JP2018000195A - Method for producing nootkatone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing nootkatone with microorganisms.SOLUTION: A method for producing nootkatone includes culturing microorganisms containing a gene encoding cytochrome P450 belonging to CYP107T in the presence of valencene.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing nootkaton using microorganisms, and a microorganism having the ability to produce nootkaton.

  Nootkaton is a sesquiterpene ketone compound present in the skin of grapefruit and the like, and is one of the scent components of grapefruit. Nootkatone is a plant secondary metabolite and is known to be produced by oxidation of valencene. On the other hand, there has been no report that nootkaton has been found as a microbial metabolite.

  Nootkaton is attracting attention as a natural fragrance, but stable supply of nootkaton is difficult due to factors such as the fact that only a small amount is contained in grapefruit and the production depends on the cultivation conditions of grapefruit. Yes and expensive. Therefore, methods for chemically synthesizing Nootkaton and methods for producing microorganisms or plants have been studied. For example, in Patent Document 1, valencene is given to a microorganism belonging to the genus Rhodococcus having a specific mycological property collected from soil to convert it into nootkatone. In Non-Patent Document 1, valencene is converted into nootkatone by recombinant Escherichia coli expressing cytochrome CYP109B1 derived from Bacillus subtilis in a two-liquid phase reaction system composed of an organic phase and an aqueous phase. .

Japanese Patent No. 3245254

Microbial Cell Factories, 2009, 8:36

  The production of nootkatone for use as a natural fragrance requires not using chemical synthesis but using a biocatalyst such as a microorganism or a plant or a natural enzyme. As described above, several methods for producing Nootkaton using microorganisms have been reported, but all require special bacteria and reaction systems. Accordingly, there is a need for a simpler and cheaper method for producing nootkatone using a biocatalyst such as a microorganism.

  As a result of diligent research to solve the above problems, the present inventors have surprisingly found that it is possible to produce nootkatone from Valensen using a specific cytochrome P450 enzyme system originally possessed by microorganisms. The headline and the present invention were completed.

That is, the present invention
[1] A method for producing Nootkaton, comprising culturing a microorganism containing a gene encoding cytochrome P450 belonging to CYP107T in the presence of valencene,
[2] Cytochrome P450 belonging to CYP107T includes the amino acid sequence represented by SEQ ID NO: 1, or at least one amino acid is deleted, inserted, added and / or substituted in the amino acid sequence represented by SEQ ID NO: 1. A deletion of 0 to several amino acids compared to SEQ ID NO: 1 in the regions corresponding to positions 231-235, 270-273, and 335-344 in SEQ ID NO: 1, The method according to [1], comprising an amino acid sequence comprising insertions, additions and / or substitutions;
[3] The method according to [2], wherein the cytochrome P450 belonging to CYP107T comprises the amino acid sequence represented by SEQ ID NO: 1.
[4] The method according to any one of [1] to [3], wherein the microorganism is actinomycete,
[5] The method according to [4], wherein the microorganism is Streptomyces actinomycetes,
[6] A microorganism into which a gene encoding cytochrome P450 belonging to CYP107T has been introduced,
[7] Cytochrome P450 belonging to CYP107T includes the amino acid sequence represented by SEQ ID NO: 1, or at least one amino acid is deleted, inserted, added and / or substituted in the amino acid sequence represented by SEQ ID NO: 1. A deletion of 0 to several amino acids compared to SEQ ID NO: 1 in the regions corresponding to positions 231-235, 270-273, and 335-344 in SEQ ID NO: 1, The microorganism of [6], comprising an amino acid sequence comprising an insertion, addition, and / or substitution;
[8] The microorganism according to [7], wherein cytochrome P450 belonging to CYP107T comprises the amino acid sequence represented by SEQ ID NO: 1,
[9] The microorganism according to any one of [6] to [8], wherein the microorganism is actinomycete, and [10] the microorganism according to [9], wherein the microorganism is a Streptomyces genus To do.

  ADVANTAGE OF THE INVENTION According to this invention, the method of manufacturing a nootkaton more simply and cheaply using microorganisms is provided. The noot katon produced by the method of the present invention can be used as a natural fragrance in a wide range of fields including foods and cosmetics.

It is a schematic diagram which shows the vector for gene overexpression used in the Example. In the figure, “PLDter” is a phospholipase D gene terminator region derived from Streptomyces cinnamoneus, “Target Gene” is a target gene region, “SSMPpro” is a Streptomyces septatas (Streptomyces septatum) Protease promoter, “xylApro” is a Streptomyces avermitilis xylose isomerase promoter, “tsr” is a thiostrepton resistance gene, and “rep pIJ101” is a replication region of actinomycetes. The gas chromatography (GC) / mass spectrometry (MS) analysis result of the SCO3770 overexpression strain is shown. It is a schematic diagram which shows the SCO3770 gene disruption vector used in the Example. In the figure, “oriT” and “oriV” are replication origins of Escherichia coli, “Km” is a kanamycin resistance gene, melC1C2 is a melC operon region derived from Streptomyces antibiotics, and “ΔSCO3770” is a SCO3770 gene. A partial sequence is shown. The gas-chromatography analysis result of the culture supernatant extract of 4 types of strain | stump | stocks is shown. The gas chromatographic analysis result of the microbial cell extract of 4 types of strain | stump | stocks is shown. The gas chromatographic analysis result of the culture supernatant extract of a strain overexpressing SLNWT — 3333 is shown. The gas chromatographic analysis result of the microbial cell extract of a SLNWT_3333 overexpression strain is shown.

  Cytochrome P450 is a group of heme-containing enzymes that catalyze oxidation reactions of a wide variety of substrates, and is present in almost all living organisms including bacteria (excluding some), plants, and mammals. Cytochrome P450s are classified into many families and subfamilies based on their sequence similarity. In the present invention, a microorganism containing a gene encoding cytochrome P450 belonging to subfamily CYP107T is used. The amino acid sequence or gene sequence of cytochrome P450 belonging to subfamily CYP107T is, for example, those described in Cytochrome P450 Homepage (http://drnelson.uthsc.edu/CytochromeP450.html), or the result of BLAST search in the site You can select from the top hits. When performing a BLAST search, as a prerequisite, the query sequence must be a cytochrome P450 sequence. Whether or not the query sequence is a cytochrome P450 sequence is, for example, whether or not a cytochrome P450 motif (PF00067) exists in the query sequence in Pfam (protein domain family database: http://pfam.xfam.org/) or the like. Can be determined by confirming.

  Examples of “cytochrome P450 belonging to CYP107T” include CYP107T1 derived from Streptomyces coelicolor A3 (2). The amino acid sequence encoding CYP107T1 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 4. Therefore, as an example of “cytochrome P450 belonging to CYP107T” in the present invention, cytochrome P450 including the amino acid sequence represented by SEQ ID NO: 1; 55% or more, 60% or more, 65% with respect to the amino acid sequence represented by SEQ ID NO: 1 Cytochrome P450 comprising an amino acid sequence having a sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more; cytochrome comprising the amino acid sequence represented by SEQ ID NO: 1 P450; and 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more with respect to the amino acid sequence shown in SEQ ID NO: 1 And cytochrome P450 consisting of an amino acid sequence having the following sequence identity.

  Although the amino acid sequence of cytochrome P450 is very diverse, it is known that there is a well-conserved region (consensus sequence) common to all cytochrome P450s (for example, J. Biol. Chem., 2002). Jul 5; 277 (27), pp.24000-24005). That is, EXXXR motif in K-helix (E is glutamic acid residue, R is arginine residue, X is any amino acid residue), cysteine residue in heme binding site and two glycine residues upstream thereof (Heme binding motif) and the threonine residue in the I-helix. In the “cytochrome P450 belonging to CYP107T” in the present invention, the consensus sequence site is preferably conserved.

  Therefore, in the present invention, “cytochrome P450 belonging to CYP107T” is an amino acid in which at least one amino acid is deleted, inserted, added and / or substituted in the amino acid sequence represented by SEQ ID NO: 1 in addition to the above-described example. A sequence comprising 0 to several amino acid deletions and insertions compared to SEQ ID NO: 1 in the regions corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1, A cytochrome P450 comprising an amino acid sequence comprising additions and / or substitutions; and an amino acid sequence wherein at least one amino acid has been deleted, inserted, added and / or substituted in the amino acid sequence shown in SEQ ID NO: 1. In regions corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1 Respectively, deletion of 0 to several amino acids as compared to SEQ ID NO: 1, insertion, addition, and / or include cytochrome P450 comprising the amino acid sequence comprising a substitution. The number of “at least one amino acid” to be deleted, inserted, added and / or substituted in the amino acid sequence shown in SEQ ID NO: 1 is the number of amino acid sequences containing the deleted, inserted, added and / or substituted. As long as it codes cytochrome P450 belonging to CYP107T, it is not particularly limited.

  Furthermore, as an example of “cytochrome P450 belonging to CYP107T” in the present invention, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 1. , 85% or more, 90% or more, or 95% or more of amino acid sequences each having a sequence identity corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1. And cytochrome P450 comprising an amino acid sequence comprising deletion, insertion, addition and / or substitution of 0 to several amino acids compared to SEQ ID NO: 1. In addition, as a further example of “cytochrome P450 belonging to CYP107T” in the present invention, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% with respect to the amino acid sequence shown in SEQ ID NO: 1. An amino acid sequence having a sequence identity of 85% or more, 90% or more, or 95% or more in the region corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1. Examples include cytochrome P450 consisting of an amino acid sequence that includes deletion, insertion, addition, and / or substitution of 0 to several amino acids compared to SEQ ID NO: 1, respectively.

  Positions 231-235, 270-273, and 335-344 in SEQ ID NO: 1 correspond to the consensus sequence sites in the I-helix, K-helix, and heme binding sites, respectively. The number of amino acid deletions, insertions, additions, and / or substitutions contained in the “region corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1” is from 0 to a number. For example, 0, 1, 2, 3, 4, 5, 6, or 7, preferably 0. For example, the number of amino acid deletions, insertions, additions and / or substitutions contained in the region corresponding to positions 231-235 of SEQ ID NO: 1 is 0, 1, 2, 3, or 4, preferably 0. For example, the number of amino acid deletions, insertions, additions, and / or substitutions contained in the region corresponding to positions 270-273 of SEQ ID NO: 1 is 0, 1, 2, or 3, preferably 0 It is. For example, the number of amino acid deletions, insertions, additions, and / or substitutions contained in the region corresponding to positions 335-344 of SEQ ID NO: 1 is 0, 1, 2, 3, 4, 5, 6, or 7. The number is preferably 0. More preferably, when “cytochrome P450 belonging to CYP107T” in the present invention includes an amino acid sequence in which at least one amino acid is deleted, inserted, added, and / or substituted in the amino acid sequence represented by SEQ ID NO: 1, or 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the amino acid sequence shown in SEQ ID NO: 1 In the region corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1, and amino acid deletion, insertion, addition, and / or Or a region other than the region corresponding to positions 231-235, 270-273, and 335-344 of SEQ ID NO: 1 without substitution. To deletions of amino acids, insertion, addition, and / or substitutions.

  When the region corresponding to positions 231-235 of SEQ ID NO: 1 contains amino acid deletions, insertions, additions, and / or substitutions, the threonine residue (T) at position 235 of SEQ ID NO: 1 is maintained. preferable. When the region corresponding to positions 270-273 of SEQ ID NO: 1 contains amino acid deletions, insertions, additions, and / or substitutions, the glutamic acid residue (E) at position 270 and the arginine residue at position 273 of SEQ ID NO: 1 (R) is preferably maintained. When the region corresponding to positions 335-344 of SEQ ID NO: 1 includes amino acid deletions, insertions, additions, and / or substitutions, the glycine residues (G) at positions 336 and 338 of SEQ ID NO: 1 and positions 342 It is preferred that the cysteine residue (C) is maintained.

  In the present invention, the amino acid substitution may be a conservative amino acid substitution with an amino acid having a side chain of similar properties.

  The nucleotide sequence of “a gene encoding cytochrome P450 belonging to CYP107T” may be determined based on the amino acid sequence of “cytochrome P450 belonging to CYP107T”. For example, but not limited to, a nucleotide sequence comprising SEQ ID NO: 4; 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more with respect to the nucleotide acid sequence shown in SEQ ID NO: 4 % Nucleotide sequence comprising a sequence having a sequence identity of greater than or equal to 85%, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95%; a nucleotide sequence consisting of SEQ ID NO: 4; Examples include nucleotide sequences having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity.

  The type of microorganism used in the present invention is not particularly limited as long as it is a microorganism having a cytochrome P450 redox system. For example, actinomycetes, bacilli, eubacteria, rhizobia, bacteria and the like can be mentioned. Preferably, the genus Streptomyces, Nocardia, Rhodococcus, Saccharopolyspora, Micromonospora, Pseudomonas, Bradyrizobium, Mycobacterium, Bacillus, Rhizobium and the like, more preferably Streptomyces coelicolor, Streptomyces lividans, Streptomyces violaceort, Streptomyces cerevisiae striatums grieus), Streptomyces venezuelae ( Streptomyces venezuelae), Streptomyces cinnamoneus, Streptomyces albus, Streptomyces albus, Streptomyces alestomyce Kas (Streptomyces hygroscopicus), Streptomyces antibiotics, Streptomyces erythraea, Streptomyces erthraea Streptomyces fradiae, Rhodococcus fascians, Psudomonas putida, Psudomonas incogita, um, and B.

  In the present invention, the microorganism containing a gene encoding cytochrome P450 belonging to CYP107T may be a microorganism containing a gene encoding cytochrome P450 belonging to endogenous CYP107T, or encodes cytochrome P450 belonging to exogenous CYP107T. Or a microorganism containing both endogenous and exogenous genes. In the present invention, a microorganism overexpressing a gene encoding cytochrome P450 belonging to CYP107T (hereinafter sometimes referred to as “overexpression strain”) is preferably used. Here, overexpression means that a gene encoding cytochrome P450 belonging to CYP107T is contained more than the wild type, or that the expression level of cytochrome P450 belonging to CYP107T is high. Such an overexpressing strain may be generated spontaneously or may be artificially created. For example, a microorganism containing a gene encoding cytochrome P450 belonging to the exogenous CYP107T or a microorganism containing both the exogenous and endogenous genes, or the above-described artificial overexpression strain may be, for example, a cytochrome P450 belonging to CYP107T. It can be created by introducing a gene coding for. The gene encoding cytochrome P450 belonging to CYP107T can be prepared by a conventional method such as PCR or hybridization using a primer or probe designed based on known base sequence information.

  The gene encoding cytochrome P450 belonging to CYP107T can be introduced by a conventional method. For example, a gene encoding cytochrome P450 belonging to the CYP107T is inserted into an appropriate vector, and a microorganism is transformed with the vector. The vector is appropriately selected from plasmid vectors, phage vectors, shuttle vectors, and the like. The vector may contain an appropriate selection marker such as a suitable regulatory factor such as a promoter, terminator, operator or enhancer, a thiostrepton resistance gene, or an ampicillin resistance gene. Transformation may be performed by a method usually used in the art. Therefore, the present invention also provides a vector containing a gene encoding cytochrome P450 belonging to the CYP107T.

  The present invention also provides a method for producing a microorganism containing a gene encoding cytochrome P450 belonging to CYP107T, which comprises introducing a gene encoding cytochrome P450 belonging to CYP107T into the microorganism. For example, there is provided a method for producing a microorganism overexpressing a gene encoding cytochrome P450 belonging to CYP107T, which comprises introducing a gene encoding cytochrome P450 belonging to CYP107T into the microorganism.

  In the present invention, a nootkatone is produced by culturing a microorganism containing a gene encoding cytochrome P450 belonging to CYP107T in the presence of valencene (hereinafter sometimes referred to as “main culture”). In some cases, nootkatol is produced as a precursor of nootkatone. “In the presence of valencene” in the main culture may be achieved by adding valencene to the culture medium, or a microorganism containing a gene encoding cytochrome P450 belonging to CYP107T and producing valencene. Alternatively, it may be achieved by using a microorganism into which a gene has been introduced. The medium for the main culture is not particularly limited as long as it contains a nutrient necessary for the growth of the microorganism to be used. Preferably a liquid medium is used. The medium contains components usually used for culturing microorganisms, a carbon source, a nitrogen source, and valencene, and may further contain minerals, vitamins, and the like as necessary. As the medium, for example, a commercially available medium such as TSB medium (tryptic soy broth medium) added with a carbon source, a nitrogen source, and valencene can be used. As the carbon source, saccharides or sugar alcohols that can be assimilated by the microorganism used, for example, glucose, xylose, arabinose, starch, glycerol, and the like can be used. As the nitrogen source, inorganic or organic ammonium salts, tryptone, peptone, gravy extract, yeast extract, whey, casein hydrolyzate, soybean powder, fish meal and the like can be used. The amount of the carbon source and nitrogen source or the ratio of the carbon source and nitrogen source contained in the medium is not particularly limited and can be determined as appropriate. The amount of valencene contained in the medium is also not particularly limited. The amount of valencene added to the medium can be appropriately determined according to the desired production amount of Nootkaton, the culture environment, and the like. For example, the amount is such that the concentration of valencene in the medium is 0.0001 to 5% (w / v), preferably 0.01 to 0.5% (w / v). When a microorganism into which a gene has been introduced so as to produce valencene is used as the microorganism, it is not necessary to add valencene to the medium. When a microorganism artificially introduced with a gene encoding cytochrome P450 belonging to CYP107T is used as the microorganism, an antibiotic for selection of a transformant (for example, thiostrepton, ampicillin, etc.) is further added to the main culture medium. Etc.) may be added.

  The culture conditions of the main culture, for example, the number of microorganisms at the start of culture, the culture time, the culture temperature, the pH of the medium, etc. are not particularly limited as long as the microorganisms grow well and produce Nootkaton, and the microorganisms used It can be determined as appropriate according to the type, the production amount of the desired Nootkaton, or the culture environment. The main culture is performed, for example, at 20 to 35 ° C., preferably at 28 to 30 ° C., for example, at pH 5 to 9, preferably at pH 6 to 8, for example, 3 days or more, preferably 5 to 20 days.

  A microorganism containing a gene encoding cytochrome P450 belonging to CYP107T may be cultured in advance before the main culture to increase the number of cells (hereinafter, the culture before the main culture is referred to as “seed culture”). May be noted). In inoculum culture, a medium usually used as a growth medium for microorganisms is used, and valencene is not added to the medium. When a microorganism artificially introduced with a gene encoding cytochrome P450 belonging to CYP107T is used as the microorganism, an antibiotic for selecting a transformant (eg, thiostrepton, ampicillin, etc.) in the inoculum culture medium Etc. may be added. The culture conditions such as culture time and culture temperature are not particularly limited as long as the above-mentioned microorganisms grow well, and can be appropriately determined. Inoculum culture is performed, for example, at 28-30 ° C., pH 6-8, for 2-5 days.

  After the main culture, the culture solution is separated into cells and culture supernatant, and nootkatone (or a mixture of nootkaton and nootkatol) is recovered from the cells and / or culture supernatant. Extraction and purification of nootkatone from the microbial cells and culture supernatant may be performed according to conventional methods. For example, the culture solution can be separated into cells and culture supernatant by centrifugation, and the cells or culture supernatant can be treated with an organic solvent such as methanol and hexane to extract nootkatone. Furthermore, the said extract can be refine | purified using well-known means, such as a chromatography, and a nootkatone with high purity can be obtained.

  EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Screening of Nootkaton Production Candidate Gene Group (1) Gene Cloning Based on the genomic information (Genbank No. NC_003888) of Streptomyces coelicolor A (3) 2, SCO5223, SCO3099, SCO3770, And primers for amplifying the SCO3636 gene region were designed (Table 1). The SCO5223, SCO3099, SCO3770, and SCO3636 gene regions were found to encode cytochrome P450 belonging to CYP170A, CYP107U, CYP107T, and CYP107P, respectively. A Streptomyces coelicolor genome was used as a template, the designed primers were used as a forward primer and a reverse primer, and a PCR reaction was performed using a KOD-Plus-Neo ^{(registered trademark)} kit manufactured by Toyobo Co., Ltd. to amplify a target gene fragment. The PCR reaction was performed under the condition of 30 cycles of “94 ° C. for 2 minutes” after 1 cycle and “98 ° C. for 10 seconds, 68 ° C. for 1 minute 30 seconds”. The sequences of the genes corresponding to SCO5223, SCO3099, SCO3770, and SCO3636 obtained by such amplification correspond to SEQ ID NOs: 2, 3, 4, and 20, respectively.

  In order to overexpress the target protein, a Streptomyces septatus-derived metalloendoprotease (SSMP) promoter (SEQ ID NO: 11) (see JP 2009-65837 A) was used. The amplified fragment together with this promoter was ligated to a vector having pIJ101 as a replication origin to obtain a target gene overexpression vector. The structure of the vector is shown in FIG.

(2) Transformation of actinomycetes Streptomyces lividans TK64 strain (National Institute of Technology and Evaluation, Biotechnology Headquarters, Biogenetic Resources Division (NBRC): NBRC No. 15678) transformed with a gene overexpression vector did. The transformation of actinomycetes is described in “Genetic Manipulation of Streptomyces” (Hopwood, DA, et al., 1985, Genetic Manipulation of Streptomyces: a Laboratory of Streptomyces: The procedure was as described. The obtained transformants were designated as SCO5223, SCO3099, SCO3770, and SCO3636 overexpressing strains.

(3) Culture method SCO5233, SCO3099, SCO3770, and SCO3636 overexpressing strains prepared as described above are first used as seed cultures, TSB medium (Bacto ^(trademark) Tryptic manufactured by Becton Dickinson) containing thiostrepton (50 μg / mL). Soy Broth (Soybean-Casein Digest Medium), product number: 21825) was cultured with shaking in a test tube at 28 ° C. for 72 hours in 5 mL. Then, as the main culture, put 50 mL of SSMP medium (Table 2) containing thiostrepton (10 μg / mL) into a 500 mL baffle flask, inoculate 3% (1.5 mL) of the inoculum culture solution, and shake at 28 ° C. Cultured at last. 72 hours after the start of culture, a glucose solution was added to a final concentration of 10%, and valencene (manufactured by SIGMA-ALDRICH, product number: 75056-10G-F) was added to a final concentration of 0.025%. Cultured with shaking.

pH = 7.0

(4) Detection method of Nootkaton After completion of the culture, 2 mL of the bacterial cell culture solution was recovered and then separated by centrifugation (14,000 rpm, 20 minutes) to recover the bacterial cells. 1.5 mL methanol was added and mixed by shaking. To 1 mL of the supernatant, 0.25 mL distilled water and 0.5 mL hexane were added and mixed by shaking. The hexane layer was recovered by centrifugation. Gas chromatograph GC-2010 and mass spectrometer manufactured by Shimadzu Corporation using InertCap 5 (inner diameter: 0.25 mm, length: 30 m, film thickness: 0.40 μm) of GL Sciences Co., Ltd. were used as the Nootkaton in the obtained hexane solution. Analysis was performed using GCMS-QP2010Plus (Table 3).

  From the results of gas chromatography / mass spectrometry analysis, it was not possible to confirm the production of nootkatone in the SCO5223, SCO3099, and SCO3636 overexpressing strains. On the other hand, the sample of the SCO3770 overexpressing strain contains a peak eluted by gas chromatography at the same retention time as that of the Nootkaton standard (manufactured by Tokyo Chemical Industry Co., Ltd., product number: N0920). As shown in FIG. 2, nootkatone production was confirmed in the SCO3770 overexpressing strain.

Example 2: Detailed analysis of SCO3770 function (1) Preparation of SCO3770 gene disruption strain Primers for amplifying the 97th to 1090th regions of the SCO3770 gene (SEQ ID NO: 4) were designed (Table 4). A Streptomyces coelicolor genome was used as a template, the designed primers were used as a forward primer and a reverse primer, and a PCR reaction was performed using PrimeSTAR ^{(registered trademark)} GXL DNA Polymerase kit manufactured by Takara Bio Inc. to amplify the target gene fragment. The PCR reaction was carried out under the conditions of “cycle at 98 ° C. for 5 minutes” after 1 cycle and “98 ° C. for 30 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute” for 30 cycles. The sequence obtained by such amplification (hereinafter sometimes referred to as “ΔSCO3770 sequence”) is shown in SEQ ID NO: 12.

  In order to delete the gene on the Streptomyces lividans TK64 genome corresponding to the SCO3770 gene, the amplified fragment was used as a replication origin from pGM160 (Muth et.al, 1989, Mol. Gen. Genet., 219, 341-348). The SCO3770 gene disruption vector was ligated to the vector. The structure of the vector is shown in FIG. The sequence of the melC operon region in the vector is shown in SEQ ID NO: 15.

  Streptomyces lividans strain TK64 was transformed with the SCO3770 gene disruption vector. As a result, the SCO3770 disruption strain (hereinafter referred to as the SCO3770 disruption strain in which the SCO3770 gene disruption sequence (ΔSCO3770 sequence) was inserted into the gene corresponding to the SCO3770 gene on the genome by homologous recombination and the function of the gene corresponding to the SCO3770 gene was destroyed. In some cases, “ΔSCO3770 strain” is also obtained.

(2) Construction of SCO3770 gene reversion strain In order to confirm the function of the SCO3770 gene, the SCO3770 gene-disrupted strain described in Example 2 (1) was transformed with the SCO3770 gene overexpression vector described in Example 1 (1). Converted. The obtained transformant was designated as an SCO3770 overexpression (ΔSCO3770) strain.

(3) Production measurement of Nootkaton Four types of strains (Streptomyces lividans TK64 strain, SCO3770 overexpressing strain, SCO3770-destroying strain, SCO3770 overexpressing (ΔSCO3770) strain) were prepared by the procedure described in Example 1 (3). After culturing, 2 mL of the bacterial cell culture solution was collected and then separated by centrifugation (14,000 rpm, 20 minutes), and 1 mL of the supernatant and the bacterial cells were separately collected.
The collected supernatant was mixed with shaking after adding 1/2 volume of hexane. The hexane layer was recovered by centrifugation (14,000 rpm, 1 minute). On the other hand, the collected microbial cells were mixed by shaking with addition of 1.5 mL methanol. To 1 mL of the supernatant, 0.25 mL distilled water and 0.5 mL hexane were added and mixed by shaking. The hexane layer was recovered by centrifugation. The amount of Nootkaton in the hexane solution obtained from the supernatant and the bacterial cells was measured using a gas chromatograph manufactured by Shimadzu Corporation using InertCap 5 (inner diameter 0.25 mm, length 30 m, film thickness 0.40 μm) from GL Sciences. Analysis was performed using a graph GC-2014 (Table 5). Under this condition, the Noot Katon is eluted around 12 minutes. Moreover, the amount of Nootkaton is calculated from a standard curve of β-caryophyllene standard product in the chromatogram.

  When the amount of nootkatone in the culture supernatant and the bacterial cells was measured, Streptomyces lividans TK64 strain (former strain) did not produce nootkatone, whereas SCO3770 overexpressing strains confirmed the production of nootkatone ( FIG. 4, FIG. 5, Table 6). On the other hand, Noutkaton was not produced in the ΔSCO3770 strain. In the SCO3770 overexpression (ΔSCO3770) strain in which the SCO3770 gene was overexpressed with respect to this strain, production of nootkatone could be confirmed. From these results, it was revealed that the SCO3770 gene product was converted from Valensen to Nootkaton.

Concentration (mg / L) in terms of 1 mL of culture solution. Average of 3 samples.

Example 3 Screening of Nootkaton Production Candidate Genes (1) Gene Cloning Streptomyces albus (ATCC 21838 strain (National Institute of Technology and Evaluation, Biotechnology Headquarters, Biogenetic Resources Division (NBRC)): NBRC Based on the sequence information of SLNWT — 3333, which is a sequence derived from No. 107858), an artificial gene was created using an artificial gene synthesis technique (SEQ ID NO: 16), and SLNWT — 3333 has a sequence homology with SCO3770 (SEQ ID NO: 1) at the amino acid level. 69%, and has one amino acid substitution in the region corresponding to positions 335 to 344 in SEQ ID NO: 1 compared to SEQ ID NO: 1, and positions 231 to 235 and 270 to 273 in SEQ ID NO: 1 are conserved. Has been In addition, SLNWT — 3333 maintains the glycine residues at positions 336 and 338 of SEQ ID NO: 1 and the cysteine residue at position 342.

  In order to overexpress the protein of interest, the Streptomyces avermitilis-derived xylose isomerase (xylA) promoter (SEQ ID NO: 17) (disclosed in J. Microbiol. Biotechnol. 2008 May; 18 (5): 837-44) is used. did. The artificial gene together with this promoter was linked to a vector having pIJ101 as a replication origin to obtain an overexpression vector of the target gene. The structure of the vector is shown in FIG.

(2) Transformation of Actinomycetes Streptomyces lividans TK64 strain was transformed with a gene overexpression vector in the same manner as in Example 1- (2). The obtained transformant was designated as SLNWT — 3333 overexpressing strain.

(3) Culture method The SLNWT — 3333 overexpression strain prepared as described above was cultured in the same manner as in Example 1- (3). However, the culture time after addition of valencene was 432 hours.

(4) Measurement of Noutkaton Production After completion of culture, the amount of SLNWT — 3333 overexpressing strain in the culture broth and the amount of nootkatone in the cells were measured in the same manner as in Example 2- (3). (FIG. 6, FIG. 7, Table 7).

  ADVANTAGE OF THE INVENTION According to this invention, the method of manufacturing a nootkaton more simply and cheaply using microorganisms is provided. The noot katon produced by the method of the present invention can be used as a natural fragrance in a wide range of fields including foods and cosmetics.

Claims (10)

  A method for producing a nootkaton, comprising culturing a microorganism containing a gene encoding cytochrome P450 belonging to CYP107T in the presence of valencene.   An amino acid sequence in which cytochrome P450 belonging to CYP107T includes the amino acid sequence represented by SEQ ID NO: 1, or at least one amino acid is deleted, inserted, added, and / or substituted in the amino acid sequence represented by SEQ ID NO: 1. In the region corresponding to positions 231-235, 270-273, and 335-344 in SEQ ID NO: 1, 0 to several amino acid deletions, insertions, additions compared to SEQ ID NO: 1, respectively. And / or an amino acid sequence comprising a substitution.   The method according to claim 2, wherein the cytochrome P450 belonging to CYP107T comprises the amino acid sequence represented by SEQ ID NO: 1.   The method according to any one of claims 1 to 3, wherein the microorganism is actinomycete.   The method according to claim 4, wherein the microorganism is Streptomyces actinomycetes.   A microorganism into which a gene encoding cytochrome P450 belonging to CYP107T has been introduced.   An amino acid sequence in which cytochrome P450 belonging to CYP107T includes the amino acid sequence represented by SEQ ID NO: 1, or at least one amino acid is deleted, inserted, added, and / or substituted in the amino acid sequence represented by SEQ ID NO: 1. In the region corresponding to positions 231-235, 270-273, and 335-344 in SEQ ID NO: 1, 0 to several amino acid deletions, insertions, additions compared to SEQ ID NO: 1, respectively. And / or an amino acid sequence comprising a substitution.   The microorganism according to claim 7, wherein the cytochrome P450 belonging to CYP107T comprises the amino acid sequence represented by SEQ ID NO: 1.   The microorganism according to any one of claims 6 to 8, wherein the microorganism is actinomycetes.   The microorganism according to claim 9, wherein the microorganism is an actinomycete belonging to the genus Streptomyces.