucleotides
  • It may be an expression unit (ie, an expression unit that allows expression of polycistronic mRNA).
  • the expression unit is a genomic region (eg, a natural genomic region which is a natural locus where a polynucleotide encoding the above protein is inherently present, or a non-natural genomic region which is not the natural locus) in a microorganism (host cell), or a nongenomic It can be contained in an area (eg, in the cytoplasm).
  • the expression unit may be contained in the genomic region at one or more (e.g., 1, 2, 3, 4 or 5) different positions.
  • Specific forms of expression units contained in non-genomic regions include, for example, plasmids, viral vectors, phages, and artificial chromosomes.
  • the promoter constituting the expression unit is not particularly limited as long as it can express in a host cell the protein encoded by the polynucleotide linked downstream thereof.
  • the promoter may be homologous or heterologous to the host cell, but is preferably heterologous.
  • constitutive or inducible promoters commonly used for the production of recombinant proteins can be used.
  • a promoter for example, PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose inducible promoter, cold
  • the shock promoter includes a tetracycline inducible promoter.
  • a promoter having strong transcription activity in host cells can be used. Promoters having strong transcription activity in host cells include, for example, promoters of genes highly expressed in host cells, and promoters derived from viruses.
  • a transformed microorganism having improved activity of the protein as compared to a wild-type microorganism comprises (i) a microorganism comprising a heterologous expression unit comprising a polynucleotide encoding the protein and a promoter operably linked thereto.
  • heterologous expression unit means that the expression unit is heterologous to the host cell.
  • at least one element constituting the expression unit is heterologous to the host cell. Examples of elements constituting the expression unit that are heterologous to the host cell include, for example, the elements described above.
  • one or both of the polynucleotide encoding the protein of interest, or the promoter constituting the heterologous expression unit is heterologous to the host cell. Therefore, in the present invention, one or both of the polynucleotide encoding the protein of interest, or the promoter is an organism other than the host cell (eg, prokaryote and eukaryote, or microorganism, insect, plant, mammal, etc.) Animals) or derived from viruses or artificially synthesized.
  • the heterologous expression unit is preferably a heterologous expression unit in which at least one element constituting the expression unit is heterologous to the host cell.
  • the protein constituting the expression unit may be heterologous to the host cell.
  • Such microorganisms include, for example, (i-1) (A ') a protein containing the amino acid sequence of SEQ ID NO: 30, (B') a substitution or deletion of one or several amino acids in the amino acid sequence of SEQ ID NO: 30
  • the transformed microorganism in which the activity of the protein is improved compared to a wild type microorganism is (ii) a non-naturally occurring expression unit comprising a polynucleotide encoding the protein and a promoter operably linked thereto. It may be a microorganism contained in a genomic region or a non-genomic region.
  • the transformed microorganism whose activity of the above-mentioned protein is improved as compared to a wild-type microorganism is (iii) a microorganism comprising a polynucleotide encoding the above-mentioned protein in an expression unit in multiple copy numbers, It is also good.
  • the number of copies may be, for example, 2 or more, preferably 3 or more, more preferably 4 or more, still more preferably 5 or more, particularly preferably 10 or more.
  • the transformed microorganism in which the activity of the above-mentioned protein is improved compared to a wild-type microorganism is (iv) mutated in a unique expression unit (eg, promoter region) so as to enhance the expression of the protein
  • a unique expression unit eg, promoter region
  • Non-natural expression in which a mutation is introduced by a technique such as genome editing to a microorganism containing the introduced non-natural expression unit, or (v) the polynucleotide encoding the protein such that the activity of the protein is improved It may be a microorganism containing a unit.
  • the transformed microorganism in which the activity of the above-mentioned protein is improved as compared to a wild-type microorganism is any one of (i) to (iii).
  • host cells used as transformed microorganisms include, for example, bacteria such as bacteria belonging to Enterobacteriaceae and fungi.
  • the bacteria may also be gram positive or gram negative.
  • Gram-positive bacteria include, for example, bacteria of the genus Bacillus, bacteria of the genus Corynebacterium.
  • Bacillus bacteria Bacillus subtilis is preferable.
  • Corynebacterium (Corynebacterium) genus bacteria Corynebacterium glutamicum (Corynebacterium glutamicum) is preferable.
  • Examples of gram-negative bacteria include Escherichia bacteria and Pantoea bacteria.
  • Escherichia bacteria Escherichia coli is preferable.
  • Pantoea ananatis is preferred as the Pantoea genus bacteria.
  • fungi microorganisms of the genus Saccharomyces (Saccharomyces) and the genus Schizosaccharomyces (Schizosaccharomyces) are preferred.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Schizosaccharomyces pombe is preferable.
  • the transformed microorganism may be a microorganism that produces monooxygenase that can convert valencene to nooctol.
  • the transformed microorganism may be a host cell comprising expression units (preferably heterologous expression units) of such monooxygenases.
  • cytochrome P450 can be mentioned as a monooxygenase having the ability to convert valencene to nooctol. Cytochrome P450 is known from its structural features to be membrane-bound cytochrome P450 widely distributed in eukaryotes and cytosolic soluble cytochrome P450 widely distributed in prokaryotes.
  • CYP cytochrome P450 oxidase
  • SrKO Stevia rebaudiana Kaurene oxidase
  • CYP706M1 from Callitropsis nootkatensis
  • CYP71D55 from Hyoscyamus muticus
  • International Publication No. 2016/029187 See Katarina et al., FEBS Lett., 2014; 588: 1001-1007; Takahashi et al., J. Biol. Chem., 2007; 282: 31744-31752).
  • CPR cytochrome P450 reductase
  • cytochrome P450 derived from prokaryote examples include P450BM3 derived from Bacillus megaterium, and a mutant of P450cam derived from Pseudomonas putida (Rebecca JS et al., Org. Biomol. Chem., 2005; 3: 57-64).
  • Cytochrome P450 derived from organisms such as other microorganisms, plants, animals (eg, mammals) and the like can also be used in the present invention.
  • the transformed microorganism used may be a microorganism that produces a protein coupled to the enzyme.
  • Such proteins include ferredoxin reductase (eg, putidaredoxin reductase (CamA) from Pseudomonas putida, Ferredoxin reductase (FNR; locus no., TTC0096) from Thermus thermophilus), ferredoxin (eg, putidaredoxin (CamB from Pseudomonas putida) (CamB) And ferredoxin (Fdx; locus no., TTC 1809), and variants thereof (WO 2016/029187, Rebecca JS et al., Org. Biomol. Chem., 2005; 3).
  • the transformed microorganism may also be a microorganism that produces farnesyl diphosphate synthase (EC: 2.5. 1. 10) and valencene synthase (EC: 4. 2. 3. 73).
  • the transformed microorganism may be a host cell comprising expression units (preferably heterologous expression units) of farnesyl diphosphate synthase and valencene synthase.
  • the expression units of farnesyl diphosphate synthase and valencene synthase may be two independent expression units expressing monocistronic mRNA or a single expression unit expressing polycistronic mRNA .
  • E. coli E. coli-derived farnesyl diphosphate synthase ispA (SEQ ID NO: 68), Saccharomyces cerevisiae-derived farnesyl diphosphate synthase ERG20 (NCBI accession P08524), and variants thereof (WO 2016/029187, Frohwitter et al. J Biotechnol., 2014; 191: 205-213).
  • Farnesyl diphosphate synthase derived from organisms such as other microorganisms, plants, animals (eg, mammals) and the like can also be used in the present invention.
  • valencene synthase for example, valencene synthase derived from Cupressus nootkatensis (SEQ ID NO: 64; GeneBank: AFN 21429.1), valencene synthase derived from Vitis vinifera, valencen synthase derived from Citrus sinensis, valencen synthase derived from Callitropsis nootkatensis, and these Variants may be mentioned (WO 2016/029187, Frohwitter et al., J Biotechnol., 2014; 191: 205-213, Beekwilder et al., Plant Biotechnol J., 2014; 12 (2): 174- See 182). Valencene synthases derived from organisms such as other microorganisms, plants, animals (eg, mammals) and the like can also be used in the present invention.
  • the transformed microorganism may further be a microorganism capable of supplying isopentenyl diphosphate (IPP) from a carbon source used as a medium component.
  • IPP isopentenyl diphosphate
  • Such microorganisms are microorganisms having either the MEP pathway or the MVA pathway, or both. Microorganisms usually have any of these pathways.
  • bacteria of the genus Escherichia such as Escherichia coli
  • Saccharomyces genus yeasts such as Saccharomyces cerevisiae can inherently have the ability to synthesize dimethylallyl diphosphate by the MVA pathway.
  • Such a transformed microorganism is an expression unit (preferably heterologous expression) of one or more (e.g.
  • Such transformed microorganisms may also be host cells that contain an expression unit (preferably a heterologous expression unit) of isopentenyl diphosphate delta isomerase which has the ability to convert IPP to dimethylallyl diphosphate (DMAPP). Good.
  • expression units may be independent plural expression units expressing monocistronic mRNA or may be a single expression unit expressing polycistronic mRNA.
  • 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7, Example 1, Dxs, ACCESSION ID NP — 414954; Example 2, AT3G21500, ACCESSION Example 3, AT4G15560, ACCESSION ID NP_193291; Example 4, AT5G11380, ACCESSION ID NP_001078570), 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (EC: 1.1.1.267; Example 1 Dxr, ACCESSION ID NP_414715; Example 2, AT5G62790, ACCESSION ID NP_001190600), 4-Diphosphocytidyl-2-C-Me 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC: 2.7.7.60; Example 1, IspD, ACCESSION ID NP_417227
  • Examples of an enzyme involved in the MVA pathway include mevalonate kinase (EC: 2.7.1.36; Example 1, Erg12p, ACCESSION ID NP 013935; Example 2, AT5G27450, ACCESSION ID NP 001190411), phosphomevalonate kinase (EC: 2.7.4.2; Example 1, Erg8p, ACCESSION ID NP_013947; Example 2, AT1G31910, ACCESSION ID NP_001185124), diphosphomevalonic acid decarboxylase (EC: 4.1.1.33; Example 1, Mvd1p, ACCESSION ID NP_014441 Example 2, AT2G38700, ACCESSION ID NP_181404; Example 3, AT3G54250, ACCESSIO ID NP_566995), acetyl-CoA-C-acetyltransferase (EC: 2.3.1.9; Example 1, Erg10p, ACCESSION ID NP_015297; Example 2, AT
  • Example 10 Example 1, Erg13p, ACCESSION ID NP_013580; Example 2, AT4G11820, ACCESSION ID NP_192919; Example 3, MvaS, ACCESSION ID AAG02438), hydroxymethylgluta Ryl-CoA reductase (EC: 1.1.1.34; example , Hmg2p, ACCESSION ID NP_013555; Example 2, Hmg1p, ACCESSION ID NP_013636; Example 3, AT1G76490, ACCESSION ID NP_177775; Example 4, AT2G17370, ACCESSION ID NP_179329, EC: 1.1.1.88, eg, MvaA, ACCESSION ID P13702), acetyl-CoA-acetyltransferase / hydroxymethylglutaryl-CoA reductase (EC: 2.3.1. 9.1.1. 1. 34, eg, MvaE, ACCESSION ID AAG02439).
  • Idi1p ACCESSION ID NP_015208
  • AT3G02780 ACCESSION ID NP_186927
  • AT5G16440 ACCESSION ID NP_197148
  • Idi ACCESSION ID NP_417365
  • the transformed microorganism used in the present invention can be produced by any method known in the art.
  • a transformed microorganism as described above can be produced by a method using an expression vector (eg, competent cell method, electroporation method), or genome modification technology.
  • the expression vector is an integrative vector that produces homologous recombination with the host cell's genomic DNA
  • the expression unit can be integrated into the host cell's genomic DNA by transformation.
  • the expression vector is a non-integrating vector that does not generate homologous recombination with the host cell genomic DNA
  • the expression unit is not integrated into the host cell genomic DNA by transformation, and the expression vector As it is, it can exist independently from genomic DNA.
  • genome editing technology eg, CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)
  • integration of the expression unit into host cell genomic DNA eg, integration of the expression unit into host cell genomic DNA, and modification of the expression unit inherent in the host cell It is possible.
  • the expression vector may further contain, in addition to the minimal unit described above as an expression unit, elements such as a terminator that functions in a host cell, a ribosome binding site, and a drug resistance gene.
  • elements such as a terminator that functions in a host cell, a ribosome binding site, and a drug resistance gene.
  • drug resistant genes include resistant genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin and phosphinothricin.
  • the expression vector may also further comprise a region that allows homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA.
  • the expression vector may be designed such that the expression unit contained therein is located between a pair of homologous regions (eg, homology arms homologous to a specific sequence in the genome of the host cell, loxP, FRT) .
  • the genomic region (target of the homologous region) of the host cell into which the expression unit is to be introduced is not particularly limited, but may be the locus of a gene that is expressed at a high level in the host cell.
  • the expression vector may be a plasmid, a viral vector, a phage, or an artificial chromosome.
  • the expression vector may also be an integral or non-integrative vector.
  • the integrating vector may be a type of vector which is integrated into the genome of the host cell.
  • the integrative vector may be of a type in which only a portion (e.g., an expression unit) is integrated into the genome of the host cell.
  • the expression vector may further be a DNA vector or an RNA vector (eg, a retrovirus).
  • the expression vector may also be a commonly used expression vector.
  • expression vectors examples include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC (eg, Examples include pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof.
  • pUC eg, pUC19, pUC18
  • pSTV eg, pBR322
  • pHSG eg, pHSG299, pHSG298, pHSG399, pHSG398)
  • RSF eg, RSF10101010
  • pACYC eg, Examples include pACYC177, pACYC184
  • pMW
  • nootkatol which is a substrate used in the method of the present invention
  • a reaction system containing the alcohol dehydrogenase eg, an aqueous solution containing the alcohol dehydrogenase, a culture solution containing a transformed microorganism producing the alcohol dehydrogenase.
  • nootkatol produced in the reaction system can also be used as a substrate.
  • noutokatol a substrate in a reaction system
  • a protein as described above eg, monooxygenase and its conjugate protein, farnesyl diphosphate synthase, valencene synthase, an enzyme involved in the MVA pathway or MEP pathway, isopentenyl It can be produced by utilizing phosphate delta isomerase).
  • noutokatole may be produced from a carbon source in the culture medium in which transformed microorganisms expressing proteins as described above are cultured.
  • nootkatone can be produced from a carbon source via noutokatol.
  • an aqueous solution containing the above-mentioned alcohol dehydrogenase can be used as a reaction system.
  • a buffer is preferred.
  • the buffer include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer.
  • the pH is, for example, about 5-9.
  • the amounts of alcohol dehydrogenase and noutokatole (substrate) in the reaction system, and the reaction time can be appropriately adjusted according to the amount of nootkatone to be produced.
  • the reaction temperature is not particularly limited as long as the reaction proceeds, but 20 to 40 ° C. is preferable.
  • the method of the present invention can be carried out by cultivating the transformed microorganism using a culture medium containing the transformed microorganism as a reaction system. It can be carried out.
  • a culture medium those described above can be used.
  • the culture medium preferably contains a carbon source.
  • the carbon source for example, carbohydrates such as monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc .; invert sugar obtained by hydrolyzing sucrose; glycerol; carbon number such as methanol, formaldehyde, formate, carbon monoxide, carbon dioxide 1 compound (hereinafter referred to as C1 compound); oil such as corn oil, palm oil and soybean oil; acetate; animal oil and fat; animal oil; fatty acid such as saturated fatty acid and unsaturated fatty acid; lipid; phospholipid; Glycerine fatty acid esters such as monoglycerides, diglycerides and triglycerides; polypeptides such as microbial proteins and vegetable proteins; renewable carbon sources such as hydrolysed biomass carbon sources; yeast extract; or combinations thereof
  • the nitrogen source inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia
  • the organic trace nutrient source it is desirable to contain an appropriate amount of a requirement substance such as vitamin B1, L-homoserine, or a yeast extract.
  • a small amount of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like may be added as needed.
  • the medium used in the present invention may be any of a natural medium and a synthetic medium, as long as the medium contains a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic trace components.
  • the culture conditions of the transformed microorganism are not particularly limited, and standard cell culture conditions can be used.
  • the culture temperature is preferably 20 to 40 ° C., and more preferably 30 to 37 ° C.
  • the CO 2 concentration is preferably about 6% to about 84%, and the pH is preferably about 5 to 9. It is also preferable to culture under aerobic, anoxic or anaerobic conditions, depending on the nature of the host cell.
  • any appropriate method can be used as a culture method.
  • culture methods include batch culture methods, fed-batch culture methods, and continuous culture methods.
  • an inducer such as IPTG (isopropyl- ⁇ -thiogalactopyranoside) in the culture medium May be added to induce expression of the protein.
  • Confirmation of the production of nootkatone can be made as appropriate.
  • such confirmation can be performed by extracting nootkatone from the reaction system with an organic solvent, and subjecting the extract to gas chromatography or mass spectrometry.
  • Recovery and purification of nootkatone from culture medium can also be performed as appropriate.
  • recovery and purification of nootkatone are carried out by extraction / fractionation with an organic solvent, and a method using an inclusion compound (by making an inclusion compound such as cyclodextrin or the like contact the inclusion complex, A method of removing nootkatone from the inclusion complex.
  • Nootkatone recovery and purification may also be performed by methods of separation by precision distillation, as with common perfumes.
  • Example 1 Identification of Noutokatol Oxidase Oxidizing in Pantoea ananatis 1-1) Construction of Pichia pastoris-derived alcohol dehydrogenase expression plasmid Pichia pastoris-derived alcohol dehydrogenase (designated as ADH3P) is reported as an enzyme that oxidizes Noutokatole (Wriessengger et al., Metab Eng., 2014, Jul; 24: 18-29). Using this protein as a control, P. An attempt was made to identify the nootkatol oxidase present in Ananatis. An expression plasmid for ADH3P, pSTV28-P tac -ADH3P, was constructed according to the following procedure.
  • PUC57-ADH3P in which the ADH3P gene was cloned into pUC57 was obtained using GenScript artificial gene synthesis service.
  • the nucleotide sequence and amino acid sequence of the synthesized ADH3P are shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • PCR (Prime star GXL (registered trademark) 94 ° C ⁇ 10 sec., 54 ° C ⁇ 20 sec) using the combination of primers shown by ADH3P-F (SEQ ID NO: 3) and ADH3P-R (SEQ ID NO: 4) with pUC57-ADH3P as a template , 68 ° C., 120 sec., 35 cycles).
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system (manufactured by Promega, A9281) to obtain a gene fragment of ADH3P.
  • pSTV28-P tac -T trp (WO 2013/069634) as a new template
  • PCR PCR
  • Prime star GXL registered trademark
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of pSTV28-P tac -T trp .
  • the gene fragments of ADH3P and pSTV28-P tac -T trp were ligated with In-Fusion® HD cloning Kit (Clontech, 639648), and transformed into strain JM109. After seeding on LB medium containing 40 mg / L chloramphenicol, overnight culture was performed at 37 ° C. to obtain a transformed microorganism. Appeared transformed microorganisms is performed colony PCR with a combination of primers of at ADH3P-F and ADH3P-R, ADH3P under the control of the tac promoter plasmid expressing acquired the pSTV28-P tac -ADH3P.
  • PCR (Prime star GXL (registered trademark) 94 ° C ⁇ 10 sec., 54 ° C ⁇ 20 sec., 68 ° C ⁇ 180 sec., using the genomic DNA of the ananatis AJ 13355 strain as a template and the combination of primers shown by PAJ_XXXX-F and PAJ_XXXX-R cycle) was carried out.
  • the primer sequence numbers of PAJ_XXXX-F and PAJ_XXXX-R used in the experiment are summarized in Table 2.
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a PAJ_XXXX gene fragment.
  • pSTV28-P tac -T trp as a new template, a combination of primers shown by pSTV-F (SEQ ID NO: 5) and pSTV-R (SEQ ID NO: 6) performs PCR (Prime Star GXL (registered trademark) 94 ° C. 10 sec., 54 ° C., 20 sec., 68 ° C., 240 sec., 35 cycles) were performed. The obtained PCR product was similarly purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a pSTV28-P tac -T trp gene fragment.
  • PCR Primary Star GXL (registered trademark) 94 ° C. 10 sec., 54 ° C., 20 sec., 68 ° C., 240 sec., 35 cycles
  • the gene fragments of PAJ_XXXX and pSTV28-P tac -T trp were ligated with In-Fusion® HD cloning Kit (Clontech, 639648), and transformed into strain JM109.
  • the resulting transformed microorganism was subjected to colony PCR using a combination of primers shown by PAJ_XXXX-F and PAJ_XXXX-R to obtain a plasmid expressed by PAJ_XXXX under the control of tac promoter, pSTV28-P tac -PAJ_XXXX.
  • strains were respectively designated as SC17 (0) / pSTV28-P tac -ADH3P strain, SC17 (0) / pSTV28-P tac -PAJ_XXXX strain, and SC17 (0) / pSTV28-P tac -T trp strain.
  • MS-PIPES-Nootkatol medium containing about 20 mg / L nootkator (manufactured by Sundia), and shake culture was performed in a test tube at 37 ° C. for about 16 hours.
  • the culture solution was diluted 51 times with pure water, and the OD 600 value was measured using a U-2001 Spectrometer (manufactured by Hitachi) and the glucose concentration by BF-5 (manufactured by Able Biot).
  • the composition of the MS-PIPES-Nootkatol medium is shown below.
  • a stock solution 40 g of glucose and 1 g of MgSO 4 ⁇ 7H 2 O were dissolved in pure water and the solution was adjusted to 400 mL, then 115 ° C., 10 min.
  • the autoclave was sterilized under the conditions of B stock solution; 5 g (NH 4 ) 2 SO 4 , 1 g KH 2 PO 4 , 2 g Bacto-yeast extract, 10 mg FeSO 4 ⁇ 7 H 2 O, 10 mg MnSO 4 ⁇ 5 H 2 O dissolved in pure water, pH adjusted to KOH After adjusting to 7.0 with, the volume was increased to 400 mL. Thereafter, at 115 ° C. for 10 minutes. Autoclave sterilization.
  • Nootkatol and nootkatone contained in the culture solution were quantitatively analyzed by the following procedure. 200 ⁇ L of the culture broth was well suspended in 800 ⁇ L of 99.5% ethanol, and the cells were removed by centrifugation (4 ° C., 15,000 rpm, 10 min.). The obtained supernatant fluid was analyzed as an analysis sample. The analysis sample was measured using GC-2010 Plus (manufactured by Shimadzu Corporation) under the following conditions. The column used was DB-5; total length 30 m, inner diameter 0.25 mm, membrane pressure 0.25 ⁇ m (manufactured by Agilent Technologies).
  • Nootkatone standard solutions were prepared by dissolving commercial reagents (manufactured by Sigma) in 99.5% ethanol to prepare 10, 100, and 1000 mg / L standard solutions.
  • a commercial reagent manufactured by Sundia
  • Detailed analysis conditions are shown below (vaporization chamber temperature: 250 ° C., injection amount: 1.0 ⁇ L, carrier gas: He, column oven temperature: 65 ° C. to 300 ° C., column temperature rising condition 65 ° C. to 210 ° C .; 15 ° C. / Min., 210 ° C.
  • Example 2 Identification of nootkatol oxidase in Escherichia coli 2-1) Search for nootkatol oxidase in Escherichia coli A protein showing high identity with a protein encoded by PAJ_3430 is selected from E. coli. The genome information (NC_000913) of the coilMG1655 strain was searched. The nucleotide sequence of PAJ_3430 is shown in SEQ ID NO: 29, and the amino acid sequence is shown in SEQ ID NO: 30. Based on the amino acid sequence of PAJ_3430, a homology search was performed by blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
  • E. coli Construction of expression plasmid of E. coli-derived YahK According to the method described above, E. coli.
  • E. PCR Primary Star GXL (registered trademark) 94 ° C., 10 sec., 54 ° C.) using the genomic DNA of E. coli MG1655 strain as a template and the combination of the primers shown by b0325-F (SEQ ID NO: 33) and b0325-R (SEQ ID NO: 34). 20 sec., 68 ° C., 120 sec., 35 cycles) were carried out.
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain the yahK gene fragment.
  • Wizard registered trademark
  • SV Gel and PCR Clean-UP system
  • pSTV28-P tac -T trp WO 2013/069634
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of pSTV28-P tac -T trp .
  • the gene fragments of yahK and pSTV28-P tac -T trp were ligated with In-Fusion® HD cloning Kit and transformed into strain JM109. After seeding on LB medium containing 40 mg / L chloramphenicol, overnight culture was performed at 37 ° C. to obtain a transformed microorganism.
  • the transformed microorganism that has appeared is subjected to colony PCR using a combination of primers shown by M13 Primer M4 (SEQ ID NO: 35) and M13 Primer RV (SEQ ID NO: 36), and E. coli under control of the tac promoter.
  • the obtained cells were inoculated into MS-PIPES-Nootkatol medium containing about 40 mg / L nootkatol, and shake culture was performed at 37 ° C. for about 16 hours.
  • the OD 600 value, the glucose concentration, the nootkatol concentration, and further the nootkatone concentration were measured according to the method described above.
  • the culture results are shown in Table 4.
  • the SC17 (0) / pSTV28-P tac -T trp strain which is a control, produced 5.1 mg / L nootka ton, whereas the SC17 (0) / pSTV28-P tac -PAJ_3430 strain produced 17.4 mg / p.
  • Example 3 Mevalonic acid pathway enhanced. Construction of ananatis IP03 strain 3-1) P.1. Construction of P. ananatis SC 17 (0) ⁇ L-ldh :: att L ⁇ 80-Km R- att R ⁇ 80 The effects of PAJ_3430 and b0325 in ananatis were examined. First, P. pylori with enhanced mevalonic acid pathway. ananatis IP03 strain (acetoacetyl-CoA thiolase from enterococcus faecalis, an enzyme involved in the mevalonic acid pathway, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (MvaE), 3-hydroxy-3- from E.
  • HMG-CoA 3-hydroxy-3-methylglutaryl-CoA reductase
  • HMG-CoA Methylglutaryl CoA synthase
  • MvaS Mevalonic acid kinase
  • PMK phosphomevalonic acid kinase
  • MVD diphosphomevalonic acid decarboxylase
  • yIDI isoprenyl pyrophosphate isomerase
  • the expression cassette attL ⁇ 80-Km R -attR ⁇ 80 of the kanamycin resistance gene was introduced into the L-ldh gene (PAJ_p0276) located on the ananatis chromosome.
  • the genomic sequence (Genbank: AP012032) of the Ananatis AJ 13355 strain has been published, and based on the base sequence (SEQ ID NO: 37) of the L-ldh gene, L-ldh_Km ⁇ 80_F (SEQ ID NO: SEQ ID NO) having a sequence homologous to the same at the 5 'end. 38) and a primer for L-ldh_Km ⁇ 80_R (SEQ ID NO: 39) were designed.
  • L-ldh_Km ⁇ 80_F and L-ldh_Km ⁇ 80_R (Prime star GXL (registered trademark) 94 ° C ⁇ 10sec., 54 ° C ⁇ 20sec , 68 sec., 120 sec., 35 cycles)
  • the obtained PCR product is purified with Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain homologous sequences with the L-ldh gene at both ends.
  • the attL ⁇ 80-Km R -attR ⁇ 80 gene fragment was obtained.
  • AttL ⁇ 80-Km R -attR ⁇ 80 gene fragments 600 ng SC17 (0) obtained according to the / PRSFRedTER strain was introduced by electroporation and seeded in LB medium containing 50 mg / L kanamycin.
  • L-ldh-F SEQ ID NO: 40
  • L-ldh-R SEQ ID NO: 41
  • substitution was performed with the -attR ⁇ 80 sequence.
  • the transformed microorganism was designated as SC17 (0) ⁇ L-ldh :: attL ⁇ 80-Km R -attR ⁇ 80 / pRSFRedTER strain.
  • kanamycin resistance gene (kan gene) was removed from the SC17 (0) ⁇ L-ldh :: att L ⁇ 80-Km R- att R ⁇ 80 strain. It was carried out using pAH129-cat according to the method of the previous report (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60).
  • SC17 (0) the ⁇ L-ldh :: attL ⁇ 80-Km competent cells of R -AttRfai80 strain was prepared and pAH129-cat was introduced by electroporation method.
  • pAH129 was removed from SC17 (0) ⁇ L-ldh :: attB ⁇ 80 / pAH129-cat strain
  • the strain was cultured overnight at 34 ° C. on an LB plate.
  • the resulting cells were replated on 1 mL of LB medium and cultured at 34 ° C. for 3 hours. Thereafter, the temperature was raised to 42 ° C., and culture was further performed for 1 hour.
  • the culture solution was seeded on an LB plate so that single colonies appeared, and cultured overnight at 37 ° C.
  • the resulting transformed microorganism was designated as SC17 (0) ⁇ L-ldh :: att B ⁇ 80 / pAH123-cat strain.
  • competent cells of SC17 (0) ⁇ L-ldh :: attB ⁇ 80 / pAH123-cat strain are prepared and reported according to the previous report (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60).
  • the CRIM plasmid, pAH162- PphoC- mvaES ( WO 2015/080273) was introduced by electroporation. Thereafter, 1 mL of SOC medium was added, and recovery culture was performed at 34 ° C.
  • the culture solution was applied to an LB plate containing 25 mg / L of tetracycline and cultured at 34 ° C. overnight.
  • the resulting transformed microorganism was P. Ananatis SWITCH-PphoC ⁇ gcd ⁇ L-ldh :: It was named as pAH162-P phoC- mvaES strain.
  • the recovered cells were seeded on LB plates containing 60 mg / L chloramphenicol and cultured overnight at 37 ° C.
  • the resulting transformed microorganisms were plated on LB plates containing 60 mg / L chloramphenicol and 0.2 M arabinose to form single colonies.
  • the obtained colonies were respectively inoculated on LB plates containing 25 mg / L of tetracycline and LB plates containing 60 mg / L of chloramphenicol, and cultured at 34 ° C. for 16 hours.
  • IP03 / pRSFParaIX strain A strain exhibiting this phenotype was newly designated as IP03 / pRSFParaIX strain.
  • the plate was plated on an LB plate containing 10% sucrose and 1 mM IPTG, and cultured at 34 ° C. for 16 hours. The resulting single colony was inoculated on LB medium containing 60 mg / L chloramphenicol, and it was confirmed that growth was not possible. Ananatis IP03 strain was obtained.
  • Example 4 Construction of Expression Plasmid of Cytochrome P450 Mutant (CamC * AB) from Pseudomonas putida Modified to Recognize Valensen as a Substrate 4-1) Construction of Cytochrome P450 camC (CamC) Expression Plasmid from Pseudomonas putida From Valensen A cytochrome P450 expression plasmid was constructed to catalyze the hydroxylation reaction to nootkatone. The genomic sequence of Pseudomonas putida KT2440 has been published (NC_002947).
  • Cytochrome P450camC (CamC) derived from Pseudomonas putida is known to function as a cytochrome P450 oxidase (Rebecca JS et al., Org. Biomol. Chem., 2005; 3: 57-64).
  • the gene synthesis of the base sequence of CamC was performed using the artificial gene synthesis consignment service provided by GenScript.
  • GenScript The nucleotide sequence and amino acid sequence of the synthesized CamC are shown in SEQ ID NO: 42 and SEQ ID NO: 43.
  • PCR (Prime star GXL (registered trademark) 94 ° C with the combination of primers shown by pSol-P450CamC-CF (SEQ ID NO: 44) and pSol-P450CamC-CR (SEQ ID NO: 45) using pUC57-CamC cloned with CamC as a template 10 sec., 54 ° C., 20 sec., 68 ° C., 150 sec., 35 cycles) were performed.
  • the obtained PCR product was purified by Wizard SV Gel and PCR Clean-UP system to obtain a CamC gene fragment.
  • pSol-His (Lucigen 49060-1) and a gene fragment of CamC were ligated with In-Fusion® HD cloning Kit and transformed into strain JM109.
  • CamC * a quadruple mutant expression plasmid of cytochrome P450 camC derived from Pseudomonas putida A mutant (CamC *) of CamC that recognizes valencene as a substrate has been reported so far (Rebecca JS et al) , Org. Biomol. Chem., 2005; 3: 57-64).
  • CamC * a mutation of F87A / Y96F / L244A / V247L has been introduced to wild-type CamC.
  • An expression plasmid for CamC *, pSol-CamC * was constructed according to the following procedure.
  • PCR (Prime star GXL (registered trademark) 94 ° C ⁇ using the combination of primers shown by CamC-F87A / Y96F-CF (SEQ ID NO: 46) and CamC-F87A / Y96F-CR (SEQ ID NO: 47) with pSol-CamC as a template 10 sec., 54 ° C., 20 sec., 68 ° C., 300 sec., 35 cycles) were performed.
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a plasmid containing the CamC (F87A / Y96F) gene in which a mutation has been introduced into the CamC gene.
  • PCR Primary Star
  • CamC-L244A / V247L-CF SEQ ID NO: 48
  • CamC-L244A / V247L-CR SEQ ID NO: 49
  • GXL (registered trademark) 94 ° C., 10 sec., 54 ° C., 20 sec., 68 ° C., 240 sec., 35 cycles) were performed.
  • the PCR product obtained was purified with Wizard (registered trademark) SV Gel and PCR Clean-UP system, and a CamC * (F87A / Y96F / L244A / V247L) gene fragment in which a mutation was further introduced to the CamC (F87A / Y96F) gene Was obtained.
  • the same plasmid was transformed into the JM109 strain, and the gene mutation site of the obtained plasmid pSol-CamC * was sequence-analyzed with the combination of the primers shown by pRham Forward and pETite Reverse to confirm that the mutation was introduced.
  • the plasmid that CamC * expresses under the control of the rhamnose promoter was named pSol-CamC *.
  • Cytochrome P450 camC * AB (CamC * AB) Expression Plasmid Derived from Pseudomonas putida pSol-CamC * AB was constructed according to the following procedure.
  • the genomic sequence of Pseudomonas putida KT2440 has been published (NC_002947).
  • Putidaredoxin reductase (CamA) and putidaredoxin (CamB) from Pseudomonas putida are known to function in conjunction with the CamC protein (Rebecca JS et al., Org. Biomol. Chem., 2005; 3: 57-64).
  • the nucleotide sequence and amino acid sequence of CamA are shown in SEQ ID NO: 50 and SEQ ID NO: 51, and the nucleotide sequence and amino acid sequence of CamB are shown in SEQ ID NO: 52 and SEQ ID NO: 53.
  • Gene synthesis of an artificial operon in which the CamA gene and the CamB gene were linked was performed using an artificial gene synthesis contract service provided by GenScript.
  • the base sequence of the artificial operon CamA-CamB consisting of the CamA gene and the CamB gene is shown in SEQ ID NO: 54.
  • PCR Primary star GXL (registered trademark) 94 ° C 10 sec., 54 ° C., 20 sec., 68 ° C., 150 sec., 35 cycles
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of CamA-CamB.
  • PCR (Prime star GXL (registered trademark) 94) is performed using the combination of primers shown by pSol-CamC * as a template and pSol-CamC * -F (SEQ ID NO: 57) and pSol-CamC * -R (SEQ ID NO: 58). C. ⁇ 10 sec., 54 ° C. ⁇ 20 sec., 68 ° C. ⁇ 300 sec., 35 cycles) were carried out. The obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain the pSol-CamC * gene fragment.
  • Wizard registered trademark
  • Example 5 Varensen producing ability was imparted Construction of ananatis IP03 VI strain 5-1) P. a. Construction of P. ananatis SC17 (0) ⁇ pflA :: att L ⁇ 80-Km R- att R ⁇ 80 P. ananatis IP03 strain to which P. valens producing ability was imparted. Ananatis IP03VI strain was constructed according to the following procedure. The SC17 (0) ⁇ pflA :: attL ⁇ 80-Km R -attR ⁇ 80 strain was constructed according to the following procedure. Using pMWattphi (Minaeva NI et al., BMC Biotechnol.
  • PCR Prime star GXL (registered trademark) 94 ° C., 10 sec., 54 ° C., 20 sec., 68 ° C., 180 sec., 35 cycles) were performed.
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment retaining a homologous region to pflA gene (Locus tag; PAJ_0669) at both ends.
  • the culture solution was applied to an LB plate containing chloramphenicol 60 mg / L and cultured at 30 ° C. overnight. It was confirmed that the emerging chloramphenicol resistant strain exhibited kanamycin sensitivity, and thus it was designated as SC17 (0) ⁇ pflA :: attB ⁇ 80 / pAH129-cat strain. Subsequently, pAH129-cat was removed from SC17 (0) ⁇ pflA :: attB ⁇ 80 / pAH129-cat strain. The SC17 (0) ⁇ pflA :: att B ⁇ 80 / pAH129-cat strain was inoculated in LB medium and cultured at 34 ° C. for 3 hours. Then, it culture
  • the culture solution was applied to an LB plate to obtain single colonies.
  • a clone which can not grow on an LB plate containing chloramphenicol was obtained and designated as strain SC17 (0) ⁇ pflA :: attB ⁇ 80.
  • Valencen synthase (VlnSCN) derived from Cupressus nootkatensis and E. coli. Construction of CRIM plasmid pAH162-P tac -VlnSCN-ispA containing the expression cassette of farnesyl diphosphate synthase (IspA) from E. coli pAH162-P tac -VlnSCN-ispA was constructed according to the following procedure. The base sequence (GeneBank: AFN 21429.1) of valencene synthase (VlnSCN) derived from Cupressus nootkatensis has already been published.
  • VlnSCN Gene synthesis of the base sequence of VlnSCN was performed using an artificial gene synthesis trust service provided by GenScript.
  • the nucleotide sequence and amino acid sequence of the synthesized VlnSCN are shown in SEQ ID NO: 63 and SEQ ID NO: 64.
  • PCR Prime star (registered trademark) GXL 94 ° C, combining pUC57-VlnSCN in which VlnSCN has been cloned, with the combination of the primers shown in P tac- VlnSCN-F (Sequence field number 65) and VlnSCN-R (SEQ ID NO: 66) 10 sec., 54 ° C., 20 sec., 72 ° C., 120 sec., 35 cycles) were performed.
  • P tac- VlnSCN-F Sequence field number 65
  • VlnSCN-R SEQ ID NO: 66
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a VlnSCN gene fragment.
  • E.I. Primers for amplifying IspA were designed based on the genome sequence (NC_000913) of E. coli MG1655 strain. The nucleotide and amino acid sequences of IspA are shown in SEQ ID NO: 67 and SEQ ID NO: 68.
  • IspA-F SEQ ID NO: 69
  • IspA-R SEQ ID NO: 70
  • 72 ° C., 90 sec., 35 cycles The obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of IspA.
  • VlnSCN gene fragment and the IspA gene fragment as a template, a combination of the primers shown by P tac -VlnSCN-F (SEQ ID NO: 65) and IspA-R (SEQ ID NO: 70) 94 ° C., 10 sec., 54 ° C., 20 sec., 72 ° C., 240 sec., 35 cycles) were carried out.
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a VlnSCN-IspA gene fragment in which the VlnSCN gene and the IspA gene were linked.
  • pAH162-Ptac (WO 2017/022856) digested with restriction enzymes PstI and BamHI and the gene fragment of VlnSCN-ispA were ligated using In-Fusion® HD cloning Kit (Clontech, 639648) , PIR2 strain (ThermoFisher, C111110).
  • the transformed microorganism which has appeared is subjected to colony PCR using a combination of primers shown by pAH162-Gene-F2 (SEQ ID NO: 71) and pAH162-Gene-R (SEQ ID NO: 72), and the VlnSCN-ispA operon is expressed under the control of tac promoter pAH162-P tac -VlnSCN-ispA was obtained.
  • P.I. Construction of P. ananatis IP03 VI strain pAH123-cat was introduced into the ananatis SC17 (0) ⁇ pflA :: attB ⁇ 80 strain by electroporation. Thereafter, 1 mL of SOC medium was added and recovery culture was performed at 34 ° C. for 2 hours. The cells after recovery culture were seeded on LB plates containing 60 mg / L chloramphenicol and cultured overnight at 37 ° C. The resulting transformed microorganism was P. Ananatis SC17 (0) ⁇ pflA :: named att B ⁇ 80 / pAH123-cat strain. Then, P.
  • Competent cells of the ananatis SC17 (0) ⁇ pflA :: attB ⁇ 80 / pAH123-cat strain were prepared, and according to the previous report (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60), pAH162-P 200 ng of tac- VlnSCN-ispA was introduced by electroporation. Then, after shaking culture at 34 ° C. for 2 hours using SOC medium, culture was performed at 42 ° C. for 1 hour. A portion of the culture was applied to an LB plate containing Tet 25 mg / L and Cm 60 mg / L and cultured overnight at 37 ° C.
  • the resulting transformed microorganism was designated as strain SC17 (0) ⁇ pflA :: pAH162-P tac -Vln SCN-ispA.
  • P.I. Genomic DNA was prepared from the Ananatis SC17 (0) ⁇ pflA :: pAH162-P tac -Vln SCN-ispA strain. Thereafter, competent cells of IP03 strain were prepared, and P. 600 ng of genomic DNA extracted from A. ananatis SC17 (0) ⁇ pflA :: pAH162-P tac -Vln SCN-ispA strain was introduced. Thereafter, 1 mL of SOC medium was added, and recovery culture was performed at 34 ° C. for 2 hours.
  • the culture solution was applied to an LB plate containing 25 mg / L of tetracycline and cultured at 34 ° C. overnight.
  • the resulting transformed microorganism was designated as IP03 ⁇ pflA :: pAH162-P tac -VlnSCN-ispA strain.
  • the tetracycline resistance gene was removed from the strain.
  • Competent cells of the IP03 ⁇ pflA :: pAH162-P tac -VlnSCN-ispA strain were prepared, and pRSFParaIX was introduced by electroporation. Thereafter, recovery culture was performed at 34 ° C. for 2 hours in SOC medium.
  • the recovered cells were seeded on LB plates containing 60 mg / L chloramphenicol and cultured overnight at 37 ° C.
  • the resulting transformed microorganisms were plated on LB plates containing 60 mg / L chloramphenicol and 0.2 M arabinose to form single colonies.
  • the obtained colonies were respectively inoculated on LB plates containing 25 mg / L of tetracycline and LB plates containing 60 mg / L of chloramphenicol, and cultured at 34 ° C. for 16 hours.
  • IP03VI / pRSFParaIX strain A strain exhibiting this phenotype was newly designated as IP03VI / pRSFParaIX strain.
  • the plate was plated on an LB plate containing 10% sucrose and 1 mM IPTG, and cultured at 34 ° C. for 16 hours. The resulting single colony was inoculated on LB medium containing 60 mg / L chloramphenicol, and it was confirmed that growth was not possible. Ananatis IP03 VI strain was obtained.
  • Example 6 Construction of IP03VI strain in which various ADH gene expression cassettes were introduced into the chromosome 6-1)
  • expression cassettes for various ADH genes were introduced into the chromosome.
  • the attB ⁇ 80 site for introduction into a chromosome was constructed at the locus of the mgsA gene (Locus tag; PAJ_0722). P.
  • the genome sequence (GenBank: AP012032.2) of the ananatis AJ13355 strain has been published, and based on the base sequence (SEQ ID NO: 73) of the mgsA gene, mgsA_Km ⁇ 80_F (SEQ ID NO: 74) having a sequence homologous to the same gene at the 5 'end. And primers of mgsA_Km ⁇ 80_R (SEQ ID NO: 75) were designed. PCR was performed using pMWattphi (Minaeva NI et al., BMC Biotechnol.
  • pAH162-P tac -ADH3P pAH162-P tac -ADH3P was constructed according to the following procedure. Using the pSTV28-P tac -ADH3P constructed above as a template, PCR (Prime star GXL (registered trademark)) by combining primers shown by pAH162-ADH3P-F (SEQ ID NO: 78) and pAH162-ADH3P-R (SEQ ID NO: 79) 94 ° C., 10 sec., 54 ° C., 20 sec., 68 ° C., 120 sec., 35 cycles) were carried out.
  • PCR Primary star GXL (registered trademark)
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of ADH3P.
  • pAH162-P tac was digested with restriction enzymes PstI and BamHI, and the obtained plasmid fragment was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system.
  • gene fragments of ADH3P and pAH162 were ligated with In-Fusion (registered trademark) HD cloning Kit and transformed into PIR2 strain.
  • Colony PCR was performed with the combination of the primers shown by pAH162-Gene-F2 (SEQ ID NO: 71) and pAH162-Gene-R (SEQ ID NO: 72) using the emerging transformed microorganism, and ADH3P was cloned into pAH162-P tac. I confirmed that it was.
  • the plasmid expressed by ADH3P under the control of the tac promoter was named pAH162-P tac -ADH3P.
  • pAH162-P tac -yahK (b0325)
  • pAH162-P tac -b0325 was constructed according to the following procedure.
  • E. PCR Primary star GXL (registered trademark) 94 ° C., 10 sec.
  • pAH162-yahKE-F SEQ ID NO: 80
  • pAH162-yahKE-R SEQ ID NO: 81
  • the obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system.
  • the gene fragment of E. coli-derived yahK (b0325) was obtained.
  • pAH162 was digested with restriction enzymes PstI and BamHI, and the obtained plasmid fragment was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system. Thereafter, the gene fragments of yahK (b0325) and pAH162 were ligated with In-Fusion® HD cloning Kit, and transformed into PIR2 strain.
  • the transformed microorganism that has appeared is subjected to colony PCR using a combination of primers shown by pAH162-Gene-F2 (SEQ ID NO: 71) and pAH162-Gene-R (SEQ ID NO: 72), and E. coli under control of the tac promoter.
  • a plasmid pAH162-P tac -yahk (b0325) was obtained which was expressed by the E. coli-derived yahK (b0325) gene.
  • PAH162-P tac -ADH3P and pAH162-P tac -yahK were introduced by the reaction method. Then, after shaking culture at 34 ° C. for 2 hours using SOC medium, culture was performed at 42 ° C. for 1 hour. A portion of the culture was applied to an LB plate containing Tet 25 mg / L and Cm 60 mg / L and cultured overnight at 37 ° C. The resulting transformed microorganisms were designated as strain SC17 (0) ⁇ mgsA :: pAH162-P tac -ADH3P and strain SC17 (0) ⁇ mgsA :: pAH162-P tac -yahK (b0325).
  • IP03VI ⁇ mgsA :: pAH162-P tac -ADH3P strain and IP03 ⁇ mgsA :: pAH162-P tac -yahK (b0325) strain SC17 (0) ⁇ mgsA :: pAH162-P tac -ADH3P strain and SC17 (0) ⁇ mgsA
  • the pAH162-P tac -yahK (b0325) strain was uniformly applied to LB plates containing 25 mg / L of tetracycline and cultured at 34 ° C. for 16 hours. Genomic DNA was extracted from the cells obtained according to the method described above.
  • competent cells are prepared from the IP03VI strain, and the SC17 (0) ⁇ mgsA :: pAH162-P tac -ADH3P strain and the SC17 (0) ⁇ mgsA :: pAH162-P tac -yahK (b0325) strain are electroporated. 600 ng of genomic DNA extracted from each was introduced. Recovery culture performed recovery culture at 34 ° C. for 2 hours in SOC medium. The culture solution was applied to an LB plate containing 25 mg / L of tetracycline and cultured at 34 ° C. overnight.
  • IP03 ⁇ mgsA :: pAH162-P tac -ADH3P strain
  • IP03VI ⁇ mgsA :: pAH162-Ptac-yahK (b0325) strain.
  • IP03VI ⁇ mgsA :: P tac -ADH3P strain and IP03 ⁇ mgsA :: P tac -yahK (b0325) strain
  • IP03VI ⁇ mgsA :: pAH162-P tac -ADH3P strain
  • IP03 ⁇ mgsA :: pAH162-P tac -yahK (b0325) strain
  • PRSFParaIX was electroporated into both the strains. Then, SOC medium was added to 1 mL, and recovery culture was performed at 34 ° C. for 2 hours. The recovered cells were seeded on LB plates containing 60 mg / L chloramphenicol and cultured overnight at 37 ° C.
  • the resulting transformed microorganisms were plated on LB plates containing 60 mg / L chloramphenicol and 0.2 M arabinose to form single colonies.
  • the obtained colonies were respectively inoculated on LB plates containing 25 mg / L of tetracycline and LB plates containing 60 mg / L of chloramphenicol, and cultured at 34 ° C. for 16 hours.
  • By obtaining a strain that can not grow only on LB plates containing tetracycline it was confirmed that the tetracycline resistance gene had been shed.
  • IP03VI ⁇ mgsA P tac -ADH3P / pRSFParaIX strain
  • IP03VI ⁇ mgsA P tac -yahK (b0325) / pRSFParaIX strain, respectively.
  • the plate was plated on an LB plate containing 10% sucrose and 1 mM IPTG, and cultured at 34 ° C. for 16 hours.
  • the resulting single colonies were inoculated in LB medium containing chloramphenicol 60mg / L, IP03VI ⁇ mgsA pRSFParaIX by making sure that can not be growth dropped out :: P tac -ADH3P stocks and IP03 ⁇ mgsA :: P tac - YahK (b0325) strain was obtained.
  • the genomic sequence (GenBank: AP012032.2) of the Ananatis AJ 13355 strain has been published, and based on the sequence (SEQ ID NO: 82) of the promoter region of the yahK gene (PAJ_3430), a sequence homologous to the promoter region of the same gene at the 5 'end Primers of Ptac-yahK1-F (SEQ ID NO: 83) and Ptac-yahK1-R (SEQ ID NO: 84) having the following were designed.
  • PCR was performed using ⁇ attL-Km R - ⁇ attR-Ptac (WO 2008/090770) as a template with Ptac-yahK1-F and Ptac-yahK1-R primers, and both ends were homologous to the promoter region of the yahK gene
  • a ⁇ attL-Km R - ⁇ attR-P tac gene fragment having a sequence was obtained. 600 ng of the resulting ⁇ att L-Km R - ⁇ att R -P tac gene fragment was introduced into the SC17 (0) / pRSFRedTER strain by electroporation and seeded in LB medium containing 50 mg / L kanamycin.
  • colony PCR is carried out using the obtained transformed microorganism with the primers shown by yahK1-CF (SEQ ID NO: 85) and yahK1-CR (SEQ ID NO: 86), and the promoter region of the yahK (PAJ_3430) gene is ⁇ attL-Km R It was confirmed that substitution was performed with- ⁇ attR-P tac sequence.
  • the transformed microorganism is P. Ananatis SC17 (0) ⁇ att L-Km R- ⁇ att R -P tac- yahK / pRSFRedTER strain was designated.
  • the plate was plated on an LB plate containing 10% sucrose and 1 mM IPTG, and cultured at 34 ° C. for 16 hours.
  • the obtained single colony is inoculated on LB medium containing 60 mg / L of chloramphenicol, and it is confirmed that it can not grow, thereby a SC17 (0) ⁇ att L-K m R- ⁇ att R- ⁇ att R -P tac- yah K (PAJ_3430) strain I got
  • IP03VI P tac -yahK (PAJ_3430) strain SC17 (0) ⁇ att L-Km R- ⁇ att R- ⁇ tac- yah K (PAJ_3430) strain was uniformly applied to LB plates containing 50 mg / L kanamycin. And cultured at 34.degree. C. for 16 hours. Genomic DNA was extracted from the cells obtained according to the method described above. Thereafter, competent cells were prepared from the IP03VI strain, and 600 ng of genomic DNA extracted from the SC17 (0) ⁇ att L-Km R - ⁇ att R- ⁇ tac -yahK (PAJ_3430) strain was introduced by electroporation.
  • IP03VI P tac -yahK PAJ_3430
  • pRSFParaIX pRSFParaIX
  • the plate was plated on an LB plate containing 10% sucrose and 1 mM IPTG, and cultured at 34 ° C. for 16 hours.
  • the obtained single colony was inoculated on LB medium containing 60 mg / L of chloramphenicol, and it was confirmed that growth was not possible, thereby obtaining IP03VI P tac -yahK (PAJ_3430) strain from which pRSFParaIX was eliminated.
  • Example 7 Construction of valencene-converted bacteria into which various ADH gene expression cassettes have been introduced and nootkaton conversion test from valencene 7-1) Construction of valencene-converted bacteria into which various ADH gene expression cassettes have been introduced IP03VI strain, IP03VI ⁇ mgsA :: attB ⁇ 80 PSol-CamC * AB was introduced into the strains IP03VI ⁇ mgsA :: P tac -ADH3P and IP03 VI ⁇ mgsA :: P tac -yahK (b0325), and further into the IP03VI P tac -yahK (PAJ_3430) strain by electroporation.
  • a stock solution 40 g of glycerol and 1 g of MgSO 4 ⁇ 7H 2 O were dissolved in pure water and the solution was adjusted to 400 mL, then 115 ° C., 10 min. Autoclave sterilization.
  • B stock solution 5 g (NH 4 ) 2 SO 4 , 1 g KH 2 PO 4 , 2 g Bacto-yeast extract, 10 mg FeSO 4 ⁇ 7 H 2 O, 10 mg MnSO 4 ⁇ 5 H 2 O dissolved in pure water, pH adjusted to KOH After adjusting to 7.0 with, the volume was increased to 400 mL. Thereafter, at 115 ° C. for 10 minutes. Autoclave sterilization.
  • the medium was added to a final concentration of 2.0 g / L.
  • a 1 M solution of ZuSO 4 ⁇ 7 H 2 O; ZnSO 4 ⁇ 7 H 2 O was dissolved in pure water to prepare 1 M ZnSO 4 ⁇ 7 H 2 O. Then, it was sterile filtered with a 0.22 ⁇ m filter. During culture, the medium was added to a final concentration of 5 mM.
  • IP03VI ⁇ mgsA :: attB ⁇ 80 / pSol-CamC * AB strain produced 18.9 mg / L nootkatone
  • IP03VI ⁇ mgsA :: P tac -ADH3P / pSol-CamC * AB strain produced 26.0 mg / mg.
  • An accumulation of L was observed.
  • E.I. An accumulation of 53.1 mg / L was observed in the IP03VI ⁇ mgsA :: P tac -yahK (b0325) / pSol-CamC * AB strain into which the expression cassette of the yahK (b0325) gene derived from E. coli MG1655 was introduced.
  • the present invention is useful for producing nootkatone which can be used for products such as beverages and cosmetics.
  • SEQ ID NO: 1 shows the base sequence of Pichia pastoris-derived alcohol dehydrogenase (ADH3P).
  • SEQ ID NO: 2 shows the amino acid sequence of alcohol dehydrogenase from Pichia pastoris (ADH3P).
  • SEQ ID NOs: 3 to 28 show the nucleotide sequences of the primers.
  • SEQ ID NO: 29 is P.
  • the nucleotide sequence of Y. anhatis derived YahK (PAJ_3430) gene is shown.
  • SEQ ID NO: 30 is P.I.
  • FIG. 7 shows the amino acid sequence of Y. ananatis-derived YahK (PAJ_3430) protein.
  • SEQ ID NO: 31 corresponds to E.I.
  • SEQ ID NO: 32 corresponds to E.I.
  • Fig. 6 shows the amino acid sequence of the E. coli YahK (b0325) protein.
  • SEQ ID NOs: 33 to 36 show the nucleotide sequences of the primers.
  • SEQ ID NO: 37 is P.
  • the nucleotide sequence of L-ldh gene (PAJ_p0276) derived from Ananatis AJ 13355 strain is shown.
  • SEQ ID NOs: 38 to 41 show the nucleotide sequences of the primers.
  • SEQ ID NO: 42 shows the nucleotide sequence of synthesized Pseudomonas putida-derived cytochrome P450camC (CamC).
  • SEQ ID NO: 43 shows the amino acid sequence of cytochrome P450 camC (CamC) derived from Pseudomonas putida.
  • SEQ ID NOs: 44 to 49 show the nucleotide sequences of the primers.
  • SEQ ID NO: 50 shows the nucleotide sequence of cytochrome P450 camA (CamA) derived from Pseudomonas putida.
  • SEQ ID NO: 51 shows the amino acid sequence of cytochrome P450 camA (CamA) derived from Pseudomonas putida.
  • SEQ ID NO: 52 shows the nucleotide sequence of cytochrome P450 cam B (Cam B) derived from Pseudomonas putida.
  • SEQ ID NO: 53 shows the amino acid sequence of cytochrome P450 cam B (Cam B) derived from Pseudomonas putida.
  • SEQ ID NO: 54 shows the base sequence of an artificial operon CamA-CamB consisting of a CamA gene and a CamB gene.
  • SEQ ID NOs: 55 to 62 show the nucleotide sequences of the primers.
  • SEQ ID NO: 63 shows the base sequence of valencene synthase (VlnSCN) derived from Cupressus nootkatensis.
  • SEQ ID NO: 64 shows the amino acid sequence of valencene synthase (VlnSCN) derived from Cupressus nootkatensis.
  • SEQ ID NOs: 65 and 66 show the nucleotide sequences of the primers.
  • SEQ ID NO: 67 corresponds to E.I. 1 shows the nucleotide sequence of farnesyl diphosphate synthase (IspA) derived from E. coli.
  • SEQ ID NO: 68 is an E.I.
  • Fig. 2 shows the amino acid sequence of farnesyl diphosphate synthase (IspA) derived from E. coli.
  • SEQ ID NOs: 69 to 72 show the nucleotide sequences of the primers.
  • SEQ ID NO: 73 is P.I.
  • the nucleotide sequence of the mgsA gene (PAJ_p0276) derived from the Ananatis AJ13355 strain is shown.
  • SEQ ID NOs: 74 to 81 show the nucleotide sequences of the primers.
  • SEQ ID NO: 82 is P.
  • the nucleotide sequence of the promoter region of the ananatis-derived yahK gene is shown (atg at the 3 'end indicates the start codon of the yahK gene).
  • SEQ ID NOs: 83 to 86 show the nucleotide sequences of the primers.

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Abstract

L'invention fournit un procédé de fabrication efficace de nootkatone selon un procédé biologique. Plus précisément, l'invention fournit un procédé de fabrication de nootkatone qui inclut une étape au cours de laquelle un nootkatol est converti en nootkatone en présence (i) d'un microorganisme transformé présentant un alcool déshydrogénase amélioré en comparaison avec un microorganisme sauvage, (ii) et d'un alcool déshydrogénase. L'alcool déshydrogénase consiste en : (A) une protéine contenant une séquence d'acides aminés de SEQ ID No 30 ou 32 ; (B) une protéine qui contient une séquence d'acides aminés incluant la substitution, la délétion, l'insertion ou l'addition d'un ou plusieurs acides aminés, dans la séquence d'acides aminés de SEQ ID No 30 ou 32, et qui possède une activité d'alcool déshydrogénase ; ou (C) une protéine qui contient une séquence d'acides aminés possédant 90% d'identité ou plus avec la séquence d'acides aminés de SEQ ID No 30 ou 32, et qui possède une activité d'alcool déshydrogénase.
PCT/JP2018/034996 2017-09-22 2018-09-21 Procédé de fabrication de nootkatone WO2019059337A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480512A (zh) * 2021-12-13 2022-05-13 华南理工大学 氧化还原酶及其突变体在生物合成圆柚酮中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016029187A2 (fr) * 2014-08-21 2016-02-25 Givaudan Sa Procédés de production de terpènes oxygénés
EP3196312A1 (fr) * 2016-01-25 2017-07-26 Metabolic Explorer Conversion efficace de méthylglyoxal en hydroxyacétone à l'aide de nouvelles enzymes et leurs utilisations
KR20170141384A (ko) * 2016-06-15 2017-12-26 경상대학교산학협력단 터펜 알코올 또는 그 유도체의 제조 방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016029187A2 (fr) * 2014-08-21 2016-02-25 Givaudan Sa Procédés de production de terpènes oxygénés
JP2017525395A (ja) * 2014-08-21 2017-09-07 マナス バイオシンセシス インコーポレイテッド 含酸素テルペンの産生方法
EP3196312A1 (fr) * 2016-01-25 2017-07-26 Metabolic Explorer Conversion efficace de méthylglyoxal en hydroxyacétone à l'aide de nouvelles enzymes et leurs utilisations
KR20170141384A (ko) * 2016-06-15 2017-12-26 경상대학교산학협력단 터펜 알코올 또는 그 유도체의 제조 방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Database UniProtKB 7 June 2017 (2017-06-07), HARA, Y. ET AL.: "SubName: Full=Zinc-type alcohol dehydrogenase-like protein YahK {ECO:0000313|EMBL:BAK13510.1", XP055584897, retrieved from Uniprot Database accession no. A0A0H3L9E7 *
PICK, A. ET AL.: "Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 97, no. 13, July 2013 (2013-07-01), pages 5815 - 5824, XP055584900, ISSN: 0175-7598, DOI: 10.1007/s00253-012-4474-5 *
WRIESSNEGGER, T. ET AL.: "Production of the sesquiterpenoid (+)-nootkatone by metabolic endineering of Pichia pastoris", METABOLIC ENGINEERING, vol. 24, 2014, pages 18 - 29, XP055211611, DOI: doi:10.1016/j.ymben.2014.04.001 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480512A (zh) * 2021-12-13 2022-05-13 华南理工大学 氧化还原酶及其突变体在生物合成圆柚酮中的应用
WO2023109530A1 (fr) * 2021-12-13 2023-06-22 华南理工大学 Utilisation de l'oxydoréductase et de son mutant dans la biosynthèse de la nootkatone

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