WO2019059337A1 - Nootkatone production method - Google Patents

Abstract

The present invention provides an efficient method for producing nootkatone via a biological method. More specifically, the present invention provides a nootkatone production method including converting nootkatole to nootkatone in the presence of (i) a transformant microorganism having improved alcohol dehydrogenase activity over a wild-type organism, or (ii) alcohol dehydrogenase, the alcohol dehydrogenase being: (A) a protein having the amino acid sequence of SEQ ID NO. 30 or 32; (B) a protein having the amino acid sequence of SEQ ID NO. 30 or 32 with one or more amino acid substitutions, deletions, insertions, or additions, and having alcohol dehydrogenase activity; or (C) a protein having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO. 30 or 32, and having alcohol dehydrogenase activity.

Description

Method of manufacturing Nootkaton

The present invention relates to a method of producing nootkatone.

Many organisms (eg, microorganisms, plants, animals) express a series of enzymes involved in either the methyl erythritol 4-phosphate (MEP) pathway or the mevalonic acid (MVA) pathway, or both. ing. These pathways are responsible for the supply of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are also known as intermediates required for terpenoid production. For example, when nootkatone is biosynthesized as a terpenoid, farnesyl diphosphate synthase (FPP) is biosynthesized from IPP and DMAPP by farnesyl diphosphate synthase, and then valencene, which is a raw material of nootkatone from FPP, is valensen synthase. It is synthesized.

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).

Several findings have been reported for the production of nootkatone by biological methods.
U.S. Pat. In E. coli, a method for producing nootkatone is described by utilizing cytochrome P450 derived from Stevia rebaudiana.
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.
In 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.

International Publication No. 2016/029187

Wriessnegger T et al. , Metab Eng. 2014 Jul; 24: 18-29 Sowden RJ et al. , Org Biomol Chem. 2005 Jan 7; 3 (1): 57-64 Pick A et al. , Appl Microbiol Biotechnol. 2013 Jul; 97 (13): 5815-24

The object of the present invention is to provide an efficient method of producing nootkatone by biological methods.

As a result of intensive studies, 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.

That is, the present invention is as follows.
[1] A method for producing nootkatone,
(I) converting nootkatole to nootkatone in the presence of (i) a transformed microorganism whose activity of alcohol dehydrogenase is improved compared to that of a wild-type microorganism, or (ii) alcohol dehydrogenase,
Alcohol dehydrogenase is as follows:
(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 method comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 30 or 32 and being a protein having alcohol dehydrogenase activity.
[2] The method of [1], wherein the transformed microorganism is an expression unit comprising a polynucleotide encoding the alcohol dehydrogenase and a promoter operably linked thereto.
[3] The method of [2], wherein 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.
[7] The method according to any one of [2] to [6], wherein the transformed microorganism produces monooxygenase having the ability to convert valencene to nooctol.
[8] The method of any of [2] to [7], wherein the transformed microorganism produces farnesyl diphosphate synthase and valencene synthase.
[9] The method according to any one of [2] to [8], wherein the transformed microorganism has either the methyl erythritol 4-phosphate pathway or the mevalonic acid pathway, or both.

According to the method of the present invention, 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.

In the protein (B), 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).

In protein (C), the percent identity with the amino acid sequence of SEQ ID NO: 2 or 5 is 90% or more. Preferably, 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. The percent identity can be determined as follows.
Calculation of% identity of a polypeptide (protein) can be performed by the algorithm blastp. More specifically, the calculation of% identity of the polypeptide is calculated using the default setting Scoring Parameters (Matrix: BLOSUM 62; Gap Costs: Existence = 11 Extension) in algorithm blastp provided in the National Center for Biotechnology Information (NCBI). Compositional Adjustments can be performed using Conditional Compositional Score Matrix Adjustment.
The calculation of% identity of polynucleotides (genes) can be performed by the algorithm blastn. More specifically, the calculation of% identity of polynucleotide is carried out using the default setting Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) in algorithm blastn provided in NCBI. It can be carried out.

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., 100%) may have more active. In addition, (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 (i.e., 100%) may have more active. The following conditions can be adopted as such specific measurement conditions. First, a transformed microorganism that expresses a target protein is inoculated on LB medium and cultured overnight at 34 ° C. Next, 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. After completion of the culture, 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.) .

In the proteins (B) and (C), 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.

When the amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution. As used herein, the term "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. For example, as such a family, 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), β branched side chain 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) -containing side chains (eg, Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, 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. As such 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.

Examples of 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).

In one embodiment, 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. As 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. When an inducible promoter is used to express a target protein, 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. When the target protein is secreted and expressed, 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.

In another embodiment, 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. In the context of the present invention, the term "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.

Preferably, 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 of

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.

In the polynucleotide (b) above, the term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, as stringent conditions, hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate) followed by 50 ° -65 ° C. in 0.2 × SSC, 0.1% SDS And one or more washes.

In the above polynucleotide (c), 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.

In the above polynucleotide (d), 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.

Preferably, 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. When 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. For example, in E. coli, the low frequency codons shown in Table A are known.

Thus, the present invention can utilize degenerate variants that match the codon usage of the host cell as described below. For example, 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. Preferably, 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).
If the degenerate variant is RNA, 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.

Identification of low frequency codons can be easily performed based on any host cell type and genomic sequence information by utilizing techniques known in the art. Thus, degenerate variants may include changes to low frequency codons to non-low frequency codons (eg, high frequency codons). In addition, since 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. Thus, 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.

In the context of the present invention, 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 smallest unit that enables production. 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 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) and a promoter operably linked thereto 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. For example, the promoter may be homologous or heterologous to the host cell, but is preferably heterologous. For example, constitutive or inducible promoters commonly used for the production of recombinant proteins can be used. As such 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. Preferably, 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.

In one embodiment, 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. It may be The term "heterologous expression unit" means that the expression unit is heterologous to the host cell. Thus, in the present invention, 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. Preferably, 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.

In the microorganism (i), 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 A protein having an alcohol dehydrogenase activity, or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 30, and comprising an amino acid sequence containing deletion, insertion, or addition; A microorganism comprising an expression unit comprising a polynucleotide encoding a protein having alcohol dehydrogenase activity and a promoter operably linked thereto (but excluding Pantoea microorganism, preferably Pantoea ananatis), (i-2) ( A ′ ′) a protein comprising the amino acid sequence of SEQ ID NO: 32 (B ′ ′) a protein comprising an amino acid sequence containing one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO: 32 and having alcohol dehydrogenase activity; or (C ′ ′ An expression unit comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 32, and comprising a polynucleotide encoding a protein having alcohol dehydrogenase activity and a promoter operably linked thereto; And microorganisms containing the above (except for Escherichia coli, preferably Escherichia coli).

In another embodiment, 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.

In yet another embodiment, 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.

In yet another embodiment, 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 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.

Preferably, 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).

In the present invention, 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. As Bacillus bacteria, Bacillus subtilis is preferable. As Corynebacterium (Corynebacterium) genus bacteria, Corynebacterium glutamicum (Corynebacterium glutamicum) is preferable. Examples of gram-negative bacteria include Escherichia bacteria and Pantoea bacteria. As the Escherichia bacteria, Escherichia coli is preferable. Pantoea ananatis is preferred as the Pantoea genus bacteria. As fungi, microorganisms of the genus Saccharomyces (Saccharomyces) and the genus Schizosaccharomyces (Schizosaccharomyces) are preferred. As a microorganism of the genus Saccharomyces (Saccharomyces), Saccharomyces cerevisiae (Saccharomyces cerevisiae) is preferable. As a microorganism of the genus Schizosaccharomyces, Schizosaccharomyces pombe is preferable.

The transformed microorganism may be a microorganism that produces monooxygenase that can convert valencene to nooctol. Thus, the transformed microorganism may be a host cell comprising expression units (preferably heterologous expression units) of such monooxygenases. As a monooxygenase having the ability to convert valencene to nooctol, for example, cytochrome P450 can be mentioned. 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. For example, as eukaryote-derived cytochrome P450 oxidase (CYP), Stevia rebaudiana Kaurene oxidase (SrKO), CYP706M1 from Callitropsis nootkatensis, CYP71D55 from Hyoscyamus muticus, and variants thereof can be mentioned (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). Moreover, CPR (Genbank number NM — 118585.3) derived from Arabidopsis thaliana, SrCPR derived from Stevia rebaudiana, and variants thereof can be mentioned as cytochrome P450 reductase (CPR) which functions as a conjugate function as these cytochrome P450 oxidases (Wriessnegger et al., Metabolic Engineering, 2014; 24: 18-29; WO 2016/029187). On the other hand, examples of cytochrome P450 derived from prokaryote 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.

When expressing a prokaryote-derived cytochrome P450 oxidase such as Pseudomonas putida-derived cytochrome P450camC (CamC), 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). : 57-64, Mandai et al., EBS J., 2009; 8: see 2416-2429). The above-mentioned proteins 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 also be a microorganism that produces farnesyl diphosphate synthase (EC: 2.5. 1. 10) and valencene synthase (EC: 4. 2. 3. 73). Thus, 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 .

As farnesyl diphosphate synthase, for example, 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.

As 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. Such microorganisms are microorganisms having either the MEP pathway or the MVA pathway, or both. Microorganisms usually have any of these pathways. For example, bacteria of the genus Escherichia, such as Escherichia coli, may inherently have the ability to synthesize dimethylallyl diphosphate by the MEP pathway. In addition, Saccharomyces genus yeasts such as Saccharomyces cerevisiae can inherently have the ability to synthesize dimethylallyl diphosphate by the MVA pathway. For the MEP pathway, see, for example, Kuzuyama T and Seto H, Proc Jpn Acad Ser B Phys Biol Sci. 88, 41-52 (2012); Grawert T et al. , Cell Mol Life Sci. 68, 3797-3814 (2011). Moreover, for the MVA pathway, for example, Kuzuyama T and Seto H, Proc Jpn Acad Ser B Phys Biol Sci. 88, 41-52 (2012); Miziorko HM, Arch Biochem Biophys. 505, 131-143 (2011). Such a transformed microorganism is an expression unit (preferably heterologous expression) of one or more (e.g. 1, 2, 3, 4, 5 or 6) enzymes involved in either the MEP pathway or the MVA pathway, or both A host cell comprising the unit). 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. These expression units may be independent plural expression units expressing monocistronic mRNA or may be a single expression unit expressing polycistronic mRNA.

As an enzyme involved in the MEP pathway, for example, 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; Example 2, AT2G02500, ACCESSION ID NP_565286) (EC: 2.7.1.148; Example 1, IspE, ACCESSION ID NP_415726; Example 2, AT2G26930, ACCESSION ID NP_180261), 2-C-Methyl-D-Erythritol-2,4-Cycloniphosphate Synthase (EC: 4.6.1.12; Example 1, IspF, ACCESSION ID NP_417226; Example 2, AT1G63970, ACCESSION ID NP_564819), 1-hydroxy -2-Methyl-2- (E) -butenyl-4-niphosphate synthase (EC: 1.17.7.1; Example 1, IspG, ACCESSION ID NP_417010; Example 2, AT5G60600, ACCESSION NP_001119467), 4- Hydroxy-3-methyl-2-butenyl diphosphate reductase (EC: 1.17.1.2; Example 1, IspH, ACCESSION ID NP_414570; Example 2, AT4G34350, ACCESSION ID NP_567965).

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, AT5G47720, ACCESSION ID NP_001032028; Example 3, AT5G48230, ACCESSION ID NP_568694) , Hydroxymethylglutaryl-CoA synthase (EC: 2.2.3. 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).

Examples of isopentenyl diphosphate delta isomerase (EC: 5.3.3.2) include Idi1p (ACCESSION ID NP_015208), AT3G02780 (ACCESSION ID NP_186927), AT5G16440 (ACCESSION ID NP_197148), and Idi (ACCESSION ID NP_417365). Can be mentioned.

The transformed microorganism used in the present invention can be produced by any method known in the art. For example, 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. If 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. On the other hand, when 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. Alternatively, according to genome editing technology (eg, CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)), 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. Examples of 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. For example, 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. Alternatively, 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. Examples of such expression vectors 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.

It is possible to add nootkatol, which is a substrate used in the method of the present invention, to 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). it can. Alternatively, in the method of the present invention, nootkatol produced in the reaction system can also be used as a substrate. For example, noutokatol, a substrate in a reaction system, is 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). Preferably, noutokatole may be produced from a carbon source in the culture medium in which transformed microorganisms expressing proteins as described above are cultured. When a transformed microorganism expressing the above-mentioned alcohol dehydrogenase is used in addition to the protein as described above, nootkatone can be produced from a carbon source via noutokatol.

When the method of the present invention is carried out using the above-mentioned alcohol dehydrogenase itself, an aqueous solution containing the above-mentioned alcohol dehydrogenase can be used as a reaction system. As an aqueous solution, a buffer is preferred. Examples of 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.

When the method of the present invention is carried out in the presence of the above-mentioned transformed microorganism producing alcohol dehydrogenase, 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. As the culture medium, those described above can be used. The culture medium preferably contains a carbon source. As 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 Be As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. As 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. In addition to these, 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. As the gas composition, 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. Examples of such culture methods include batch culture methods, fed-batch culture methods, and continuous culture methods. When the expression of a specific protein produced by a transformant microorganism is under the control of an inducible promoter such as a lac promoter, an inducer such as IPTG (isopropyl-β-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. For example, 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. For example, 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. For confirmation of production of nootkatone, and recovery and purification, see, for example, WO 2016/029187, US Patent Application Publication 2012/0246767, JP-A 2002-47239, Xie et al. , Food Chem. , 2009; 117: 375-380.

EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

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. Similarly, using pSTV28-P _tac -T _trp (WO 2013/069634) as a new template, a combination of pSTV-F (SEQ ID NO: 5) and pSTV-R (SEQ ID NO: 6) with PCR (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 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.

1-2) P.I. Search for a nootkatol oxidase endogenous to Ananatis p. An attempt was made to identify a nootkatol oxidase endogenous to Ananatis. An alcohol dehydrogenase that recognizes nouctol as a substrate was searched from the database. P. The genomic sequence (GenBank: AP012032.2) of the Ananatis AJ 13355 strain has already been published. Using the KEGG database (Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg), 11 genes hit with the keyword alcohol dehydrogenase were extracted. The extracted genes encoding 11 alcohol dehydrogenases are summarized in Table 1.

1-3) P.I. Construction of the alcohol dehydrogenase expression plasmid derived from Ananatis. For the ananatis-derived alcohol dehydrogenase, an expression plasmid for each gene, pSTV28-P _tac -PAJ_XXXX (PAJ_XXXX stands for Locus tag number) was constructed according to the following procedure. P. 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. Similarly, using 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. 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.

1-4) P.I. Introduction of alcohol dehydrogenase expression plasmid into B. ananatis SC17 (0) strain An electrocompetent cell of ananatis SC17 (0) strain (Katashkana JI et al., BMC Mol Biol., 2009; 10: 34) is prepared, and pSTV28-P _tac -ADH3P, pSTV28-P _tac -PAJ_XXXX, or pSTV28-P. _tac -T _trp was introduced by electroporation. Then, the plate was seeded on an LB plate containing 40 mg / L chloramphenicol. Then, the culture was performed at 34 ° C. overnight to obtain a transformed microorganism. The obtained 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.

1-5) Conversion test from nootkatol to nootkaton using various alcohol dehydrogenase expression-enriched strains The SC17 (0) / pSTV28-P _tac -ADH3P strain, SC17 (0) / pSTV28-P _tac -PAJ_XXXX strain constructed above And SC17 (0) / pSTV28-P _tac -T _trp strain were inoculated on LB medium containing 40 mg / L chloramphenicol, and then cultured overnight at 34 ° C. Thereafter, the obtained cells were inoculated into 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. After completion of the culture, 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.
1M PIPES; 60.48 g of PIPES was dissolved in pure water, the pH was adjusted to 7.0 with KOH, and the volume was increased to 200 mL. Then, it was sterile filtered with a 0.22 μm filter.
2 g / L Nootkatol; Noutokator (manufactured by Sundia) was dissolved in 99.5% ethanol to a final concentration of 2 g / L.

Nootkatol and nootkatone contained in the culture solution were quantitatively analyzed by the following procedure. 200 μL of the culture broth was well suspended in 800 μL 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 μm (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. As a nootkatol standard solution, a commercial reagent (manufactured by Sundia) was dissolved in 99.5% ethanol to prepare 10, 100, and 1000 mg / L standard solutions. Detailed analysis conditions are shown below (vaporization chamber temperature: 250 ° C., injection amount: 1.0 μL, 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. to 215 ° C .; 0.5 ° C./min., 215 ° C. to 300 ° C .; 15 ° C./min., Detector temperature: 300 ° C., detector: FID, hydrogen flow rate: 40 mL / min. Total empty; 400 mL / min). The results of the conversion test from nootkatol to nootkatone are shown in Table 3. The SC17 (0) / pSTV28-P _tac -T _trp strain, which is a control, produced nootkatone of 2.8 mg / L, whereas the SC17 (0) / pSTV28-P _tac -ADH3P strain produced 11.2 mg / L. Nootkatone accumulation of L was observed. On the other hand, among the 11 extracted genes, the highest Nootkatone accumulation was observed in the SC17 (0) / pSTV28-P _tac -PAJ_3430 strain. The strain accumulated 12.7 mg / L nootkatone in the culture medium, and showed a higher value than the SC17 (0) / pSTV28-P _tac -ADH3P strain. PAJ_3430 encodes Zinc-type alcohol dehydrogenase-like protein YahK and found that the same protein catalyzes the oxidation reaction of Noutokatol.

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). As a result, YahK encoded by Locus tag b0325 showed 66% identity. E. The nucleotide sequence of E. coli-derived YahK is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32.

2-2) E. Construction of expression plasmid of E. coli-derived YahK According to the method described above, E. coli. An expression plasmid for E. coli-derived YahK, pSTV28-P _tac -b0325, was constructed according to the following procedure. E. PCR (Prime 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. Similarly, using pSTV28-P _tac -T _trp (WO 2013/069634) as a new template, a combination of pSTV-F (SEQ ID NO: 5) and pSTV-R (SEQ ID NO: 6) with PCR (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 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. A plasmid expressed by E. coli MG1655 strain-derived yahk gene, pSTV28-P _tac -b0325, was obtained.

2-3) P.I. Construction of Ananatis SC17 (0) / pSTV28-P _tac -b0325 Strain An electrocompetent cell of SC17 (0) strain was prepared, and pSTV28-P _tac -b0325 was introduced by electroporation. Thereafter, the cells were inoculated on LB medium containing 40 mg / L chloramphenicol, and cultured overnight at 34 ° C. to obtain transformed microorganisms. The obtained strain was P. Ananatis SC17 (0) / pSTV28-P _tac -b0325 strain was designated.

2-4) E. A conversion test from nootkatol to nootkaton using E. coli -derived yahk gene expression-enriched strain A conversion test from noutokatol to nootkaton was carried out in the same manner as described above using SC17 (0) / pSTV28-P _tac -b0325 strain. The SC17 (0) / pSTV28-P _tac -PAJ_3430 strain, the SC17 (0) / pSTV28-P _tac -b0325 strain, and the SC17 (0) / pSTV28-P _tac -T _trp strain, 40 mg / L chloramphenicol After seeding in LB medium containing L. and cultured overnight at 34.degree. Thereafter, 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. After completion of the culture, 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. Nootkatone accumulation of L was observed. In addition, nootkatone accumulation of 8.9 mg / L higher than that of the control was also observed in SC17 (0) / pSTV28-P _tac -b0325 strain. From the above results, E.I. By enhancing the expression of the E. coli-derived yahk gene, we found that the accumulation of nootkatone increased.

Example 3: Mevalonic acid pathway enhanced. Construction of ananatis IP03 strain 3-1) P.1. Construction of P. ananatis SC 17 (0) ΔL-ldh :: att L φ 80-Km ^R- att ^R φ 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. faecalis Methylglutaryl CoA (HMG-CoA) synthase (MvaS), Mevalonic acid kinase (Mvk) derived from Methanocella paludicola, phosphomevalonic acid kinase (PMK) derived from S. cerevisiae, diphosphomevalonic acid decarboxylase (MVD) derived from S. cerevisiae, S. cerevisiae Was introduced a gene encoding isoprenyl pyrophosphate isomerase (yIDI) derived from ) Was constructed in accordance with the following procedure. P. The expression cassette attLφ80-Km ^R -attRφ80 of the kanamycin resistance gene was introduced into the L-ldh gene (PAJ_p0276) located on the ananatis chromosome. P. 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_Kmφ80_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_Kmφ80_R (SEQ ID NO: 39) were designed. Using pMWattphi (Minaeva NI et al., BMC Biotechnol. 2008; 8: 63) as a template and primers L-ldh_Kmφ80_F and L-ldh_Kmφ80_R (Prime star GXL (registered trademark) 94 ° C · 10sec., 54 ° C · 20sec , 68 sec., 120 sec., 35 cycles), and 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 attLφ80-Km ^R -attRφ80 gene fragment was obtained. Thereafter, previously described (Katashkina JI et al, BMC Mol Biol, 2009; 10:.. 34). AttLφ80-Km R -attRφ80 gene fragments 600 ng SC17 (0) obtained according to the / PRSFRedTER strain (Katashkina JI et al, BMC Mol Biol., 2009; 10: 34) was introduced by electroporation and seeded in LB medium containing 50 mg / L kanamycin. Thereafter, colony PCR is carried out with the primers shown by L-ldh-F (SEQ ID NO: 40) and L-ldh-R (SEQ ID NO: 41) for the resulting transformed microorganism, and the L-ldh gene is attLφ80-Km ^R It was confirmed that substitution was performed with the -attRφ80 sequence. The transformed microorganism was designated as SC17 (0) ΔL-ldh :: attLφ80-Km ^R -attRφ80 / pRSFRedTER strain. Next, in order to remove chloramphenicol resistant pRSFRedTER from the same strain, it 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 P. pylori could not be grown. Ananatis SC17 (0) ΔL-ldh :: attLφ80-Km ^R -attRφ80 strain was obtained.

3-2) P.I. Next, the kanamycin resistance gene (kan gene) was removed from the SC17 (0) ΔL-ldh :: att L φ 80-Km ^R- att ^R φ 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). To remove the kan gene, SC17 (0) the ^ΔL-ldh :: attLφ80-Km competent cells of R -AttRfai80 strain was prepared and pAH129-cat was introduced by electroporation method. Thereafter, 1 mL of SOC medium was added, and recovery culture was performed at 34 ° C. for 2 hours, and then the temperature was raised to 42 ° C., and culture was further performed for 1 hour. The cells after recovery culture were seeded on an LB plate containing 60 mg / L of chloramphenicol and cultured at 37 ° C. overnight, and then cultured overnight at 30 ° C. While confirming that the obtained transformed microorganism can not grow on a plate containing kanamycin 50 mg / L, using primers indicated by L-ldh-F (SEQ ID NO: 40) and L-ldh-R (SEQ ID NO: 41) Colony PCR was performed to confirm that the L-ldh gene had been replaced by the attBφ80 sequence. This strain was newly designated as SC17 (0) ΔL-ldh :: att Bφ80 / pAH129-cat strain. According to the method of the previous report (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60), pAH129 was removed from SC17 (0) ΔL-ldh :: attBφ80 / pAH129-cat strain The 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. Next, the culture solution was seeded on an LB plate so that single colonies appeared, and cultured overnight at 37 ° C. By confirming that the emerged transformed microorganism can not grow on the LB plate containing 60 mg / L chloramphenicol, P. p. Ananatis SC17 (0) ΔL-ldh :: attBφ80 strain was obtained.

3-3) P.I. Construction of the ananatis SC17 (0) ΔL-ldh :: pAH162-P _phoC- mvaES / pAH123-cat strain SC17 (0) ΔL-ldh :: attBφ80 strain pAH123-cat (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60) were introduced by electroporation. Then, SOC medium was added to 1 mL, 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 designated as SC17 (0) ΔL-ldh :: att Bφ80 / pAH123-cat strain. Subsequently, competent cells of SC17 (0) ΔL-ldh :: attBφ80 / 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. for 2 hours, and then the temperature was raised to 42 ° C., and culture was further performed for 1 hour. The cells after recovery culture were seeded on an LB plate containing 25 mg / L of tetracycline, cultured at 37 ° C. overnight, and further cultured overnight at 30 ° C. The resulting transformed microorganism was P. _Ananatis SC17 (0) ΔL-ldh :: pAH162-P _phoC- mvaES strain was designated.

3-4) P.I. according to _{ananatisSWITCH-PphoCΔgcd ΔL-ldh :: P} phoC -mvaES strain the approach building Genomic DNA was prepared from _{SC17 (0) ΔL-ldh ::} pAH162-P phoC -mvaES strain. Then, after preparing competent cells from SWITCH-PphoCΔgcd strain (WO 2017/051930), genomic DNA extracted from SC17 (0) ΔL-ldh :: pAH162-P _phoC- mvaES strain by _{electroporation} , Introduced 600ng. The transformed cells were cultured in 1 mL of SOC medium at 34 ° C. for 2 hours for recovery culture. 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-PphoCΔgcdΔL-ldh :: It was named as pAH162-P _phoC- mvaES strain.

3-5) P.I. Construction of S. ananatis IP03 Strain The tetracycline resistance gene was removed from the SWITCH-PphoCΔgcdΔL-ldh :: pAH162-P _phoC- mvaES strain. P. pRSFParaIX (Tajima Y. et al., Appl Environ Microbiol. 2015; 81 (3): 929-937) was electroporated into the ananatis SWITCH-PphoCΔgcdΔL-ldh :: pAH162-P _phoC- mvaES strain. 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 be grown only on LB plates containing tetracycline, it was confirmed that the tetracycline gene was shed. A strain exhibiting this phenotype was newly designated as IP03 / pRSFParaIX strain. Next, in order to remove chloramphenicol-resistant pRSFParaIX from the 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. 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. The transformed microorganism that has appeared appears to be a colony PCR (Sapphire Amp® Fast PCR Master mix 94 ° C. · 10 sec., 54 ° C./20 sec./72° C. ·) by the combination of the primers shown in the kit and indicated by pRham Forward and pETite Reverse. 25 sec., 35 cycles) were carried out to obtain a plasmid pSol-CamC which is expressed by CamC under the control of the rhamnose promoter.

4-2) _Construction of a quadruple mutant (CamC *) 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). In 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. The same plasmid was transformed into JM109 strain, and the gene mutation site of the obtained plasmid pSol-CamC (F87A / Y96F) was sequence-analyzed with the primers indicated by pRham Forward and pETite Reverse to confirm that the mutation was introduced. Next, PCR (Prime star) is carried out using a combination of primers shown by CamC-L244A / V247L-CF (SEQ ID NO: 48) and CamC-L244A / V247L-CR (SEQ ID NO: 49) using pSol-CamC (F87A / Y96F) as a template. 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 *.

4-3) _{Construction of} 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. Using primers pUC57-CamA-CamB chemically synthesized by GenScript as a template and primers shown by CamAB-F (SEQ ID NO: 55) and CamAB-R (SEQ ID NO: 56), PCR (Prime star GXL (registered trademark) 94 ° C 10 sec., 54 ° C., 20 sec., 68 ° C., 150 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 of CamA-CamB. Next, 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. The gene fragments of pSol-CamC * and CamAB were ligated with In-Fusion® HD cloning Kit and transformed into strain JM109. Transformed microorganisms that appeared appeared colony PCR (Sapphire Amp (registered trademark) Fast PCR Master mix 94 ° C · 10 sec., 54 ° C · 20 sec., 72 ° C · 40 sec., 35 cycles) by the combination of the primers shown by pRham Forward and pETite Reverse. ) Was performed to obtain a plasmid pSol-CamC * AB which is expressed by CamC *, CamA and CamB under the control of the rhamnose promoter.

Example 5: Varensen producing ability was imparted Construction of ananatis IP03 VI strain 5-1) P. a. Construction of P. ananatis SC17 (0) Δ pflA :: att Lφ80-Km ^R- att Rφ80 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) ΔpflA :: attLφ80-Km ^R -attRφ80 strain was constructed according to the following procedure. Using pMWattphi (Minaeva NI et al., BMC Biotechnol. 2008; 8: 63) as a template, a combination of pflA-phi80-attR (SEQ ID NO: 59) and a primer shown in pflA-phi80-attL (SEQ ID NO: 60) PCR (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. 600 ng of the same gene fragment was transformed into SC17 (0) / pRSFRedTER strain to obtain a strain showing kanamycin resistance. Colony PCR was performed using the primers shown by pflA-check1 (SEQ ID NO: 61) and pflA-check2 (SEQ ID NO: 62) to confirm that the pflA gene was replaced with the attLφ80-Km ^R -attRφ80 gene.

5-2) P.I. Construction of P. ananatis SC17 (0) Δ pflA :: att Bφ80 Removal of the kan gene from the Ananatis SC17 (0) ΔpflA :: attLφ80-Km ^R -attRφ80 strain was carried out according to the following procedure. The same strain was uniformly applied to an LB plate containing 50 mg / L kanamycin and cultured at 34 ° C. for 16 hours. The cells were scraped from the plate, competent cells were prepared, and pAH129-cat was introduced by electroporation. Thereafter, 1 mL of SOC medium was added and shake culture was performed at 34 ° C. for 2 hours, and shake culture was further performed at 42 ° C. for 1 hour. 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) ΔpflA :: attBφ80 / pAH129-cat strain. Subsequently, pAH129-cat was removed from SC17 (0) ΔpflA :: attBφ80 / pAH129-cat strain. The SC17 (0) ΔpflA :: att Bφ80 / pAH129-cat strain was inoculated in LB medium and cultured at 34 ° C. for 3 hours. Then, it culture | cultivated at 42 degreeC for 1 hour. The culture solution was applied to an LB plate to obtain single colonies. Among the transformed microorganisms, a clone which can not grow on an LB plate containing chloramphenicol was obtained and designated as strain SC17 (0) ΔpflA :: attBφ80.

5-3) 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. 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. The obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a VlnSCN gene fragment. Next, 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. E. PCR (Prime Star (registered trademark) GXL 94 ° C., 10 sec., 54 ° C., 20 sec. using a combination of primers shown by IspA-F (SEQ ID NO: 69) and IspA-R (SEQ ID NO: 70) , 72 ° C., 90 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 gene fragment of IspA. After that, using the 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. Next, 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.

5-4) P.I. Construction of P. ananatis IP03 VI strain pAH123-cat was introduced into the ananatis SC17 (0) ΔpflA :: attBφ80 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) ΔpflA :: named att Bφ80 / pAH123-cat strain. Then, P. Competent cells of the ananatis SC17 (0) ΔpflA :: attBφ80 / 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) ΔpflA :: pAH162-P _tac -Vln SCN-ispA. According to the method described above, P.I. Genomic DNA was prepared from the Ananatis SC17 (0) ΔpflA :: 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) ΔpflA :: 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 IP03ΔpflA :: pAH162-P _tac -VlnSCN-ispA strain. Subsequently, the tetracycline resistance gene was removed from the strain. Competent cells of the IP03ΔpflA :: 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. 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. A strain exhibiting this phenotype was newly designated as IP03VI / pRSFParaIX strain. Next, in order to remove chloramphenicol-resistant pRSFParaIX from the 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) P.I. Construction of Ananatis SC17 (0) ΔmgsA :: attLφ80-Km ^R -attRφ80 Strain For the IP03VI strain, expression cassettes for various ADH genes were introduced into the chromosome. First, the attBφ80 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_Kmφ80_F (SEQ ID NO: 74) having a sequence homologous to the same gene at the 5 'end. And primers of mgsA_Kmφ80_R (SEQ ID NO: 75) were designed. PCR was performed using pMWattphi (Minaeva NI et al., BMC Biotechnol. 2008; 8: 63) as a template with the primers mgsA_Kmφ80_F and mgsA_Kmφ80_R, and attLφ80-Km ^R -attRφ80 having homologous sequences with the mgsA gene region at both ends The gene fragment was obtained. 600 ng of the obtained att L 80-Km ^R- att ^R 80 gene fragment was introduced into the SC17 (0) / pRSFRedTER strain by electroporation and seeded in LB medium containing 50 mg / L kanamycin. Then, colony PCR was performed with primers shown in a transformed microorganism obtained mgsA-F (SEQ ID NO: 76) and mgsA-R (SEQ ID NO: 77), mgsA gene is replaced with ^attLφ80-Km R -attRφ80 sequence I confirmed that. The transformed microorganism is P. Ananatis SC17 (0) ΔmgsA :: attLφ80-Km ^R -attRφ80 / pRSFRedTER strain was designated. Then, in order to remove chloramphenicol-resistant pRSFRedTER from the 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 P. pylori could not be grown. Ananatis SC17 (0) ΔmgsA :: attLφ80-Km ^R -attRφ80 strain was obtained.

6-2) P.I. Construction of P. ananatis SC17 (0) ΔmgsA :: att Bφ80 The kanamycin resistance gene (kan gene) was removed from the Ananatis SC17 (0) ΔmgsA :: attLφ80-Km ^R -attRφ80 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). In order to remove the kan gene, P. pAH129-cat was electroporated into ananatis SC17 (0) ΔmgsA :: attLφ80-Km ^R -attRφ80 strain. Thereafter, 1 ml of SOC medium was added, and recovery culture was performed at 34 ° C. for 2 hours, and then the temperature was raised to 42 ° C., and culture was further performed for 1 hour. The cells after recovery culture were seeded on an LB plate containing 60 mg / L chloramphenicol and cultured overnight at 37 ° C., followed by further overnight culture at 30 ° C. While confirming that the obtained transformed microorganism can not grow on a plate containing kanamycin 50 mg / L, colony PCR was performed using the primers shown by mgs AF (SEQ ID NO: 76) and mgs AR (SEQ ID NO: 77). It implemented and confirmed that the mgsA gene was substituted by the attBφ80 sequence. A new share of P. ananatis SC17 (0) ΔmgsA :: attBφ80 / pAH129-cat strain. Subsequently, removal of pAH129 from strain SC17 (0) ΔmgsA :: attBφ80 / pAH129 was performed according to the following procedure. The SC17 (0) ΔmgsA :: att Bφ80 / pAH129 strain was inoculated into 1 mL of LB medium and cultured at 34 ° C. for 3 hours. Then, it culture | cultivated at 42 degreeC for 1 hour. The culture solution was applied to an LB plate to obtain single colonies. It was confirmed that the obtained clone was not viable at 60 mg / L chloramphenicol, and was used as strain SC17 (0) ΔmgsA :: attBφ80.

6-3) Construction of CRIM Plasmid, 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. The obtained PCR product was purified by Wizard (registered trademark) SV Gel and PCR Clean-UP system to obtain a gene fragment of ADH3P. Next, 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. Thereafter, 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.

6-4) Construction of CRIM Plasmid, pAH162-P _tac -yahK (b0325) pAH162-P _tac -b0325 was constructed according to the following procedure. E. PCR (Prime star GXL (registered trademark) 94 ° C., 10 sec.) is carried out using the genomic DNA of E. coli MG1655 strain as a template and the combination of primers shown by pAH162-yahKE-F (SEQ ID NO: 80) and pAH162-yahKE-R (SEQ ID NO: 81). , 54 ° C., 20 sec., 68 ° C., 120 sec., 35 cycles). 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. Next, 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.

_{6-5) SC17 (0) ΔmgsA ::} pAH162-P tac -ADH3P shares and _{SC17 (0) ΔmgsA :: pAH162-} P tac -yahK (b0325) Construction of strain _{SC17 (0) ΔmgsA :: pAH162-} P tac - The ADH3P strain and the SC17 (0) ΔmgsA :: pAH162-P _tac -yahK (b0325) strain were constructed according to the following procedure. A competent cell solution of SC17 (0) ΔmgsA :: attBφ80 strain was prepared, and pAH123-cat was introduced by electroporation. Thereafter, 1 mL of SOC medium was added 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 microorganism was designated as SC17 (0) ΔmgsA :: attBφ80 / pAH123-cat strain. Subsequently, competent cells of SC17 (0) ΔmgsA :: attBφ80 / pAH123-cat strain are prepared and electroporated according to the previous report (Andreeva IF et al., FEMS Microbiol Lett. 2011; 318 (1): 55-60). PAH162-P _tac -ADH3P and pAH162-P _tac -yahK (b0325) 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) ΔmgsA :: pAH162-P _tac -ADH3P and strain SC17 (0) ΔmgsA :: pAH162-P _tac -yahK (b0325).

6-6) Construction of IP03VIΔmgsA :: pAH162-P _tac -ADH3P strain and IP03 ΔmgsA :: pAH162-P _tac -yahK (b0325) strain SC17 (0) ΔmgsA :: pAH162-P _tac -ADH3P strain and SC17 (0) ΔmgsA 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. Then, competent cells are prepared from the IP03VI strain, and the SC17 (0) ΔmgsA :: pAH162-P _tac -ADH3P strain and the SC17 (0) ΔmgsA :: 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. The resulting transformed microorganisms were respectively designated as IP03ΔmgsA :: pAH162-P _tac -ADH3P strain and IP03VIΔmgsA :: pAH162-Ptac-yahK (b0325) strain.

6-7) Construction of IP03VIΔmgsA :: P _tac -ADH3P strain and IP03ΔmgsA :: P _tac -yahK (b0325) strain IP03VIΔmgsA :: pAH162-P _tac -ADH3P strain and IP03ΔmgsA :: pAH162-P _tac -yahK (b0325) strain Removed the tetracycline resistance gene. 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. A strain exhibiting this phenotype was newly designated as IP03VIΔmgsA :: P _tac -ADH3P / pRSFParaIX strain and IP03VIΔmgsA :: P _tac -yahK (b0325) / pRSFParaIX strain, respectively. Next, in order to remove chloramphenicol-resistant pRSFParaIX from the 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 colonies were inoculated in LB medium containing chloramphenicol 60mg / L, IP03VIΔmgsA pRSFParaIX by making sure that can not be growth dropped out _:: P tac -ADH3P stocks and IP03ΔmgsA _{:: P tac} - YahK (b0325) strain was obtained.

6-8) Construction of SC17 (0) λ att L-Km ^R- λ att ^R -P _tac- yahK (PAJ_3430) Strain IP03 VI Δ mgs A :: P _tac- ADH 3 P and IP 03 VI Δ mgs A:: P _tac- y A We constructed the IP03VI P _tac -yahK (PAJ_3430) strain in which the yahK (PAJ_3430) gene is expressed under the control of the tac promoter. P. 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 λattL-Km ^R -λattR-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 λattL-Km ^R -λattR-P _tac gene fragment having a sequence was obtained. 600 ng of the resulting λ att L-Km ^R -λ 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. Thereafter, 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 λattL-Km ^R It was confirmed that substitution was performed with-λattR-P _tac sequence. The transformed microorganism is P. Ananatis SC17 (0) λatt L-Km ^R- λ att ^R -P _tac- yahK / pRSFRedTER strain was designated. Then, in order to remove chloramphenicol-resistant pRSFRedTER from the 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 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) λ att L-K m ^R- λ att ^R- λ att ^R -P _tac- yah K (PAJ_3430) strain I got

6-9) Construction of IP03VI P _tac -yahK (PAJ_3430) strain SC17 (0) λ att L-Km ^R- λ att ^R- λ _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) λ att L-Km ^R -λ att ^R- λ _tac -yahK (PAJ_3430) strain was introduced by electroporation. 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 50 mg / L of kanamycin and cultured overnight at 34 ° C. The resulting transformed microorganisms were designated as P03VI λattL-Km ^R -λattR-P _tac -yahK (PAJ_3430) strain, respectively. Subsequently, to remove the kanamycin resistance gene from _{^{IP03VI λattL-Km R -λattR-P}} tac -yahK (PAJ_3430) strain. PRSFParaIX was introduced into the strain by electroporation. Thereafter, 1 mL of SOC medium was added 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 50 mg / L kanamycin and LB plates containing 60 mg / L chloramphenicol, and cultured at 34 ° C. for 16 hours. It was confirmed that the kanamycin resistance gene was shed by obtaining a strain which can not grow only on the LB plate containing kanamycin. A strain exhibiting this phenotype was newly designated as IP03VI P _tac -yahK (PAJ_3430) / pRSFParaIX strain. Next, in order to remove chloramphenicol-resistant pRSFParaIX from the 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 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, IP03VIΔmgsA :: attBφ80 PSol-CamC * AB was introduced into the strains IP03VIΔmgsA :: P _tac -ADH3P and IP03 VI ΔmgsA :: P _tac -yahK (b0325), and further into the IP03VI P _tac -yahK (PAJ_3430) 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 an LB plate containing 50 mg / L kanamycin and cultured overnight at 37 ° C. Transformed microorganisms obtained respectively IP03VI / pSol-CamC * AB strain, IP03VIΔmgsA :: attBφ80 / pSol-CamC * AB _{strain, IP03VIΔmgsA :: P tac -ADH3P / pSol} -CamC * AB strain and IP03VIΔmgsA _{:: P tac} - It was named as yahK (b0325) / pSol-CamC * AB strain, and further as IP03VI P _tac -yahK (PAJ_3430) / pSol-CamC * AB strain.

7-2) Nootkatone conversion test from valencene Various strains were uniformly applied to LB plates and cultured at 34 ° C. for 16 hours. Thereafter, the grown cells were again applied to an LB plate containing 2.0 g / L rhamnose and cultured at 34 ° C. for 16 hours. About 1/2 plate of bacterial cells was inoculated into 5 mL of MS / Vln medium, and shake culture was performed at 30 ° C. and 120 rpm for 4 days using a thick test tube. The components of the MS / Vln medium are shown below.

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.
1M PIPES; 60.48 g of PIPES was dissolved in pure water, the pH was adjusted to 7.0 with KOH, and the volume was increased to 200 mL. Then, it was sterile filtered with a 0.22 μm filter.
100 g / L Valencene solution; valencene was dissolved in 99.5% ethanol to prepare a 100 g / L valencene solution. Thereafter, the medium was added to a final concentration of 1.0 g / L.
200 g / L rhamnose solution; rhamnose was dissolved in pure water to prepare a 200 g / L rhamnose solution. Then, it was sterile filtered with a 0.22 μm filter. At the time of culture, 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 μm filter. During culture, the medium was added to a final concentration of 5 mM.

7-3) After completion of the culture was measured _{OD 600} values in broth at the end of culture. The amount of glycerol consumed and the accumulated amount of nootkatone and nootkatole produced were measured by GC analysis. At the end of culture, 200 μL of the broth was suspended in 800 μL of ethanol and mixed for 5 minutes on an eppen shaker. Thereafter, the supernatant was centrifuged at 15,000 rpm for 10 minutes at 4 ° C., 600 μL of the supernatant was transferred to a glass glass vial for GC, and subjected to GC analysis with GC-2010 Plus (manufactured by Shimadzu Corporation). The results of Nootkatone conversion test from Valensen are shown in Table 5. The IP03VIΔmgsA :: attBφ80 / pSol-CamC * AB strain produced 18.9 mg / L nootkatone, whereas the IP03VIΔmgsA :: P _tac -ADH3P / pSol-CamC * AB strain produced 26.0 mg / mg. An accumulation of L was observed. Also, E.I. An accumulation of 53.1 mg / L was observed in the IP03VIΔmgsA :: 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 maximum nootkatone accumulation was confirmed with the IP03VIΔmgsA :: P _tac -yahK (PAJ_3430) / pSol-CamC * AB strain, reaching 148.7 mg / L. From these results, E.I. coli yahK (b0325) gene and P. coli. By enhancing the expression of the anantis-derived yahK (PAJ_3430) gene, it was confirmed that nootkatone production was increased.

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. The nucleotide sequence of the E. coli-derived yahK gene (b0325) is shown.
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.

Claims (9)

It is a manufacturing method of nootkaton,
(I) converting nootkatole to nootkatone in the presence of (i) a transformed microorganism whose activity of alcohol dehydrogenase is improved compared to that of a wild-type microorganism, or (ii) alcohol dehydrogenase,
Alcohol dehydrogenase is as follows:
(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 method comprising an amino acid sequence having 90% or more identity to the amino acid sequence of No. 30 or 32 and being a protein having alcohol dehydrogenase activity. The method according to claim 1, wherein the transformed microorganism is a microorganism comprising an expression unit comprising a polynucleotide encoding the alcohol dehydrogenase and a promoter operably linked thereto. The method according to claim 2, wherein the transformed microorganism is any 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. The method according to claim 2 or 3, wherein the transformed microorganism is a bacterium belonging to Enterobacteriaceae. The method according to any one of claims 2 to 4, wherein the transformed microorganism is Pantoea bacteria, Escherichia bacteria, or Corynebacterium bacteria. The method according to claim 5, wherein the transformed microorganism is Pantoea ananatis, Escherichia coli, or Corynebacterium glutamicum. The method according to any one of claims 2 to 6, wherein the transformed microorganism produces a monooxygenase having the ability to convert valencene to nooctol. The method according to any one of claims 2 to 7, wherein the transformed microorganism produces farnesyl diphosphate synthase and valencene synthase. The method according to any one of claims 2 to 8, wherein the transformed microorganism has either the methyl erythritol 4-phosphate pathway or the mevalonic acid pathway, or both.