JP5916564B2 - Sesquiterpene synthase gene and method for producing sesquiterpene using the gene - Google Patents

Description

The present invention relates to a protein that synthesizes β-caryophyllene, germacrene D, and β-eudesmol, which are types of physiologically active sesquiterpenes useful for humans, from farnesyl diphosphate, a gene encoding the protein, and the gene The present invention relates to a method for producing a used sesquiterpene.
Furthermore, the present invention relates to β-cubeben, β-elemene, α-neocloben and α-humulene, and / or linalool and nerolidol, which are a kind of physiologically active (sesqui) terpenes useful for humans. The present invention relates to a protein synthesized from an acid (only linalool geranyl diphosphate), a gene encoding the protein, and a sesquiterpene (monoterpene only linalool) using the gene.

Isoprenoids (also called terpenoids) are the most diverse group of compounds in nature, with over 23,000 species. And isoprenoids contain more than 3,000 sesquiterpenes. The isoprenoid contains many industrially useful products such as pharmaceuticals, agricultural chemicals, functional foods, fragrances, and their raw materials.
However, isoprenoids, especially sesquiterpenes, are rarely accumulated in nature except for some examples, and many of them require enormous costs and labor to prepare a large amount of a single product. Therefore, development research for mass production by biotechnology using genetically modified microorganisms or plants has been actively conducted.

Escherichia coli is a microorganism with the most complete technology, materials, and genetic information for gene recombination. Therefore, in recent years, research on technology development for mass production of isoprenoids using recombinant Escherichia coli has been active.
Escherichia coli does not have a mevalonate pathway and is also called the MEP pathway because it passes through the non-mevalonate pathway [2-C-methyl-D-erythritol 4-phosphate (hereinafter sometimes referred to as MEP)] Produces the first isoprenoid substrate, isopentenyl diphosphate (also called isopentenyl pyrophosphate; hereinafter sometimes referred to as IPP).
IPP is converted to dimethylallyl diphosphate (hereinafter sometimes referred to as DMAPP) by IPP isomerase (hereinafter sometimes referred to as Idi), and DMAPP is farnesyl diphosphate (hereinafter referred to as FPP). Is converted into FPP having 15 carbon atoms and geranyl diphosphate having 10 carbon atoms (hereinafter sometimes referred to as GPP) by condensing with IPP sequentially by a synthetic enzyme (synthase). A monoterpene, which is a volatile component, is branched from GPP. In addition, sesquiterpenes and triterpenes are made by branching from the FPP. FPP is further condensed with IPP by geranylgeranyl diphosphate (hereinafter sometimes referred to as GGPP) synthase to synthesize GGPP having 20 carbon atoms. Branching from this GGPP, diterpenes and carotenoids (tetraterpenes) are synthesized.

E. coli does not synthesize the above terpenes, so in order to synthesize these isoprenoids in E. coli, it is necessary to introduce and express the biosynthetic enzyme gene (s) responsible for synthesis from FPP to the isoprenoid into E. coli. .
Many plants belonging to the family Araliaceae and Zingiberaceae are edible / medicinal plants. For example, the family Araceae includes Panax ginseng , Japanese cane (A canthopanax sieboldianus ), Kosia bra ( Acanthopanax sciadophylloides Franch. Et Sav.), Arachon ( Aralia elata ), Udo (Aralia cordata), etc. is, ginger (ginger; Zingiber officinale Roscoe) Hana ginger [flower ginger; Shampoo ginger; Zingiber zerumbet Smith] , Zingiber mioga (Zingiber mioga), purple turmeric (zedoary; curcuma zedoaria Roscoe), turmeric (autumn turmeric; curcuma longa L.) Etc. are included.
Ginger (Zingiber officinale ), a representative crop of Zingiberaceae , is a perennial plant native to tropical Asia and is widely used as a food and herbal medicine. From ginger, as sesquiterpene synthase, germacrene D synthase gene ( ZoGeD ), β-bisabolene synthase gene ( ZoTps1 ), and γ-amorphene synthase (γ-amorphene synthase) synthase) gene ( ZoTps5 ) was isolated and analyzed for its structure and function (see Patent Document 1, Non-Patent Documents 1 and 2).
Also, from the same ginger plant, Zingiber zerumbet , α-humulene synthase gene ( ZzZSS1 ) and β-eudesmol synthase (β-eudesmol) synthase) gene ( ZzZSS2 ) was isolated and analyzed for its structure and function (see Non-Patent Documents 3 and 4). In ginger family plants, sesquiterpene synthase (and its gene) other than these were not known. Note that sesquiterpene synthase (and its gene) was not known at all in the Argiaceae plant.

Although Escherichia coli does not have a mevalonate pathway, studies have been carried out to introduce and express genes of enzymes of the mevalonate pathway (mevalonate pathway genes) into Escherichia coli. Suganuma et al. Have found mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, which are enzymes after D-mevalonic acid (D-mevalonate) in the mevalonate pathway. decarboxylase), and a group of genes encoding type 2 IPP isomerase (IPP isomerase) [from Streptomyces genus CL190 strain; Non-Patent Document 5; Accession no AB037666; hereinafter, simply referred to as “mevalonate pathway gene group” Together with a group of carotenoid biosynthetic genes derived from Pantoea ananatis [ crtE , crtB , crtI , crtY , crtZ ; biosynthetic genes necessary to make zeaxanthin from FPP] It was introduced into E. coli and expressed (see Non-Patent Document 6).

The recombinant Escherichia coli is efficiently cultured by adding D-mevalonate lactone (D-mevalonate lactone; hereinafter referred to as D-mevalonolactone, mevalonolactone or MVL) as a substrate to the medium, and cultivating it efficiently. It was able to synthesize (see Non-Patent Document 6). When the mevalonate pathway gene group is introduced into E. coli and expressed together with the α-humulene synthase gene (α-humulene synthase; ZzZSS1 ), the recombinant E. coli substrate contains 0.5 mg / mL of MVL as a substrate. In addition, it was shown that 1 mg of α-humulene was produced per mL by culturing (see Non-Patent Document 7).
In addition to mevalonate pathway genes and α- humulene synthase gene (ZzZSS1), Saccharomyces cerevisiae (Saccharomyces cerevisiae) 1 type of IPP isomerase from the (IPP isomerase) gene (Scidi) was introduced into E. coli When expressed, it was shown that the amount of α-humulene produced was slightly increased as compared to the case without the Scidi gene (see Non-Patent Document 7).
In addition, even if the ZzZSS1 gene was introduced into E. coli and expressed without introducing the mevalonate pathway gene group, the production of α-humulene was not detected at all (see Non-Patent Document 7). Alternatively, when the ginger β-eudesmol synthase gene ( ZzZSS2 ) is introduced and expressed together with the mevalonate pathway gene group, the recombinant E. coli is cultured by adding MVL as a substrate to the medium, and 100 μg per mL It was shown to produce β-eudesmol (Reference: Non-Patent Document 4). Therefore, by introducing the mevalonate pathway gene group (sometimes also including the Scidi gene) into E. coli and expressing it, and adding the substrate of the mevalonate pathway gene group such as MVL to the medium and culturing it, as a result It was shown that the amount of FPP produced in the recombinant E. coli was increased to a large amount.

Therefore, sesquiterpene synthase (sesquiterpene synthase; sesquiterpene cyclase) gene using FPP as a substrate, or carotenoid biosynthesis genes starting from crtE (GGPP synthase gene) are introduced and expressed at the same time. It was revealed that sesquiterpenes or carotenoids can be efficiently produced (see: Non-Patent Documents 7 and 8). Furthermore, when this system is used, it is possible to analyze the function of a newly obtained sesquiterpene synthase gene sequence whose function is unknown (see Non-patent Document 8).
That is, it is possible to examine whether the recombinant E. coli introduced to express this gene sequence is producing sesquiterpenes (Escherichia coli originally does not produce sesquiterpenes). When sesquiterpenes are produced, by analyzing the chemical structure of the sesquiterpenes, the sesquiterpene synthase gene sequence is a gene that encodes an enzyme that synthesizes a sesquiterpene product having the chemical structure from FPP. It is because it is identified. More than 7,000 sesquiterpenes have already been isolated from plants and microorganisms (mainly plants), but many sesquiterpene synthase genes are still unknown.
Based on the above, it is expected that functional analysis of the gene will proceed with this system in the future (see Non-Patent Document 8).

JP2011-125272

S. Picard, M. E. Olsson, M. Brodelius, P. E. Brodelius, Arch. Biochem. Biophys. 452: 17-28, 2006 M. Fujisawa, H. Harada, H. Kenmoku, S. Mizutani, N. Misawa, Planta, 232: 121-130; Erratum, 232: 131, 2010 F. Yu, S. Okamoto, K. Nakasone, K. Adachi, S. Matsuda, H. Harada, N. Misawa, R. Utsumi, Planta 227: 1291-1299, 2008 F. Yu, H. Harada, K. Yamasaki, S. Okamoto, S. Hirase, Y. Tanaka, N. Misawa, R. Utsumi, FEBS Lett 582: 565-572, 2008 M. Takagi, T. Kuzuyama, S. Takahashi, H. Seto, J. Bacteriol., 182: 4153-4157, 2000 K. Kakinuma, Y. Dekishima, Y. Matsushima, T. Eguchi, N. Misawa, M. Takagi, T. Kuzuyama, H. Seto, J. Am. Chem. Soc., 123: 1238-1239, 2001 H. Harada, F. Yu, S. Okamoto, T. Kuzuyama, R. Utsumi, N. Misawa, Appl. Microbiol. Biotechnol. 81: 915-925, 2009 H. Harada, N. Misawa, Appl. Microbiol. Biotechnol. 84: 1021-1031, 2009

  An object of the present invention is to obtain a novel enzyme gene necessary for the synthesis of a sesquiterpene from FPP and to provide a method for producing a sesquiterpene using the obtained novel gene.

In order to solve the above-mentioned problems, the present inventors have conducted earnest research as follows.
(I) In order to make a large amount of FPP which is a substrate of sesquiterpene from MVL, a mevalonate pathway gene group isolated from Streptomyces genus CL190 strain (Non-patent Document 5; Accession no AB037666), Plasmid pAC-Mev for expressing genes encoding mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), diphosphomevalonate decarboxylase (DPMVA decarboxylase), and IPP isomerase (IPP isomerase; type 2), and In addition to these genes, the plasmid pAC-Mev / Scidi for expressing the gene ( Scidi ) encoding IPP isomerase (IPP isomerase; type 1) isolated from Saccharomyces cerevisiae {all E. coli vector pACYC184 was used; chloramphenicol (Cm) resistance; . These plasmids were used to transform Escherichia coli, and recombinant Escherichia coli that grew on an agar medium containing Cm was obtained.
(Ii) Synthesize cDNA from total RNA extracted from Kosiabra sprout or purple turmeric rhizome, and then insert several full-length (sesqui) terpene synthase gene sequences into E. coli expression vectors Thus, plasmids (the plasmid names of one example were pET-AsTps1, pET-AsTps2a, pET-AsTps2b, pET-CzTps1a, and pET-CzTps1b; all were ampicillin (Ap) resistant) were prepared.
Each of the above plasmids is introduced into the recombinant E. coli having the plasmid prepared in (i), cultured in a medium containing MVL, Ap and Cm, and the sesquiterpene produced is analyzed to convert FPP to β -Β-caryophyllene synthase (synthase) gene for synthesizing caryophyllene, germline D synthase (synthase) gene for synthesizing germane Clen D by converting FPP, and β-eudemol synthesizing β-eudesmol by converting FPP A small synthase gene was newly found.
(Iii) Escherichia coli expression plasmids (βET-AsTps1, pET-AsTps2a, or pET-AsTps2b, respectively) of the β-caryophyllene synthase gene, the gelmacrene D synthase gene, and the β-eudesmol synthase gene obtained in (ii) PET-CzTps1a or pET-CzTps1b) is introduced into the recombinant Escherichia coli having the plasmid prepared in (i) and cultured in a medium containing MVL, Ap and Cm. It was possible to provide a method for producing caryophyllene, germacrene D, and / or β-eudesmol.
(Iv) Synthesis of cDNA from total RNA extracted from shoots of wolfberry, cypress, uddo, or kosiabura (all belonging to the family Asteraceae) and further isolated by PCR for several full-length (sesquif) ) Insert terpene synthase gene sequence into E. coli expression vector, plasmid {example of plasmid name is pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c, pET-AsiTps1d, pET-AeTps1a, pET-AeTps1b, pET- AcTPS1a, pET-AcTPS1b, pET-AcTPS1c, pET-AcTPS1d, pET-AcTPS2, and pET-AscTps3; all were ampicillin (Ap) resistant}.
Each of the above plasmids is introduced into the recombinant E. coli having the plasmid prepared in (i), cultured in a medium containing MVL, Ap and Cm, and the generated (sesqui) terpene is analyzed to convert FPP. Β-caryophyllene synthase (synthase) gene, which is different from (ii), β-cubeben synthase (synthase) gene, which converts FPP to synthesize β-cubeben β-elemene synthase (synthase) gene that synthesizes β-elemene, α-neocloben / α-humulene synthase (synthase) gene that synthesizes α-neocloben and α-humulene by converting FPP, and GPP and FPP A novel linalool / nerolidol synthase (synthase) gene that synthesizes linalool and nerolidol, respectively, was found.
(V) Escherichia coli of β-caryophyllene synthase gene, β-cubeben synthase gene, β-elemene synthase gene, α-neocloben / α-humulene synthase gene, linalool / nerolidol synthase gene obtained in (iv) Plasmids for expression (pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c or pET-AsiTps1d, pET-AeTps1a or pET-AeTps1b, pET-AcTPS1a, pET-AcTPS1b, pET-AcTPS1TP, TP1TP PET-AscTps3) is introduced into the recombinant Escherichia coli having the plasmid prepared in (i) and cultured in a medium containing MVL, Ap and Cm, so that β-caryophyllene, β- It was possible to provide a method for producing cubene, β-elemene, α-neocloben and α-humulene, and / or linalool and nerolidol.

The present invention is as follows.
“1. The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 16 or 18;
(2) In the amino acid sequence described in SEQ ID NO: 16 or 18, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 16 or 18 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate of β-eudesmol,
(3) Farnesyl diphosphate having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 16 or 18 and substantially the same quality as the amino acid sequence shown in SEQ ID NO: 16 or 18 is converted into β-eudesmol. A gene encoding a polypeptide having an activity to convert,
(4) a gene comprising a DNA comprising the base sequence set forth in SEQ ID NO: 15 or 17,
(5) Activity of hybridizing under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 15 or 17 and converting farnesyl diphosphate into β-eudesmol A gene comprising a DNA encoding a polypeptide having
(6) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 or 17, a gene comprising a DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(7) A gene comprising 90% or more homology with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 or 17.
2. Β-eudesmol synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence set forth in SEQ ID NO: 16 or 18,
(2) In the amino acid sequence described in SEQ ID NO: 16 or 18, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 16 or 18 An amino acid sequence having the activity of converting farnesyl diphosphate of β-eudesmol,
(3) Farnesyl diphosphate having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 16 or 18 and substantially the same quality as the amino acid sequence shown in SEQ ID NO: 16 or 18 is converted into β-eudesmol. Amino acid sequence having activity to convert.
3. The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53,
(2) In the amino acid sequence of SEQ ID NO: 47, 49, 51 or 53, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 47, 49, 51 or 53 A gene encoding a polypeptide having an activity to convert farnesyl diphosphate substantially identical to the amino acid sequence described above into β-elemene,
(3) Farnesyl dinucleotide having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 and substantially identical to the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 A gene encoding a polypeptide having an activity of converting phosphoric acid into β-elemente,
(4) a gene comprising a DNA comprising the base sequence set forth in SEQ ID NO: 46, 48, 50 or 52,
(5) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 46, 48, 50 or 52, and farnesyl diphosphate is converted to β-elemente A gene comprising DNA encoding a polypeptide having an activity to convert,
(6) a gene comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 46, 48, 50 or 52, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added;
(7) A gene comprising 90% or more homology with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 46, 48, 50 or 52.
4). Β-elemene synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53,
(2) In the amino acid sequence of SEQ ID NO: 47, 49, 51 or 53, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 47, 49, 51 or 53 An amino acid sequence having the activity of converting farnesyl diphosphate substantially the same amino acid sequence as described to β-elemene,
(3) Farnesyl dinucleotide having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 and substantially identical to the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 An amino acid sequence having an activity of converting phosphoric acid into β-elemene.
5. The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6, 27, 29, 31 or 33,
(2) In the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 6, 27, 29, A gene encoding a polypeptide having an activity of converting farnesyl diphosphate substantially identical to the amino acid sequence of 31 or 33 to β-caryophyllene,
(3) 90% or more homology with the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33, and substantially the same as the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33 A gene encoding a polypeptide having the activity of converting homogeneous farnesyl diphosphate into β-caryophyllene,
(4) a gene comprising a DNA comprising the base sequence set forth in SEQ ID NO: 5, 26, 28, 30 or 32,
(5) It hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence shown in SEQ ID NO: 5, 26, 28, 30 or 32, and SEQ ID NO: 6, 27, 29 A gene comprising a DNA encoding a polypeptide having an activity of converting farnesyl diphosphate substantially identical to the amino acid sequence of 31 or 33 to β-caryophyllene,
(6) a gene comprising a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 5, 26, 28, 30 or 32, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added;
(7) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 5, 26, 28, 30 or 32 and a DNA having a homology of 90% or more.
6). Β-caryophyllene synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 6, 27, 29, 31 or 33,
(2) In the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 6, 27, 29, An amino acid sequence having an activity of converting farnesyl diphosphate substantially identical to the amino acid sequence of 31 or 33 to β-caryophyllene,
(3) 90% or more homology with the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33, and substantially the same as the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33 An amino acid sequence having the activity of converting homogeneous farnesyl diphosphate into β-caryophyllene.
7). The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or 10,
(2) In the amino acid sequence shown in SEQ ID NO: 8 or 10, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence shown in SEQ ID NO: 8 or 10. A gene encoding a polypeptide having an activity of converting farnesyl diphosphate to germacrene D,
(3) Activity of converting farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 8 or 10 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 8 or 10 into Germacrene D A gene encoding a polypeptide having
(4) a gene comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 7 or 9,
(5) Farnesyl that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence described in SEQ ID NO: 7 or 9, and is substantially the same as the amino acid sequence described in SEQ ID NO: 6 A gene comprising a DNA encoding a polypeptide having an activity of converting diphosphate into β-caryophyllene,
(6) a gene comprising a DNA comprising a nucleotide sequence of SEQ ID NO: 7 or 9, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added;
(7) A gene comprising 90% or more homology with DNA comprising the nucleotide sequence of SEQ ID NO: 7 or 9.
8). Germacrene D synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence of SEQ ID NO: 8 or 10,
(2) In the amino acid sequence shown in SEQ ID NO: 8 or 10, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence shown in SEQ ID NO: 8 or 10. An amino acid sequence having the activity of converting farnesyl diphosphate to germane D
(3) Activity of converting farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 8 or 10 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 8 or 10 into Germacrene D Amino acid sequence having
9. The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 or 41,
(2) In the amino acid sequence described in SEQ ID NO: 39 or 41, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence described in SEQ ID NO: 39 or 41 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate to β-cubeben,
(3) Convert farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 39 or 41 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 39 or 41 into β-cubeben A gene encoding a polypeptide having activity,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 38 or 40,
(5) Hybridizes with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 38 or 40 under stringent conditions and has an activity of converting farnesyl diphosphate to β-cubeben A gene comprising DNA encoding a polypeptide,
(6) a DNA comprising a nucleotide sequence set forth in SEQ ID NO: 38 or 40, a gene comprising a DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(7) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 38 or 40 and having a homology of 90% or more.
10. Β-cubeben synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 39 or 41,
(2) In the amino acid sequence described in SEQ ID NO: 39 or 41, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence described in SEQ ID NO: 39 or 41 An amino acid sequence having an activity of converting farnesyl diphosphate to β-cubeben;
(3) Convert farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 39 or 41 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 39 or 41 into β-cubeben An amino acid sequence having activity.
11 The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 55,
(2) Farnesyl diphosphate in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 55, and is substantially the same as the amino acid sequence described in SEQ ID NO: 55 A gene that encodes a polypeptide having an activity of converting to α-neocloben and α-humulene,
(3) Farnesyl diphosphate having 90% or more homology with the amino acid sequence described in SEQ ID NO: 55 and substantially the same as the amino acid sequence described in SEQ ID NO: 55 is converted into α-neocloben and α-humulene A gene encoding a polypeptide having activity,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 54,
(5) Activity of hybridizing under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 54 and converting farnesyl diphosphate into α-neocloben and α-humulene A gene comprising a DNA encoding a polypeptide having
(6) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 54, a gene comprising DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(7) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 54 and having a homology of 90% or more.
12 Α-neocloben / α-humulene synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence set forth in SEQ ID NO: 55,
(2) Farnesyl diphosphate in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 55, and is substantially the same as the amino acid sequence described in SEQ ID NO: 55 An amino acid sequence having an activity to convert saponin to α-neocloben and α-humulene,
(3) Farnesyl diphosphate having 90% or more homology with the amino acid sequence described in SEQ ID NO: 55 and substantially the same as the amino acid sequence described in SEQ ID NO: 55 is converted into α-neocloben and α-humulene An amino acid sequence having activity.
13. The gene shown in any one of the following (1) to (7);
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 62;
(2) In the amino acid sequence described in SEQ ID NO: 62, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl diphosphate is substantially the same as the amino acid sequence described in SEQ ID NO: 62 And a gene encoding a polypeptide having an activity to convert farnesyl diphosphate into linalool and nerolidol, respectively
(3) Geranyl diphosphate and farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 62 and substantially the same as the amino acid sequence described in SEQ ID NO: 62, respectively, linalool and neroli A gene encoding a polypeptide having an activity of converting into a doll,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 61,
(5) It hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 61, and geranyl diphosphate and farnesyl diphosphate are respectively treated with linalool and nerolidol. A gene comprising DNA encoding a polypeptide having an activity of converting to
(6) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 61, a gene comprising DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(7) A gene comprising 90% or more homology with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 61.
14 Linalool / nerolidol synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 62,
(2) In the amino acid sequence described in SEQ ID NO: 62, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl diphosphate is substantially the same as the amino acid sequence described in SEQ ID NO: 62 And an amino acid sequence having an activity to convert farnesyl diphosphate into linalool and nerolidol, respectively.
(3) geranyl diphosphate and farnesyl diphosphate having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 62 and substantially the same as the amino acid sequence shown in SEQ ID NO: 55, respectively, linalool and neroli An amino acid sequence having the activity of converting into a doll.
15. 14. A recombinant Escherichia coli into which the gene according to any one of the preceding items 1, 3, 5, 7, 9, 11, 13 has been introduced.
16. Recombinant Escherichia coli into which the following genes (1) to (2) are introduced in addition to the gene according to any one or more of the above items 1, 3, 5, 7, 9, 11, 13;
(1) Mevalonate pathway gene group that synthesizes from mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(2) Isopentenyl diphosphate isomerase gene.
17. The preceding paragraph, wherein the mevalonate pathway gene group for synthesizing from mevalonic acid or mevalonolactone to isopentenyl diphosphate and the isopentenyl diphosphate isomerase gene are genes derived from Streptomyces genus CL190 and / or Saccharomyces cerevisiae 16. The recombinant E. coli according to 16.
18. The recombinant E. coli according to any one of items 15 to 17 above, from a culture or a cell obtained by culturing the recombinant Escherichia coli in a medium containing mevalonic acid and / or mevalonolactone, β-caryophyllene, gelmacrene D, β-eudesmol, A method for producing a sesquiterpene, comprising obtaining β-cubeben, β-elemene, α-neocloben and α-humulene, and / or linalool and nerolidol. "

In the present invention, β-caryophyllene, germacrene D, β-eudesmol, β-cubeben, β-elemene, α-neocloben and α-humulene, and / or linalool and nerolidol, and FPP (linalool only GPP) as a substrate A protein (enzyme) to be synthesized and a gene encoding the protein could be provided.
According to the present invention, β-caryophyllene, germacrene D, β-eudesmol, β-cubene, β-elemene, α-neocloben and α-humulene, and / or linalool and nerolidol are efficiently produced in Escherichia coli and the like. I was able to.

The figure showing (a) new shoots and (b) fully developed old leaves of Cosciabra ( Acanthopanax sciadophylloides Franch. Et Sav.). The bar represents 5 cm. The figure showing the amino acid sequence alignment of AsTPS1, AsTPS2a and AsTPS2b and the known ginger plant sesquiterpene synthase (synthase). ZoGDS represents a Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. The RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquitel synthase are indicated by asterisks. The figure showing the structure of plasmid pET-AsTps1, pET-AsTps2a, and pET-AsTps2b. A cDNA corresponding to the ORF of AsTps1 (1,668 bp), a cDNA corresponding to the ORF of AsTps2a (1,668 bp), and a cDNA corresponding to the ORF of AsTps2b (1,668 bp), respectively, Nde I- Xho I of the vector pETDuet-1 Is inserted in between. The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AsTps1 containing AsTps1 and plasmid pAC-Mev / Scidi. A new peak 1 is indicated by an arrow. (b) Chromatogram (control) of an ethyl acetate extract of Escherichia coli strain carrying vector pET21a not containing AsTps1 and plasmid pAC-Mev / Scidi. (c) Mass spectrum of peak 1 (upper figure) indicated by the arrow a and standard β-caryophyllene (lower figure). The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. A chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AsTps2a containing zu AsTps2a and plasmid pAC-Mev / Scidi. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AsTps2a containing AsTps2a and plasmid pAC-Mev / Scidi. A new peak 2 is indicated by an arrow. (b) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AsTps2b containing AsTps2b and plasmid pAC-Mev / Scidi. A new peak 3 is indicated by an arrow. (c) Chromatogram (control) of an ethyl acetate extract of Escherichia coli strain carrying the vector pET21a not containing AsTps2 and the plasmid pAC-Mev / Scidi. (d) Mass spectra of peaks 2 and 3 (upper and middle) indicated by arrows of (a) and (b) and a standard product of Germacrene D (lower). The figure which shows the biosynthesis from the chemical structure and farnesyl diphosphate (FPP) of (beta) -caryophyllene, germaclene D, and (beta) -eudesmol which are sesquiterpenes which can be produced by this invention. In the figure, AsTPS2 indicates AsTPS2a or AsTPS2b, and CzTPS1 indicates CzTPS1a or CzTPS1b. The figure showing the plant body and each organ [leaf (rhizome) and root (root)] of purple turmeric (Gadju; Curcuma zedoaria Roscoe). Bar represents 5 cm. The figure showing the amino acid sequence alignment of CzTPS1a and CzTPS1b and the known ginger plant sesquiterpene synthase (synthase). ZoGDS represents Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pET-CzTps1a and pET-CzTps1b. CDNA corresponding to the ORF of CzTps1a (1,644 bp) and cDNA corresponding to ORF of CzTps1b (1,644 bp), respectively, are inserted between the vector pETDuet-1 of Nde I- Kpn I. The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-CzTps1a containing CzTps1a and plasmid pAC-Mev / Scidi. A new peak 4 is indicated by an arrow. (b) Chromatogram of ethyl acetate extract of E. coli strain carrying pET-CzTps1b and plasmid pAC-Mev / Scidi. A new peak 5 is indicated by an arrow. (c) Chromatogram (control) of dodecane extract of Escherichia coli strain carrying the vector pET21a not containing CzTps1 and the plasmid pAC-Mev. (d) Mass spectra of peaks 4 and 5 (upper and middle) indicated by arrows in (a) and (b) and β-eudesmol standard (lower). It shoots a diagram representing the old leaves; (Acanthopanax sieboldianus Araliaceae) Himeukogi of Araliaceae (Araliaceae). The bar represents 5 cm. The figure showing the amino acid sequence alignment of AsiTPS1a, AsiTPS1b, AsiTPS1c and AsiTPS1d and the known ginger plant sesquiterpene synthase (synthase). ZoGDS represents Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c, and pET-AsiTps1d. AsiTps1a, AsiTps1b, cDNA corresponding to the ORF of AsiTps1c and AsiTps1d (both 1,671 bp), respectively, are inserted between the vector pETDuet-1 of Nde I- Xho I. The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Plasmid pET-AsiTps1a, (b) Plasmid pET-AsiTps1b, (c) Plasmid pET-AsiTps1c, (d) Plasmid pET-AsiTps1d Chromatogram of the extract. New peaks 6-9 are indicated by arrows. (e) AsiTps1 do not contain the vector pET21a plasmid pAC-Mev / Scidi chromatogram of ethyl acetate extracts of E. coli strains harboring (control). (f) Mass spectra of peaks 6 to 9 (the top diagram to the second diagram from the bottom) and β-caryophyllene standard products (the bottom diagram) indicated by arrows in (a) to (d). Β-Caryophyllene, β-Cubebene, β-Elemene, α-Neoclovene, and α, which are sesquiterpenes that can be produced in the present invention The figure which shows the chemical structure and biosynthesis from farnesyl diphosphate (FPP) of -humulene (α-Humulene), and also the chemical structure of linalool (Linalool) and biosynthesis from geranyl diphosphate (GPP). In the figure, AsiTPS1 indicates AsiTPS1a, AsiTPS1b, AsiTPS1c or AsiTPS1d, AeTPS1 indicates AeTPS1a or AeTPS1b, AcTPS1 indicates AcTPS1a, AcTPS1b, AcTPS1c or AcTPS3, only TPS2 The figure showing the new bud and the old leaf of Aralia elata of Araliaceae. The bar represents 5 cm. The figure showing the amino acid sequence alignment of AeTPS1a and AeTPS1b and the known ginger plant sesquiterpene synthase (synthase). ZoGDS represents Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pET-AeTps1a and pET-AeTps1b. CDNA corresponding to the ORF of AeTps1a and AeTps1b (both 1,665 bp), respectively, are inserted between the vector pETDuet-1 of Nde I- Xho I. The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AeTps1a, (b) plasmid pET-AeTps1b and further carrying plasmid pAC-Mev / Scidi. New peaks 10 and 11 are indicated by arrows. (c) AeTps1 do not contain the vector pET21a plasmid pAC-Mev / Scidi chromatogram of ethyl acetate extracts of E. coli strains harboring (control). (d) Mass spectra of peaks 10 and 11 (upper and middle) indicated by arrows in (a) and (b) and β-cubebene (database) (β-cubebene; lower). The figure showing the new bud and the old leaf of the arboraceae (Araliaceae) Udo ( Aralia cordata ). The bar represents 5 cm. The figure showing the amino acid sequence alignment of AcTPS1a, AcTPS1b, AcTPS1c, AcTPS1d, and AcTPS2 and the known ginger plant sesquiterpene synthase (synthase). ZoGDS represents Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pET-AcTps1a, pET-AcTps1b, pET-AcTps1c, pET-AcTps1d, and pET-AcTps2. AcTps1a, AcTps1b, AcTps1c and AcTps1d, and cDNA (both 1,668 bp) corresponding to the ORF of AcTps2, respectively, are inserted between the vector pETDuet-1 of Fse I- Xho I. The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Plasmid pET-AcTps1a, (b) Plasmid pET-AcTps1b, (c) Plasmid pET-AcTps1c, (d) Plasmid pET-AcTps1d, and plasmid pAC-Mev / Scidi Chromatogram of an ethyl acetate extract of Escherichia coli strains. New peaks 12-15 are indicated by arrows. (e) AcTps1 do not contain the vector pET21a plasmid pAC-Mev / Scidi chromatogram of ethyl acetate extracts of E. coli strains harboring (control). (f) Mass spectra of peaks 12 to 15 (top figure to second figure from the bottom) and β-elemene (standard product) (bottom figure) indicated by arrows in (a) to (d) . The figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AcTps2 and further carrying plasmid pAC-Mev / Scidi. New peaks 16 and 17 are indicated by arrows. (b) AcTps2 do not contain the vector pET21a plasmid pAC-Mev / Scidi chromatogram of ethyl acetate extracts of E. coli strains harboring (control). (c) Mass spectrum of peak 17 (upper figure) and α-humulene (standard product) (lower figure) indicated by arrows in (a). (d) Mass spectrum of peak 16 (upper figure) and α-neoclovene (α-neoclovene; database) (lower figure) indicated by arrows in (a). The figure which represents the amino acid sequence alignment of AscTPS3 (it describes as AsTPS3 in this figure) and the known ginger family sesquiterpene synthase (synthase). ZoGDS represents Z. officinale Roscoe-derived germane D-synthesizing enzyme (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RxR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pET-AscTps3. CDNA corresponding to the ORF of AscTps3 (both 1,749 bp), respectively, are inserted between the vector pETDuet-1 of Nde I- Kpn I. The figure showing the analysis result of the monoterpene / sesquiterpene product of Escherichia coli using GC-MS. (a) Chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying plasmid pET-AscTps3 and further carrying plasmid pAC-Mev / Scidi. New peaks 18 and 19 are indicated by arrows. (b) AscTps3 do not contain the vector pET21a plasmid pAC-Mev / Scidi chromatogram of ethyl acetate extracts of E. coli strains harboring (control). (c) Mass spectrum of peak 18 (upper figure) and linalool (standard product) (lower figure) indicated by arrows in (a). (d) Mass spectrum of peak 19 (upper figure) and nerolidol (standard product) (lower figure) indicated by arrows in (a).

  The present invention is described in detail below.

(1. Mevalonate pathway gene group)
To use D-mevalonolactone (MVL; naturally converted to D-mevalonate) added to the medium, four mevalonate pathway genes starting from mevalonate kinase using it as a substrate That is, mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), diphosphomevalonate decarboxylase (DPMVA decarboxylase) and IPP isomerase (Idi; IPP isomerase) are required.
There are two types of Idi, type 1 and type 2, which have different structures. Either Idi may be used, but the present inventors used both Idi in the following examples. In the mevalonate pathway gene group (Non-patent Document 5; Accession no AB037666) derived from the Streptomyces genus CL190 strain used in the Examples by the present inventors, MVA kinase, DPMVA decarboxylase, PMVA kinase, type 2 IPP isomerase Since the genes formed a gene group in this order, this gene group was inserted as it was into the E. coli vector pACYC184 [chloramphenicol (Cm) resistance] to create a plasmid pAC-Mev (see: non-reference) Patent Document 7).
In addition to the type 2 idi gene contained in this gene group, it is known that further addition of type 1 idi gene (for example, Scidi of Saccharomyces cerevisiae) will slightly increase FPP production in recombinant E. coli. Therefore, the plasmid pAC-Mev / Scidi in which the Scidi gene was also inserted into the plasmid pAC-Mev was prepared (Reference: Non-Patent Document 7), and this plasmid was used in the Examples.
Further, regarding the source of the mevalonate pathway gene group, the inventors used those derived from Streptomyces genus CL190 strain in the following examples, but it is also possible to use a corresponding gene group derived from yeast or other bacteria (see : Non-patent document 8).
In this example, D-mevalonolactone (MVL) was added to the medium, and four mevalonate pathway genes starting from mevalonate kinase were used. However, it is also possible to simultaneously introduce upstream mevalonate pathway genes and add upstream enzyme substrates. For example, the inventors previously introduced mevalonate pathway genes that can assimilate acetoacetate (e.g. lithium acetoacetate) cheaper than D-mevalonolactone into recombinant E. coli, and added acetoacetate to the medium, It has been confirmed that isoprenoids such as α-humulene can be efficiently produced (see Non-Patent Document 7).

{2. Sesquiterpene synthase (sesquiterpene synthase)}
More than 7,000 sesquiterpenes have already been isolated from plants and microorganisms (mainly plants), and their chemical structures are known. However, many sesquiterpene synthase (synthase) genes are unknown (see non-patent document 8). Examples of sesquiterpene synthase (and the gene that encodes it) of plants and the like whose catalytic function has been clarified include δ-selinen synthase and γ-humulene isolated from American fir (grandfir; grand fir) Synthetic enzyme genes (CL Steele, J. Crock, J. Bohlmann, R. Croteau, Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of δ- selinene synthase and γ-humulene synthase. J. Biol. Chem. 273: 2078-2089, 1998).
A sesquiterpene synthase (and its gene) derived from a plant belonging to Araliaceae has not been known. In plants belonging to the family Gingidae (Zingiberaceae), from ginger (ginger; Zingiber officinale ), as a sesquiterpene synthase, a germacrene D synthase gene (germacrene D synthase) gene ( ZoGeD ; non-patent document 1), β-bisabolene synthase ( β-bisabolene synthase) gene ( ZoTPS1 ; Patent Document 1, Non-patent Document 2) and γ-amorphene synthase gene ( ZoTPS5 ; Patent Document 1) were isolated and analyzed for their structure and function. It was broken. In addition, from the same ginger plant, Shampoo ginger ( Zingiber zerumbet ), α-humulene synthase gene ( ZzZSS1 ) and β-eudesmol synthase gene ( ZzZSS2 ) ) Was isolated and analyzed for its structure and function (see Non-Patent Documents 3 and 4). In ginger family plants, sesquiterpene synthase (and its gene) other than these was not known.
In the present invention, Koshiabura sprouts, and turmeric purple (Acanthopanax sciadophylloides Franch et Sav.. ) ( Zedoary; Curcuma zedoaria Roscoe) full-length cDNA fragments of various sesquiterpene synthase gene candidates expressing the rhizome (rhizome) of Acquired. Plasmids (for example, pET-AsTps1, pET-AsTps2a, pET-AsTps2b, pET-CzTps1a, and pET-CzTps1b) inserted with these cDNA fragments into the E. coli vector pETDuet-1 (Novagen; ampicillin (Ap) resistant) Produced.

(3. Cultivation of Escherichia coli introduced with plasmid and production of sesquiterpene)
Plasmids (for example, pET-AsTps1, pET-AsTps2a, pET-AsTps2b, pET-) in which the full-length cDNA fragment of the sesquiterpene synthase gene candidate prepared in “2” above is inserted into the E. coli vector pETDuet-1 (Novagen). Each of CzTps1a and pET-CzTps1b (Ap resistance) is introduced into Escherichia coli BL21 (DE3) together with the plasmid pAC-Mev / Scidi (Cm resistance) prepared in “1” above. Resistant recombinant E. coli was prepared.
The obtained recombinant Escherichia coli was cultured by adding D-mevalonolactone (MVL; concentration is 0.5 mg / mL in one example) to the medium. At that time, if the cDNA fragment of the sesquiterpene synthase gene candidate introduced into Escherichia coli can actually synthesize sesquiterpene synthase, the recombinant Escherichia coli will produce sesquiterpene by the action of the synthase.
Recombinant Escherichia coli having plasmid pET-AsTps1 synthesizes β-caryophyllene, that is, cDNA inserted into the plasmid (named AsTps1 gene; this nucleotide sequence is shown in SEQ ID NO: 5) is synthesized by β-caryophyllene. Synthesizing (encoding) an enzyme (AsTPS1; this amino acid sequence is shown in SEQ ID NO: 6), and that recombinant E. coli harboring the plasmid pET-AsTps2a or pET-AsTps2b synthesizes the magnolene D, ie The cDNA inserted in the plasmid (named AsTps2a or AsTps2b gene; this nucleotide sequence is shown in SEQ ID NO: 7 or 9) is the Germaclen D synthase (AsTPS2a or AsTPS2b; this amino acid sequence is shown in SEQ ID NO: 8 or 10) The recombinant E. coli carrying the plasmid pET-CzTps1a or pET-CzTps1b A cDNA inserted into the plasmid (named as CzTps1a or CzTps1b gene; the base sequence is shown in SEQ ID NO: 15 or 17) is β-eudesmol synthase (CzTPS1a or CzTPS1b; this amino acid sequence Has been found to synthesize (encode).

(4. Escherichia coli strain and genetic recombination experiment method)
In the following examples, Escherichia coli B strain BL21 (DE3) was used. However, since various strains such as E. coli K12 strain JM109 (DE3) exist in E. coli, the strain is not limited to BL21 (DE3).
In the following examples, LB medium was used as the culture medium for recombinant E. coli. However, as the culture medium for E. coli, there are many media such as 2x YT medium, TB medium, etc., and therefore, the culture medium is limited to LB medium. Not a thing.
Moreover, as a genetic recombination experiment method, many manuals exist besides the implementation manual by the manufacturer shown in the following Example. For example, Sambrook and Russel, Molecular Cloning A Laboratory Manual (Third edition) Cold Spring Harbor Laboratory Press, 2001 can be exemplified. This handbook is comprehensive and describes the types of E. coli strains, types of vectors, culture methods, etc. in addition to the usual genetic recombination experiment methods, so that experiments can be conducted with reference.

(A) β-caryophyllene synthase gene The β-caryophyllene synthase gene of the present invention comprises (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6, and (2) an amino acid sequence set forth in SEQ ID NO: 6. 1-20 amino acids, preferably 1-15, more preferably 1-10, most preferably 1-5 are substituted, deleted, inserted and / or added and are set forth in SEQ ID NO: 6 A gene encoding a polypeptide having substantially the same activity as that of converting farnesyl diphosphate having β-caryophyllene, which is possessed by a polypeptide comprising the amino acid sequence of (3), and 90% or more of the amino acid sequence described in SEQ ID NO: 6 The polypeptide having the identity and having the amino acid sequence set forth in SEQ ID NO: 6 has substantially the same activity as that of converting farnesyl diphosphate into β-caryophyllene. (4) a gene consisting of a DNA consisting of the base sequence set forth in SEQ ID NO: 5, (5) a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 5 and a stringent A gene that encodes a polypeptide that hybridizes under mild conditions and has substantially the same activity as the activity of converting farnesyl diphosphate possessed by the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 to β-caryophyllene; 6) a gene comprising DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added in the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 5; (7) from the nucleotide sequence set forth in SEQ ID NO: 5 A gene consisting of DNA having 90% or more identity with the DNA.

The gene of (1) is a gene encoding β-caryophyllene synthase obtained from Cosiabra. The β-caryophyllene synthase has an activity of converting farnesyl diphosphate into β-caryophyllene. In addition to AsTps1 , which will be described later, the gene of (1) above encodes the same polypeptide as AsTps1 , but also includes genes having different base sequences.
The gene (2) is a gene that encodes a polypeptide into which β-caryophyllene synthase obtained from Cosiabra is introduced with a mutation that does not cause loss of enzyme activity. Such mutations include artificial mutations in addition to mutations that occur in nature. Examples of means for causing artificial mutation include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually 20 amino acids or less, preferably 10 amino acids or less, more preferably 5 amino acids or less, and most preferably 3 amino acids. Whether a polypeptide into which a mutation has been introduced retains its enzymatic activity is determined by, for example, introducing a gene encoding the mutation-introduced polypeptide into E. coli, etc., and expressing it so that the E. coli can produce β-caryophyllene. You can find out if you can do it. In particular, the amino acid sequence shown in SEQ ID NO: 6 includes RxR (266-268th amino acids), DDxxD (303th to 307th amino acids), (N / D) Dxx (S / T) xxxE (448th to 455th positions). A highly conserved motif in sesquiterpene synthase. When mutations are introduced into these motifs, there is a high possibility that enzyme activity is lost. Therefore, mutations are preferably introduced into amino acids other than these motifs.
In addition, FIG. 2 shows amino acids common to β-caryophyllene synthase (AsTPS1) and a known ginger-derived sesquiterpene synthase (blacked amino acids). These amino acids are sesquiterpenes. Since mutations may play an important role in the activity of the synthase, mutations are preferably introduced into amino acids other than these amino acids.
Regarding the gene of (3) above, “the activity of converting the farnesyl diphosphate of the polypeptide having the amino acid sequence of SEQ ID NO: 6 into β-caryophyllene and substantially the same activity” means farnesyl diphosphate. It means having activity to convert to β-caryophyllene, and the degree of activity may be stronger or weaker than that of converting farnesyl diphosphate having the amino acid sequence shown in SEQ ID NO: 6 to β-caryophyllene.
Also, the identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default, that is, default parameters).
The gene of (4) above is a β-caryophyllene synthase gene obtained from Kosiabra named ApTps1.
The gene (5) is a gene obtained by utilizing hybridization between DNAs. “Stringent conditions” in this gene refers to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually hybridization at 37 ° C. in a buffer containing 5 × SSC and 1% SDS and washing treatment at 37 ° C. with a buffer containing 1 × SSC and 0.1% SDS. Preferably, conditions such as hybridization at 42 ° C. in a buffer containing 5 × SSC and 1% SDS and washing treatment at 42 ° C. with a buffer containing 0.5 × SSC and 0.1% SDS, more preferably , Hybridization at 65 ° C. in a buffer containing 5 × SSC and 1% SDS, and washing treatment at 65 ° C. with a buffer containing 0.2 × SSC and 0.1% SDS. Whether the DNA obtained by utilizing hybridization encodes an active polypeptide is determined by, for example, introducing the DNA into E. coli, etc., and expressing it to produce β-caryophyllene. You can find out if you can do it. The DNA obtained by hybridization usually has a high identity with the gene (SEQ ID NO: 3) of (4) above. High identity refers to 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity.
The gene of (6) above has 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, most preferably 1 in DNA consisting of the base sequence shown in SEQ ID NO: 5. Preferably, it is a gene consisting of DNA in which 1 to 5 nucleotide sequences are substituted, deleted, inserted and / or added.
The gene (7) is composed of DNA having 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 98% or more identity with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 5. Including genes.

(A ′) β-caryophyllene synthase (protein having activity of converting farnesyl diphosphate to β-caryophyllene)
The β-caryophyllene synthase of the present invention has (1) the amino acid sequence described in SEQ ID NO: 6 and (2) the amino acid sequence described in SEQ ID NO: 6, preferably 1 to 20, preferably 1 to 15, more preferably 1 to The farnesyl diphosphate possessed by the polypeptide having the amino acid sequence shown in SEQ ID NO: 6 as β-caryophyllene is substituted, deleted, inserted and / or added with 10 amino acids, most preferably 1 to 5 amino acids. An amino acid sequence having an activity to convert; (3) a farnesyl diphosphate possessed by a polypeptide having an amino acid sequence of 90% or more of the amino acid sequence of SEQ ID NO: 6 and comprising the amino acid sequence of SEQ ID NO: 6; -An amino acid sequence having activity to convert to caryophyllene is included.
From the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide in the introduction of peptide mutation, for example, homologous amino acids (polar amino acids, nonpolar amino acids, Mutual substitution between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned.

(B) German Mclene D synthase gene The german Mclen D synthase gene of the present invention includes (1) a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or 10, and (2) an amino acid of SEQ ID NO: 8 or 10. In the sequence, 1 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 8 Or a gene encoding a polypeptide having substantially the same activity as the activity of converting farnesyl diphosphate having the amino acid sequence described in 10 to germacrene D, (3) 90% of the amino acid sequence described in SEQ ID NO: 8 or 10 It has the above identity and has substantially the same activity as that of converting farnesyl diphosphate having the amino acid sequence shown in SEQ ID NO: 8 or 10 into germane D A gene encoding a polypeptide, (4) a gene consisting of a DNA consisting of the base sequence described in SEQ ID NO: 7 or 9, and (5) a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in SEQ ID NO: 7 or 9 DNA encoding a polypeptide that hybridizes under stringent conditions with a polypeptide having substantially the same activity as that of converting farnesyl diphosphate having the amino acid sequence of SEQ ID NO: 8 or 10 into germane D (6) a gene consisting of DNA having 1-50 base sequences substituted, deleted, inserted and / or added in DNA consisting of the base sequence described in SEQ ID NO: 7 or 9, (7) SEQ ID NO: A gene comprising a DNA comprising a nucleotide sequence according to 7 or 9 and a DNA having 90% or more identity.
The gene of (1) above is a gene that encodes a germane D-synthesizing enzyme obtained from Kosiabra. This germaclen D synthase has the activity of converting farnesyl diphosphate to germacren D. In addition to AsTps2a or AsTps2b described later, the gene of (1) above encodes the same polypeptide as AsTps2a or AsTps2b , but also includes genes having different base sequences.
The gene of the above (2) is a gene encoding a polypeptide into which a mutation is introduced to such an extent that the enzyme activity is not lost with respect to germacren D synthase obtained from Cosiabra. The amino acid sequence shown in SEQ ID NO: 8 or 10 includes RxR (264-266th amino acid), DDxxD (301-305th amino acid), (N / D) Dxx (S / T) xxxE (445-452th) A highly conserved motif in sesquiterpene synthase. When mutations are introduced into these motifs, there is a high possibility that enzyme activity is lost. Therefore, mutations are preferably introduced into amino acids other than these motifs.
The gene of the above (4) is a germane gene synthase gene obtained from Kosiabra named AsTps2a or AsTps2b .
The gene (5) is a gene obtained by utilizing hybridization between DNAs.

(B ′) Germacrene D synthase (protein having activity of converting farnesyl diphosphate into β-caryophyllene)
Germacrene D synthase of the present invention has (1) the amino acid sequence described in SEQ ID NO: 8 or 10, and (2) the amino acid sequence described in SEQ ID NO: 8 or 10, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids are substituted, deleted, inserted and / or added, and farnesyl diphosphate having the amino acid sequence set forth in SEQ ID NO: 8 or 10 is converted to germacren D. An amino acid sequence having substantially the same activity as the activity to be converted; (3) having at least 90% identity with the amino acid sequence of SEQ ID NO: 8 or 10, and having the amino acid sequence of SEQ ID NO: 8 or 10 And an amino acid sequence having substantially the same activity as that of converting farnesyl diphosphate to germacrene D.

(C) β-eudesmol synthase gene The β-eudesmol synthase gene of the present invention includes (1) a gene encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 16 or 18, (2) SEQ ID NO: 16 or In the amino acid sequence described in 18, 18 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids are substituted, deleted, inserted and / or added, And a gene encoding a polypeptide having substantially the same activity as that of converting farnesyl diphosphate of the amino acid sequence of SEQ ID NO: 16 or 18 into β-eudesmol, (3) described in SEQ ID NO: 16 or 18 Activity of converting farnesyl diphosphate having the amino acid sequence of SEQ ID NO: 16 or 18 into β-eudesmol, which is 90% or more identical to the amino acid sequence of A gene encoding a polypeptide having the same quality of activity, (4) a gene consisting of a DNA consisting of the base sequence described in SEQ ID NO: 15 or 17, and (5) complementary to a DNA consisting of the base sequence described in SEQ ID NO: 15 or 17 It hybridizes with a DNA comprising a base sequence under stringent conditions and has substantially the same activity as that of converting farnesyl diphosphate having the amino acid sequence of SEQ ID NO: 16 or 18 into β-eudesmol. A gene comprising a DNA encoding a polypeptide; (6) a DNA comprising a nucleotide sequence set forth in SEQ ID NO: 15 or 17, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added. A gene, (7) a gene comprising 90% or more identity with the DNA comprising the nucleotide sequence of SEQ ID NO: 15 or 17.
The gene (1) is a gene encoding β-eudesmol synthase obtained from purple turmeric. This β-eudesmol synthase has the activity of converting farnesyl diphosphate into β-eudesmol. In addition to CzTps1a or CzTps1b , which will be described later, the gene (1) encodes the same polypeptide as CzTps1a or CzTps1b , but also includes genes having different base sequences.
The gene (2) is a gene that encodes a polypeptide into which β-eudesmol synthase obtained from purple turmeric has been introduced with a mutation that does not cause loss of enzyme activity. The amino acid sequence set forth in SEQ ID NO: 16 or 18 includes RxR (264-266th amino acid), DDxxD (301-305th amino acid), (N / D) Dxx (S / T) xxxE (445-452th position) A highly conserved motif in sesquiterpene synthase. When mutations are introduced into these motifs, there is a high possibility that enzyme activity is lost. Therefore, mutations are preferably introduced into amino acids other than these motifs.
The gene of (4) is a β- eudesmol synthase gene obtained from purple turmeric named CzTps1a or CzTps1b .
The gene (5) is a gene obtained by utilizing hybridization between DNAs. The DNA obtained by hybridization usually has a high identity with the gene (SEQ ID NO: 9) described above (4). High identity refers to 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity.

(C) β-eudesmol synthase (protein having activity of converting farnesyl diphosphate to β-eudesmol)
The β-eudesmol synthase of the present invention is (1) the amino acid sequence described in SEQ ID NO: 16 or 18, and (2) the amino acid sequence described in SEQ ID NO: 16 or 18, preferably 1 to 15, More preferably, 1 to 10, most preferably 1 to 5 amino acids are substituted, deleted, inserted and / or added, and farnesyl diphosphate having the amino acid sequence of SEQ ID NO: 16 or 18 is β -An amino acid sequence having substantially the same activity as that of converting to eudesmol, (3) having an identity of 90% or more with the amino acid sequence of SEQ ID NO: 16 or 18, and the sequence of SEQ ID NO: 16 or 18 An amino acid sequence having substantially the same activity as that of converting farnesyl diphosphate of the amino acid sequence into β-eudesmol is included.

(D-1) Recombinant Escherichia coli The recombinant Escherichia coli of the present invention is obtained by introducing and expressing the β-caryophyllene synthase gene, the gelmacrene D synthase gene, and / or the β-eudesmol synthase gene. It is.
In addition to the β-caryophyllene synthase gene, the gelmacrene D synthase gene, and / or the β-eudesmol synthase gene, the recombinant Escherichia coli of the present invention is preferably (1) isopentenyl from mevalonic acid or mevalonolactone. A mevalonate pathway gene group that synthesizes up to diphosphate and (2) an isopentenyl diphosphate isomerase gene are introduced and expressed.
By introducing these genes into Escherichia coli and expressing them, β-caryophyllene, gelmacrene D and β-eudesmol can be produced from mevalonic acid or mevalonolactone.

(D-2) Host other than recombinant Escherichia coli In the method for producing a sesquiterpene of the present invention, as a host other than recombinant Escherichia coli, an insect system, a yeast system, a plant cell system, a cell-free system (such as wheat germ), etc. Can be used.
In these hosts as well, β-caryophyllene, Germaclen D and / or β-eudesmol can be produced by introducing appropriate synthase genes as in the case of the recombinant E. coli system. Synthetic enzyme genes necessary for each host are as follows.
Yeast system: The β-caryophyllene synthase gene, the germacrene D synthase gene and / or the β-eudesmol synthase gene of the present invention are introduced into yeast using a yeast expression vector to express them. Terpenes can be manufactured. In addition, it is thought that the target sesquiterpene production amount can be improved by co-expressing the HMG-CoA reductase (enzyme that makes mevalonic acid from HMG-CoA) gene.
Insect system: The β-caryophyllene synthase gene, the germacren D synthase gene and / or the β-eudesmol synthase gene of the present invention are introduced into insects using an insect expression vector and expressed thereby. Terpenes can be manufactured. In addition, it is thought that the target sesquiterpene production can be improved by co-expressing the HMG-CoA reductase gene.
Plant system: The β-caryophyllene synthase gene, the germacrene D synthase gene and / or the β-eudesmol synthase gene of the present invention are introduced into a plant using a plant expression vector and expressed, thereby producing these sesquiskies. Terpenes can be manufactured. In addition, it is thought that the target sesquiterpene production can be improved by co-expressing the HMG-CoA reductase gene. In addition, when a foreign gene is introduced and expressed directly into the chloroplast by transformation of chloroplasts with particle gun, isopentenyl diphosphate (IPP) isomerase (type 1 and / or type 2) gene It is considered that the desired sesquiterpene production can be improved by co-expression.

(E-1) Mevalonate pathway gene group that synthesizes up to isopentenyl diphosphate As the mevalonate pathway gene group that synthesizes up to isopentenyl diphosphate, the mevalonate pathway derived from the aforementioned Streptomyces genus CL190 strain The gene group (Reference: Non-Patent Document 5) can be used, but other than this, the mevalonate pathway gene group derived from Saccharomyces cerevisiae (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), Mevalonate pathway genes from the bacterium Streptococcus pneumoniae (SH Yoon, YM Lee, JE Kim, SH Lee, JH Lee, JY Kim, KH Jung, YC) Shin, JD Keasling, SW Kim, Biotechnology & Bioengineering, 94: 1025-1032, 2006) can also be used.

(E-2) Isopentenyl diphosphate isomerase gene As the isopentenyl diphosphate isomerase gene, type 2 isopentenyl diphosphate (IPP) isomerase gene derived from the aforementioned Streptomyces genus CL190 strain (reference: non-patent literature) 5) and type 1 IPP isomerase gene derived from Saccharomyces cerevisiae (S. Kajiwara, PD Fraser, K. Kondo, N. Misawa, Biochemical Journal, 324: 421-426, 1997) can be used. In addition, two types of IPP isomerase genes derived from E. coli (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), green algae Hematococcus pluviaris pluvialis ) type 1 isopentenyl diphosphate isomerase gene (the above-mentioned literature by JJ Martin et al. and the above-mentioned literature by S. Kajiwara et al.) can also be used.

(F) Method for Producing Sesquiterpene The method for producing a sesquiterpene according to the present invention comprises the step of cultivating the above recombinant E. coli in a medium containing mevalonic acid and / or mevalonolactone, or β-caryophyllene, germane D and / or β-eudesmol is obtained.
The medium to be used is not particularly limited as long as it contains mevalonic acid or mevalonolactone and is suitable for culturing Escherichia coli. Use the aforementioned LB medium, 2x YT medium, TB medium, etc. with mevalonic acid or mevalonolactone added. can do.
The concentration of mevalonic acid or mevalonolactone in the medium is preferably about 0.1 to 5 g, more preferably about 0.5 to 1 g, per liter of the medium. The pH in the medium is preferably about 6.0 to 8.0, more preferably about 6.6 to 7.6. In general, the culture may be performed at about 20 to 30 ° C. for about 1 to 3 days.
By the above culture, β-caryophyllene, germacrene D, and / or β-eudesmol are generated.

  Products containing β-caryophyllene, germacrene D, and / or β-eudesmol can be purified by conventional methods. For example, if necessary, it is subjected to treatment such as centrifugation and filtration to remove suspensions such as bacterial cells, and then extracted with a general extraction solvent, for example, an organic solvent such as ethyl acetate, chloroform, methanol, etc. Can be removed under reduced pressure and purified by subjecting to a treatment such as vacuum distillation, chromatography, ion exchange resin, or adsorbent resin.

(G) Physiological activity of β-caryophyllene, germacrene D, or β-eudesmol β-caryophyllene, germanecrene D, or β-eudesmol that can be produced by the production method of the present invention has various beneficial physiological activities in humans. ing.
β-Caryophyllene (β-Caryophyllene) is also found in marijuana (cannabis) and has been used in the treatment of pain, inflammation, atherosclerosis, osteoporosis, etc. based on detailed research so far. It was almost demonstrated that it could be done. Gertsch et al. Of the Zurich University of Technology found that the natural molecule of β-caryophyllene activates a protein called “cannabinoid receptor type 2” (CB2) (J. Gertsch, M. Leonti, S. Randuner , I. Racz, JZ Chen, XQ Xie, KH Altmann, M. Karsak, A. Zimmer, PMAS, 105: 9099-9104, 2008).
Cannabinoid is a general term for chemical substances contained in cannabis. Activated CB2 calms the immune system, increases bone mass, and blocks pain signals. At this time, there is no euphoria generally considered to be the action of marijuana, nor does it affect the central nervous system.
Germacrene D (germacrene D) is known to be contained in a large amount of a ylang-ylang herb, and is known to have sedative, antiallergic, and translucent effects.
β-eudesmol (β-eudesmol) is known to have anti-angiogenic activity and to suppress gastric acid secretion (LC Chiou, JY Ling CC Chang, Eur J Pharmacol 13: 151- 156, 1995).
Recently, β-eudesmol has been shown to activate the cool receptor (human cold receptor channel TRPA1; Transient Receptor Potential Ankyrin 1) (Ichiro Ohara, Sayoko Kitao et al., Yasuyuki Okada et al., 3C29p03, Japan Agricultural Chemical Society 2012 Conference).

(H) Physiological activity of β-cubeben, β-elemene, α-neocloben, α-humulene, linalool, or nerolidol β-cubeben, β-elemene, α-neocloben, α-humulene that can be produced by the production method of the present invention , Linalool or nerolidol have various beneficial physiological activities in humans.
β-elemene has been reported to have antitumor and cancer prevention effects. For example, β-elemen has been shown to inhibit the proliferation or activation of lung and gastric cancer cells by inducing apoptosis (G. Wang, X. Li, F. Huang, J. Zhao, H. Ding, C. Cunningham, JE Coad et al, Cell Mol Life Sci 62: 881-893, 2005; J. Liu, Y. Zhang, J. Qu, L. Xu, K. Hou, J. Zhang, X. Qu, Y Liu, BMC Cancer 11: 183 2011).
It has been reported that α-humulene has an anti-inflammatory effect (AP Rogerio, EL Andrade, DFP Leite, CP Figueiredo JB Calixto, British J Pharmacology 158: 1074-1087, 2009).
Linalool has a lavender, lily of the valley, and bergamot-like fragrance and is used in large quantities as a fragrance. It is also the main scent component of beer.
Nerolidol is also a scent component.
The physiological functions of β-cubeben and α-neocloben have not been particularly reported. The main reason for this is thought to have been the lack of a source that can prepare these sesquiterpenes in large quantities. Therefore, since it can be mass-produced by the method according to the present invention, new physiological activity can be found for β-cubeben and α-neocloben by conducting various physiological function tests in the future.

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

(Determining cDNA sequence of sesquiterase synthase gene derived from Kosiabra)
Total RNA was extracted from shoots (FIG. 1a) of Cosciabra ( Acanthopanax sciadophylloides Franch. Et Sav.) Using RNeasy Plant Mini Kit (Qiagen). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)] was performed using cDNA as a template under the conditions described in Non-Patent Document 4, and an amplified fragment of 544 bp was obtained.
The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [KS2.5race: 5′-AGACGCTCAAGGTGAGAGGTGGTGAA-3 ′ (SEQ ID NO: 1) and KS2.3race: 5 ′ -Using TCGCAAGAGATAGAGTGGTGGAGTGC-3 '(SEQ ID NO: 2)] and Advantage2 Polymerase Mix (Clontech), the fragments were extended toward the 5' end and 3 'end according to the manufacturer's instructions, and the full-length cDNA sequence was determined.

(Isolation and structural analysis of three full-length sesquiterpene synthase gene cDNAs)
Oligonucleotide primer pair KS2. Nde-F {5'-AGACAGA CATAT GGCCGCGATTCTTCAGG -3 '(SEQ ID NO: 3), underline indicates Nde I recognition site} and KS2 specific for the full-length cDNA sequence obtained in Example 1 Xho-R {5′-GAGGTAC CTCGAG TTATATTCGAACAGCATCTATGAGCAC -3 ′ (SEQ ID NO: 4), underlined indicates Xho I recognition site} and Advantage2 Polymerase Mix (manufactured by Clontech), PCR using Cosiabra cDNA as a template ( Reaction composition: according to manufacturer's instructions; reaction conditions: Denaturation 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 4 minutes, 5 cycles, then 94 ° C for 30 seconds 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 4 minutes).
The obtained DNA fragment was cloned by the plasmid pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. These cDNA nucleotide sequences are shown in SEQ ID NOs: 5, 7 and 9, and the amino acid sequences of polypeptides encoded by the cDNA are shown in SEQ ID NOs: 6, 8 and 10. Further, the gene (cDNA) specified by SEQ ID NO: 5 was named AsTps 1, and the protein (polypeptide) specified by SEQ ID NO: 6 encoded by AsTps 1 was named AsTPS1. Furthermore, genes that are identified with (cDNA) designated respectively AsTps2a and AsTps2b by SEQ ID NO: 7 and 9, the proteins identified by SEQ ID NO: 8 and 10 AsTps2a and AsTps2b code to a (polypeptide) respectively, AsTPS2a and AsTPS2b Named.

AsTps1 contained a 1,668 bp open reading frame (ORF) and encoded a protein (AsTPS1) with a putative 555 amino acid residue, a molecular weight of 64.4 kDa, and a pI of 5.58. Homology searches against GenBank / EMBL / DDBJ database, AsTPS1 European grape (Vitis vinifera) from germacrene D synthase (germacrene D synthase) and 61% is known sesquiterpene synthase, the beta-caryophyllene synthase (beta- caryophyllene synthase) and 61%, kiwifruit (Actinidia deliciosa) -derived germacrene D synthase and 60% amino acid sequence identity (identity of amino acid sequence; identity).
On the other hand, AsTps2a contained an ORF of 1,668 bp and encoded a protein (AsTPS2a) with a putative 555 amino acid residue, a molecular weight of 64.8 kDa, and a pI of 5.80. As a result of homology search, AsTPS2a is known to be a known sesquiterpene synthase, European grape ( Vitis vinifera ) germacrene D synthase, 64%, β-caryophyllene synthase, 63%, It showed 61% amino acid sequence identity with germanium D synthase from Kiwifruit ( Actinidia deliciosa ).
AsTps2b encoded a protein (AsTPS2b) containing an ORF of 1,668 bp, a putative 555 amino acid residue, a molecular weight of 64.6 kDa, and a pI of 5.92. As a result of homology search, AsTPS2b is a known sesquiterpene synthase, European grape ( Vitis vinifera ) -derived germacrene D synthase, 63%, β-caryophyllene synthase, 62%, It showed 61% amino acid sequence identity with germanium D synthase from Kiwifruit ( Actinidia deliciosa ).
The homology of AsTPS1, AsTPS2a, and AsTPS2b showed amino acid sequence identity (identity) of 85% for AsTPS1 and AsTPS2a, 84% for AsTPS1 and AsTPS2b, and 99% for AsTPS2a and AsTPS2b. The difference between AsTPS2a and AsTPS2b was due to four amino acid substitutions, namely the 94th Asp to Gly substitution (Asp94Gly, the same applies hereinafter), Glu213Gly, Ala269Thr, and Leu547Ser. Due to the homology with known sesquiterpene synthases, AsTPS1, AsTPS2a and AsTPS2b are TPS-a subfamily, a group of angiosperm sesquiterpenes and diterpene synthases (J. Bohlmann, G. Meyer-Gauen, R. Croteau (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. See Proc. Natl. Acad. Sci. USA 95: 4126-4133). Furthermore, AsTPS1, AsTPS2a and AsTPS2b contained the motifs RxR, DDxxD, (N / D) Dxx (S / T) xxxE highly conserved in sesquiterpene synthase (FIG. 2).
On the other hand, unlike normal known sesquiterpene synthases, AsTPS1, AsTPS2a and AsTPS2b were predicted to have plastid transit peptides. A comparison (alignment) of amino acid sequences between these three proteins and known enzymes having high homology with these three proteins is shown in FIG.

(Functional analysis of three kinds of sesquiterpene synthase genes derived from Cosiabra)
Three full-length cDNA sequences of AsTps1 , AsTps2a , and AsTps2b , which are three types of sesquiterpene synthase genes derived from Cossiabra, were excised from the plasmid into which they were inserted using the restriction enzyme Nde I- Xho I, and the Escherichia coli vector pETDuet-1 (Novagen PET-AsTps1, pET-AsTps2a, and pET-AsTps2b were prepared by ligation to the Nde I- Xho I site of the multicloning site (MCS2) (FIG. 3). Transformation of Escherichia coli BL21 (DE3) using one of the plasmids pET-AsTps1, pET-AsTps2a, pET-AsTps2b and the plasmid pAC-Mev / Scidi (Non-patent Document 7), and the resulting set The cultured Escherichia coli was cultured, sesquiterpenes were extracted from the cultured Escherichia coli cells, and further subjected to gas chromatography-mass spectrometry (GC-MS). These methods are as described in Non-Patent Documents 2, 3, 4, and 7.

The analysis by the GC-MS, the chromatogram of the E. coli strain ethyl acetate extracts which holds the vector pET21a and plasmids pAC-Mev / Scidi containing no AsTps1 (control), also not confirmed formation of any sesquiterpene In contrast, in the experimental group using E. coli containing plasmid pET-AsTps1 and plasmid pAC-Mev / Scidi, a new peak was observed in the ethyl acetate extract from the cells. Comparison of mass spectra confirmed that this peak (peak 1) was β-caryophyllene (FIG. 4).
Therefore, when the sample was overlaid with a preparation of β-caryophyllene (Wako Pure Chemical Industries), the retention time by GC analysis was completely consistent.
From the above results, it was found that AsTps 1 is a gene encoding β-caryophyllene synthase. A sesquiterpene synthase gene that synthesizes β-caryophyllene as one of a plurality of sesquiterpene components is known (for example, ZzZZS1 in Non-Patent Document 3). There has been no known compound that can be synthesized as one main terpene component.
Similarly, the chromatogram of ethyl acetate extracts of E. coli strains harboring the vector pET21a plasmid pAC-Mev / Scidi containing no AsTps2 (control), the generation of any sesquiterpene was not confirmed to, In the experimental group using E. coli containing either plasmid pET-AsTps2a or pET-AsTps2b and plasmid pAC-Mev / Scidi, a new peak was observed in the ethyl acetate extract from the cells. By comparison of mass spectra, this peak (Peak 2 and Peak 3) was considered to be Germacrene D (FIG. 5). Then, when it overlapped with the sample of Germaclen D, the retention time by GC analysis was completely in agreement.
From the above results, it was found that AsTps2a and AsTps2b are genes encoding germacrene D synthase. The chemical structures of β-caryophyllene and germacrene D are shown in FIG.
In addition, from the above results, selective production of β-caryophyllene using E. coli into which plasmid pET-AsTps1 and plasmid pAC-Mev / Scidi were introduced, and either plasmid pET-AsTps2a or pET-AsTps2b And it was confirmed that selective production of Germacrene D was possible using Escherichia coli introduced with plasmid pAC-Mev / Scidi. In general, since sesquiterpene synthases tend to synthesize a plurality of sesquiterpenes simultaneously, enzyme genes that catalyze the production of almost single sesquiterpenes, such as AsTps1 , AsTps1b, or AsTps2b , are rare.

(Sequence determination of sesquiterpene synthase gene cDNA sequence derived from purple turmeric)
Rhizome; (Curcuma zedoaria Roscoe zedoary) purple turmeric; from (rhizome Figure 7), total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen Inc.). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)], PCR was performed using cDNA as a template under the conditions described in Non-Patent Document 4 to obtain an amplified fragment of 813 bp. The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [UN3.5race: 5′-AGCTCTTCTGCTCGAATCGACGACAC-3 ′ (SEQ ID NO: 11) and UN3.3race: 5 ′ -Using ACTTTTGCTCGAGATCGAGTGGTGGA-3 '(SEQ ID NO: 12)] and Advantage2 Polymerase Mix (manufactured by Clontech), the fragment was extended toward the 5' end and 3 'end according to the manufacturer's instructions to determine the full-length cDNA sequence.

(Isolation and structural analysis of two full-length sesquiterpene synthase gene cDNAs from purple turmeric)
A pair of oligonucleotide primers specific for the full-length cDNA sequence obtained in Example 4 UN3.Nde-F (5′-AGACAGA CATAT GGAGAAGCAATCGACCAGTACTC-3 ′ (SEQ ID NO: 13), underlined indicates Nde I recognition site) and UN3 PCR using purple turmeric cDNA as a template using Kpn -R (5'-GAGGTAC GGTACC TTAAATAGGTACTGATTCGACCAACACC -3 '(SEQ ID NO: 14), underlined indicates Kpn I recognition site) and Advantage2 Polymerase Mix (Clontech) (Reaction composition: according to manufacturer's instructions; reaction conditions: denaturation at 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 4 minutes, 5 cycles, then 94 ° C for 30 minutes Second, 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 4 minutes). The obtained cDNA was cloned using the plasmid pGEM-T Easy Vector System (Promega), and the nucleotide sequence was determined.
These cDNA nucleotide sequences are shown in SEQ ID NOs: 15 and 17, and amino acid sequences of polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 16 and 18, respectively. Further, the genes (cDNAs) specified by SEQ ID NOs: 15 and 17 are named CzTps1a and CzTps1b, and the proteins (polypeptides) specified by SEQ ID NOs: 16 and 18 encoded by CzTps1a and CzTps1b are CzTPS1a and CzTPS1b , respectively. Named.
CzTps1a contained a 1,644 bp open reading frame (ORF) and encoded a protein (CzTPS1a) with a predicted 547 amino acid residues, a molecular weight of 64.0 kDa, and a pI of 5.35. Based on homology searches against GenBank / EMBL / DDBJ databases, CzTPS1a is 66% of the known sesquiterpene synthase α-humulene synthase derived from Zingiber zerumbet , β-eudesmol synthase (Β-eudesmol synthase) and 63%, and ginger ( Zingiber officinale) -derived gelmacrene D synthase showed 59% amino acid sequence identity. In addition, the amino acid sequence coincidence of 45%, 45% and 44% with the β-caryophyllene synthase (AsTPS1) and the same Germacrene D synthase (AsTPS2a and AsTPS2b) according to the present invention was shown.
On the other hand, CzTps1b contained a 1,644 bp open reading frame (ORF) and encoded a protein (CzTPS1b) with a predicted 547 amino acid residues, a molecular weight of 64.1 kDa, and a pI of 5.41. By homology search against GenBank / EMBL / DDBJ database, CzTPS1b is 66% with α-humulene synthase derived from Zingiber zerumbet , a known sesquiterpene synthase, β-eudesmol synthase (Β-eudesmol synthase) and 63%, and ginger ( Zingiber officinale ) -derived gelmacrene D synthase and 59% amino acid sequence identity (identity). In addition, the amino acid sequence coincidence of 45%, 45% and 44% with the β-caryophyllene synthase (AsTPS1) and the same Germacrene D synthase (AsTPS2a and AsTPS2b) according to the present invention was shown. The homology between CzTPS1a and CzTPS1b was 99.6% amino acid sequence identity. The difference between the two was due to two amino acid substitutions, namely the 118th Gln to Arg substitution (Gln118Arg, and so on) and Ala418Thr. Homology with known sesquiterpene synthases indicates that CzTPS1a and CzTPS1b belong to the TPS-a subfamily, a group of angiosperm sesquiterpenes and diterpene synthases. Furthermore, CzTPS1a and CzTPS1b contained the motifs RxR, DDxxD, (N / D) Dxx (S / T) xxxE highly conserved in sesquiterpene synthase (FIG. 8).
On the other hand, as with normal known sesquiterpene synthases, CzTPS1a and CzTPS1b were predicted not to contain a plastid transit peptide. FIG. 8 shows a comparison (alignment) of amino acid sequences of these two proteins and known enzymes having high homology with them.

(Functional analysis of two sesquiterpene synthase genes derived from purple turmeric)
Two full-length cDNA sequences derived from purple turmeric sesquiterpene synthase genes CzTps1a and CzTps1b were excised from the inserted plasmids with restriction enzyme Nde I- Kpn I, and E. coli vector pETDuet-1 (manufactured by Novagen) PET-CzTps1a and pET-CzTps1b plasmids were prepared by ligation to the Nde I- Kpn I site of the multicloning site (MCS2) (FIG. 9).
E. coli BL21 (DE3) is transformed with either plasmid pET-CzTps1a or pET-CzTps1b and plasmid pAC-Mev / Scidi (Non-patent Document 7), and the resulting recombinant E. coli is cultured. Sesquiterpenes were extracted from the cultured Escherichia coli cells, and subjected to gas chromatography-mass spectrometry (GC-MS). These methods are as described in Non-Patent Documents 2, 3, 4, and 7.
As a result of analysis using GC-MS, the chromatogram (control) of the ethyl acetate extract of the Escherichia coli strain carrying the vector pET21a without CzTps 1 and the plasmid pAC-Mev / Scidi confirmed the formation of any sesquiterpene. In contrast, in the experimental section using E. coli containing either plasmid pET-CzTps1a or pET-CzTps1b and plasmid pAC-Mev / Scidi during culture, a new one was added to the ethyl acetate extract from the cells. Peak was observed. By comparison of mass spectra, this peak (peaks 4 and 5) was considered to be β-eudesmol (FIG. 10).
Therefore, when it was overlapped with a sample of β-eudesmol (Wako Pure Chemical Industries), the retention time by GC analysis was completely consistent. From the above results, it was found that CzTps1a and CzTps1b are genes encoding β-eudesmol synthase. The chemical structure of β-eudesmol is shown in FIG.
In addition, the above results indicate that β-eudesmol can be selectively produced using E. coli into which either plasmid pET-CzTps1a or pET-CzTps1b and plasmid pAC-Mev / Scidi have been introduced. It was done.
In addition, only β- eudesmol synthase gene ( ZzZSS2 ) isolated from Hana ginger has been known so far as β- eudesmol can be synthesized as a single main terpene component (see Non-Patent Document 4). ). The gene we isolated from purple turmeric ( CzTps1a or CzTps1b ) is a terpene synthase gene with the same function as ZzZSS2 , but the amino acid sequences of the proteins encoded by these two genes are quite different ( 63% amino acid identity). Rather, CzTPS1a or CzTPS1b is structurally close to α-humulene synthase ZzZSS1 (reference: Non-Patent Document 3) isolated from Japanese ginger, so the gene of the present invention ( CzTps1a or CzTps1a or The function of CzTps1b ) could not be estimated. Therefore, the gene of the present invention can be said to be a novel gene because of its structure and function.

(Determination of cDNA sequence of sesquiterpene synthase gene derived from himeukogi)
Himeukogi; from (Araliaceae Acanthopanax Sieboldianus) shoots (Fig. 11a), total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen Inc.). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)], PCR was performed using cDNA as a template under the conditions described in Non-Patent Document 4 to obtain an amplified fragment of 592 bp.
The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [surugaGDS.5race: 5′-GCGATTTAAGACGCTCAAGGTGAGAGG-3 ′ (SEQ ID NO: 21) and surugaGDS.3race: 5 ′ -Using TCGCAAGAGATAGAGTGGTGGAGTGC-3 '(SEQ ID NO: 22)] and Advantage2 Polymerase Mix (Clontech), the fragment was extended toward the 5' end and 3 'end according to the manufacturer's instructions, and the cDNA sequence was determined. Based on the obtained 5 ′ terminal sequence information, a newly synthesized oligonucleotide primer [UK1.5-RACE.326: 5′-ACGGTGTGAAGATCGTCGTCATTTCC-3 ′ (SEQ ID NO: 23)] was used to further expand the 5 ′ terminal. The full length cDNA sequence was determined.

(Isolation and structural analysis of four full-length sesquiterpene synthase gene cDNAs)
A pair of oligonucleotide primers specific for the full-length cDNA sequence obtained in Example 7 UK1. Nde-F {5'-AGACAGA CATAT GGCCACGTATCTCCAGG-3 '(SEQ ID NO: 24), underlined indicates Nde I recognition site} and UK1 Xho-R {5′-GAGGTAC CTCGAG TTATATTTGAACTGGATCTATGAGCAC -3 ′ (SEQ ID NO: 25), underlined indicates Xho I recognition site} and Advantage2 Polymerase Mix (manufactured by Clontech), PCR using Cosiabra cDNA as a template ( Reaction composition: according to manufacturer's instructions; reaction conditions: Denaturation 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, 5 cycles, then 94 ° C for 30 seconds 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 2 minutes).
The obtained DNA fragment was cloned using the plasmid pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. These cDNA nucleotide sequences are shown in SEQ ID NOs: 26, 28, 30 and 32, and the amino acid sequences of polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 27, 29, 31 and 33. Further, the genes (cDNA) specified by SEQ ID NOs: 26, 28, 30 and 32 are named AsiTps1a , AsiTps1b , AsiTps1c and AsiTps1d , respectively , and AsTps1a , AsiTps1b , AsiTps1c and AsiTps1d are encoded by SEQ ID NOs: 27, 29, 31 and The proteins (polypeptides) specified by 33 were named AsiTPS1a , AsiTPS1b , AsiTPS1c and AsiTPS1d .
AsiTps1a contained a 1,671 bp open reading frame (ORF) and encoded a protein (AsiTPS1a) with a putative 556 amino acid residue, a molecular weight of 64.7 kDa, and a pI of 5.57. Based on homology search against GenBank / EMBL / DDBJ database, AsiTPS1a is a known sesquiterpene synthase, α-copaene synthase derived from Eleutherococcus trifoliatus , 97%, European macaque derived from Vitis vinifera The amino acid sequence identity was 63% with D-synthesizing enzyme (germacrene D synthase) and 62% with β-caryophyllene synthase.
AsiTps1b contained a 1,671 bp ORF and encoded a protein (AsiTPS1b) with a putative 556 amino acid residue, a molecular weight of 64.6 kDa, and a pI of 5.64. According to homology search, AsiTPS1b has 97% amino acid sequence identity with the known sesquiterpene synthase α-copaene synthase from Mitsukoukogi, 63% with European grape-derived mamacrene D synthase, and 62% with the same β-caryophyllene synthase (Identity).
AsiTps1c encoded a protein (AsiTPS1c) containing an ORF of 1,671 bp, a putative 556 amino acid residue, a molecular weight of 64.6 kDa, and a pI of 5.57. According to homology search, AsiTPS1c is 97% of the known sesquiterpene synthase α-copaene synthase from Mitsubaukogi, 64% from European grape germin D synthase, and 63% amino acid sequence identity with β-caryophyllene synthase (Identity).
AsiTps1d encoded a protein (AsiTPS1d) containing an ORF of 1,671 bp, a putative 556 amino acid residue, a molecular weight of 64.6 kDa, and a pI of 5.62. By homology search, AsiTPS1d has 97% amino acid sequence identity with the known sesquiterpene synthase α-copaene synthase from Mitsubaukogi, 63% with European grape germin D synthase, and 62% with β-caryophyllene synthase (Identity).
AsiTPS1a, AsiTPS1b, AsiTPS1c and AsiTPS1d are as follows. AsiTPS1c and AsiTPS1d showed 99.3% amino acid sequence identity. The difference between AsiTPS1a and AsiTPS1b was due to two amino acid substitutions, ie, the substitution of the 36th Leu to Pro (Leu36Pro, and so on) and Asp42Gly. The difference between AsiTPS1a and AsiTPS1c was due to two amino acid substitutions: Asn39Ser and Phe235Leu. The difference between AsiTPS1a and AsiTPS1d was due to two amino acid substitutions, namely Met64Val and Tyr158His. Due to its homology with known sesquiterpene synthases, AsiTPS1a, AsiTPS1b, AsiTPS1c and AsiTPS1d are TPS-a subfamily, a group of angiosperm sesquiterpenes and diterpene synthases (see J. Bohlmann, G. Meyer-Gauen , R. Croteau (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. USA 95: 4126-4133).
In addition, AsiTPS1a, AsiTPS1b, AsiTPS1c and AsiTPS1d contain the motifs RxR, DDxxD, (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} that are highly conserved in sesquiterpene synthases (Figure 12).
On the other hand, AsiTPS1a, AsiTPS1b, AsiTPS1c and AsiTPS1d were predicted to have a plastid transit peptide, unlike ordinary known sesquiterpene synthases. A comparison (alignment) of amino acid sequences between these four proteins and known enzymes having high homology with these four proteins is shown in FIG.

(Functional analysis of four kinds of sesquiterpene synthase genes derived from himeukogi)
The four full-length sesquiterpene synthase genes AsiTps1a , AsiTps1b , AsiTps1c and AsiTps1d full-length cDNA sequences derived from himeukogi were excised from the plasmid into which these were inserted with the restriction enzyme Nde I- Xho I, and the E. coli vector pETDuet-1 ( Novagen) was ligated to the Nde I- Xho I site of the multiple cloning site (MCS2) to produce pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c, and pET-AsiTps1d (FIG. 13). E. coli BL21 (DE3) was transformed using any one of plasmids pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c and pET-AsiTps1d and plasmid pAC-Mev / Scidi (Non-patent Document 7). . Further, the obtained recombinant Escherichia coli was cultured, sesquiterpenes were extracted from the cultured Escherichia coli cells, and further gas chromatography mass spectrometry (GC-MS) was performed. These methods are as described in Non-Patent Documents 2, 3, 4, and 7.
The analysis by the GC-MS, the chromatogram of the E. coli strain ethyl acetate extracts which holds the vector pET21a and plasmids pAC-Mev / Scidi containing no AsiTps1 (control), also not confirmed formation of any sesquiterpene In contrast, in an experimental group using E. coli containing plasmid pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c, pET-AsiTps1d and plasmid pAC-Mev / Scidi, A new peak was seen in the ethyl extract. Comparison of mass spectra confirmed that this peak (peak 6, peak 7, peak 8, and peak 9 in FIG. 14) was β-caryophyllene.
Therefore, when overprinted with a preparation of β-caryophyllene (Wako Pure Chemical Industries), the retention time by GC analysis was completely consistent.
Therefore, it was found that AsiTps1a , AsiTps1b , AsiTps1c and AsiTps1d are genes encoding β-caryophyllene synthase. The chemical structure of β-caryophyllene is shown in FIG.
Furthermore, from the above results, using one of the plasmids pET-AsiTps1a, pET-AsiTps1b, pET-AsiTps1c and pET-AsiTps1d and Escherichia coli introduced with the plasmid pAC-Mev / Scidi, selective β-caryophyllene It was confirmed that the production was possible. In general, since sesquiterpene synthases tend to synthesize a plurality of sesquiterpenes simultaneously, enzyme genes that catalyze the production of almost a single sesquiterpene, such as AsiTps1a , AsiTps1b , AsiTps1c, and AsiTps1d , are rare.
In addition, the AsTps1 gene derived from Cosciabra ( Acanthopanax sciadophylloides ) confirmed in Example 3 is also a gene encoding β-caryophyllene synthase. Β-caryophyllene synthase encoded by AsiTps1a , AsiTps1b , AsiTps1c, and AsiTps1d genes derived from wolfberry (Hyperoptera) is 90% or more amino acid sequence identity with β-caryophyllene synthase encoded by this AsTps1 gene gene showed that. For example, AsciTPS1a differs from AsTPS1 by 42 amino acids, and the amino acid sequence identity between them was 92.4%.

(Determining cDNA sequence of sesquiterpene synthase gene derived from taranoki)
Total RNA was extracted from shoots of Aralia elata (FIG. 16a) using RNeasy Plant Mini Kit (Qiagen). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. A degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of a higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)], PCR was performed using cDNA as a template under the conditions described in Non-Patent Document 4 to obtain an amplified fragment of 588 bp. The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [Aral. Germ. D5. 5race: 5'-TCCACCCTAAGATGTGTTGCCACGTA-3 '(SEQ ID NO: 34) and Aral. Germ.D5. 3race: 5'-TGCAAGGGATAGAGTGGTGGAGTGCT-3 '(SEQ ID NO: 35)] and Advantage2 Polymerase Mix (Clontech) are used to extend the fragments toward the 5' and 3 'ends according to the manufacturer's instructions. The full-length cDNA sequence was determined.

(Isolation and structural analysis of a full-length sesquiterpene synthase gene cDNA from Tarakin)
TR1.Nde-F (5′-AGACAGA CATAT GGCTGCTTATCTTCAGGCTTC -3 ′ (SEQ ID NO: 36), underline indicates Nde I recognition site) and TR1 specific to the full-length cDNA sequence obtained in Example 10 PCR using .Tanoki cDNA as a template using .Xho-R (5′-GAGGTAC CTCGAG TTATATTGGAACAGGATCTACGAGC-3 ′ (SEQ ID NO: 37), underlined indicates Xho I recognition site) and Advantage2 Polymerase Mix (Clontech) Reaction composition: according to manufacturer's instructions; reaction conditions: Denaturation 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 4 minutes, 5 cycles, then 94 ° C for 30 seconds 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 2 minutes). The obtained cDNA was cloned using the plasmid pGEM-T Easy Vector System (Promega), and the nucleotide sequence was determined.
These cDNA nucleotide sequences are shown in SEQ ID NOs: 38 and 40, and amino acid sequences of polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 39 and 41. Further, the genes (cDNAs) specified by SEQ ID NOs: 38 and 40 are named AeTps1a and AeTps1b, and the proteins (polypeptides) specified by SEQ ID NOs: 39 and 41 encoded by AeTps1a and AeTps1b are AeTPS1a and AeTPS1b , respectively. Named.
AeTps1a contained a 1,665 bp open reading frame (ORF) and encoded a protein (AeTPS1a) with a putative 554 amino acid residue, a molecular weight of 64.3 kDa, and a pI of 5.77. Based on homology searches against the GenBank / EMBL / DDBJ database, AeTPS1a is a known sesquiterpene synthase, α-copaene synthase derived from Eleutherococcus trifoliatus , 82%, and German magnolia derived from Vitis vinifera The amino acid sequence identity was 67% with D synthase (germacrene D synthase) and 65% with β-caryophyllene synthase.
On the other hand, AeTps1b contained an ORF of 1,665 bp and encoded a protein (AeTPS1b) with a putative 554 amino acid residue, a molecular weight of 64.3 kDa, and a pI of 5.84. Based on homology searches against the GenBank / EMBL / DDBJ database, AeTPS1a is a known sesquiterpene synthase, α-copaene synthase from Mitsukogi, 82%, 67% from European grape germin D synthase, and β-caryophyllene synthase. An amino acid sequence identity of 65% was shown.
The homology between AeTPS1a and AeTPS1b was 99.8% amino acid sequence identity. The difference between the two was due to a single amino acid substitution, namely the 539th Met to Arg substitution. From the homology with known sesquiterpene synthases, it was confirmed that AeTPS1a and AeTPS1b belong to the TPS-a subfamily which is a group of angiosperm sesquiterpenes and diterpene synthases. In addition, AeTPS1a and AeTPS1b contained the motifs RxR, DDxxD, (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 17).
On the other hand, AeTPS1a and AeTPS1b were predicted to have a plastid transit peptide, unlike ordinary known sesquiterpene synthases. FIG. 17 shows a comparison (alignment) of amino acid sequences between these two proteins and known enzymes having high homology with them.

(Functional analysis of two kinds of sesquiterpene synthase genes derived from Taranoki)
Two full-length cDNA sequences of two sesquiterpene synthase genes AeTps1a and AeTps1b derived from taranoki were excised from the plasmid into which they were inserted using the restriction enzyme Nde I- Xho I, and the E. coli vector pETDuet-1 (Novagen) Ligated to the Nde I- Xho I site of the cloning site (MCS2), pET-AeTps1a and pET-AeTps1b plasmids were prepared (FIG. 18).
E. coli BL21 (DE3) is transformed with either plasmid pET-AeTps1a or pET-AeTps1b and plasmid pAC-Mev / Scidi (Non-patent Document 7), and the resulting recombinant E. coli is cultured. Sesquiterpene was extracted from the cultured Escherichia coli cells and subjected to gas chromatography mass spectrometry (GC-MS). These methods are as described in Non-Patent Documents 2, 3, 4, and 7.
The analysis by the GC-MS, the chromatogram of the ethyl acetate extracts of E. coli strains harboring the vector pET21a and plasmids pAC-Mev / Scidi containing no AeTps1 (control), also confirmed the generation of any sesquiterpene In contrast, in the experimental section using E. coli containing either plasmid pET-AeTps1a or pET-AeTps1b and plasmid pAC-Mev / Scidi during culture, a new substance was added to the ethyl acetate extract from the cells. A peak was seen. By comparison of mass spectra, this peak (peaks 10 and 11 in FIG. 19) was considered to be β-cubebene (FIG. 19).
GC retention indices (RI) of the DB-Wax column used for GC analysis is high, with 96% agreement between the mass spectrum of the peaks (peaks 10 and 11) in the GC analysis and the Kubeben mass spectrum in the database. The value (see: HS Choi, M. Sawamura (2000) Composition of the Essential Oil of Citrus tamurana Hort. Ex Tanaka (Hyuganatsu). J. Agric. Food Chem. 48: 4868-4873) The literature value for -Kubeben was almost consistent with 1552, so the peak was estimated to be β-cubeben.
Therefore, it was estimated that AeTps1a and AeTps1b are genes encoding β-cubebene synthase. The chemical structure of β-cubeben is shown in FIG.
From the above results, β-cubeben can be selectively produced using Escherichia coli introduced with either plasmid pET-AeTps1a or pET-AeTps1b and plasmid pAC-Mev / Scidi.

(Determining cDNA sequence of Udo-derived sesquiterpene synthase gene)
Total RNA was extracted from shoots of Udo ( Aralia cordata ) (FIG. 20) using RNeasy Plant Mini Kit (Qiagen). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)] was performed using cDNA as a template under the conditions described in Non-Patent Document 4 to obtain an amplified fragment of 538 bp.
The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [UD1.5race: 5′-CATGCCCTTCACATCTTCCACAATG-3 ′ (SEQ ID NO: 42) and UD1.3race: 5 ′ -Using GCCTCGTTACTCTCTTGCACGGATCA-3 '(SEQ ID NO: 43)] and Advantage2 Polymerase Mix (Clontech), the fragments were extended toward the 5' end and 3 'end according to the manufacturer's instructions, and the full-length cDNA sequence was determined.

(Isolation and structural analysis of five full-length sesquiterpene synthase gene cDNAs)
A pair of oligonucleotide primers specific for the full-length cDNA sequence obtained in Example 13 UD1. Fse-F {5′-AGACAGA GGCCGGCC ACATGGCCCTAACAGTTACATCTAATGA -3 ′ (SEQ ID NO: 44), underlined indicates FseI recognition site} and UD1. PCR (reaction) using Xho-R {5'-GAGGTAC CTCGAG TTATCCCTCAATGGGCACAGG -3 '(SEQ ID NO: 45), underlined indicates Xho I recognition site} and Advantage2 Polymerase Mix (Clontech) Composition: according to manufacturer's instructions; reaction conditions: denaturation 94 ° C. for 2 minutes, then 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 4 minutes, 5 cycles, then 94 ° C. for 30 seconds, 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 2 minutes).
The obtained DNA fragment was cloned by the plasmid pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. These cDNA nucleotide sequences are shown in SEQ ID NOs: 46, 48, 50, 52 and 54, and amino acid sequences of polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 47, 49, 51, 53 and 55. See also SEQ ID NO: AcTps1a gene (cDNA) which is identified by 46, 48, 50 and 52, AcTps1b, named AcTps1c and AcTps1d, AcTps1a, AcTps1b, SEQ ID NO: 47, 49 and 51 and 53 AcTps1c and AcTps1d code to The proteins (polypeptides) identified by are designated AcTPS1a, AcTPS1b, AcTPS1c and AcTPS1d, respectively.
Furthermore, the gene (cDNA) specified by SEQ ID NO: 54 was named AcTps2, and the protein (polypeptide) specified by SEQ ID NO: 55 encoded by AcTps2 was named AcTPS2.
AcTps1a contained a 1,668 bp open reading frame (ORF) and encoded a protein (AcTPS1a) with a putative 555 amino acid residue, a molecular weight of 64.3 kDa, and a pI of 5.30. AcTPS1a is derived from the known sesquiterpene synthase, Cichorium intybus, and 59% from the known sesquiterpene synthase (germacrene A synthase), Ixeris dentata var. Albiflora It showed 59% amino acid sequence identity with guaiadiene synthase and 58% with sunflower ( Helianthus annuus ) -derived germacrene A synthase 3 (germacrene A synthase 3).
AcTps1b contained a 1,668 bp ORF and encoded a protein (AcTPS1b) with a putative 555 amino acid residue, a molecular weight of 64.3 kDa, and a pI of 5.41. Based on homology searches against GenBank / EMBL / DDBJ databases, AcTPS1b is a known sesquiterpene synthase, 59%, and guaiadiene synthase, 59%, sunflower-derived gel maculene A. The amino acid sequence identity of enzyme 3 and 59% was shown.
AcTps1c contained an ORF of 1,668 bp and encoded a protein (AcTPS1c) with a putative 555 amino acid residue, a molecular weight of 64.2 kDa, and a pI of 5.47. According to the homology search against GenBank / EMBL / DDBJ database, AcTPS1c is a known sesquiterpene synthase, 59%, white crab-derived guaiadiene synthase (59%), 59%, sunflower-derived gel magnone A synthase (3). The amino acid sequence identity was 58%.
AcTps1d contained an ORF of 1,668 bp and encoded a protein (AcTPS1d) with a putative 555 amino acid residue, a molecular weight of 64.2 kDa, and a pI of 5.41. According to the homology search against the GenBank / EMBL / DDBJ database, AcTPS1c is a known sesquiterpene synthase, 59%, and a white crab-derived guaiadiene synthase, 59%, and a sunflower-derived gel magnone A synthase, 3 and 58. % Amino acid sequence identity.
On the other hand, AcTps2 contained an ORF of 1,668 bp and encoded a protein (AcTPS2) with a putative 555 amino acid residue, a molecular weight of 64.4 kDa, and a pI of 5.11. According to homology search, AcTPS2 is a known sesquiterpene synthase ( Cichorium intybus ) derived from macchirane A synthase and 59%, white crab ( Ixeris dentata var. Albiflora ) guaiadiene synthase It showed 59% amino acid sequence identity (germacrene A synthase 3) and 58% amino acid sequence identity (Helianthus annuus).
AcTPS1a, AcTPS1b, AcTPS1c and AcTPS1d are homologous: AcTPS1a and AcTPS1b 99.1%, AcTPS1a and AcTPS1c 98.4%, AcTPS1a and AcTPS1d 99.1%, AcTPS1b and AcTPS1c 98.9%, AcTPS1b 99.6% AcTPS1c and AcTPS1d showed 98.9% amino acid sequence identity. The difference between AcTPS1a and AcTPS1b was due to five amino acid substitutions, ie, the 17th Gly to Ser substitution (Gly17Ser, and so on), Glu131Lys, Thr322Ser, Pro508Ser, and Phe516Lys. The difference between AcTPS1a and AcTPS1c was due to nine amino acid substitutions, namely Ser126Pro, Glu131Lys, Phe174Lys, Thr322Ser, Glu481Val, Pro508Ser, Phe516Lys, Val525Ile, Val543Ile. The difference between AcTPS1a and AcTPS1d was due to five amino acid substitutions, namely Glu131Lys, Met151Thr, Thr322Ser, Pro508Ser and Phe516Lys. The difference between AcTPS1b and AcTPS1c was due to six amino acid substitutions, namely Ser17Gly, Ser126Pro, Phe174Lys, Glu481Val, Val525Ile, and Val543Ile. The difference between AcTPS1b and AcTPS1d was due to two amino acid substitutions, Ser17Gly and Met151Thr. The difference between AcTPS1c and AcTPS1d was due to six amino acid substitutions, namely Pro126Ser, Met151Thr, Lys174Phe, Val481Glu, Ile525Val, and Ile543Val. On the other hand, regarding the homology between AcTPS1a, AcTPS1b, AcTPS1c and AcTPS1d and AcTPS2, AcTPS1a and AcTPS2 are 94.1%, AcTPS1b and AcTPS2 are 94.2%, AcTPS1c and AcTPS2 are 93.9%, and AcTPS1d and AcTPS2 are 4.2% amino acids. The sequence identity was shown. The difference between AcTPS1a, AcTPS1b, AcTPS1c, AcTPS1d and AcTPS2 is 31 amino acid substitutions, i.e. ), Gln335Glu, Phe367Leu, His368Gln, Ser390Ile, His410Asp, Ile412Val, Gly421Asp, Ser446Ala, Val453Ala, Ser455Arg, Pro456Gln, Lys459Gln, Lys461Arg, Thr465Ile, Ser519GTP , By Leu532Phe, Thr533Ala. Due to its homology with known sesquiterpene synthases, AcTPS1a, AcTPS1b, AcTPS1c, AcTPS1d and AcTPS2 are TPS-a subfamily, a group of angiosperm sesquiterpenes and diterpene synthases (see J. Bohlmann, G. Meyer). -Gauen, R. Croteau (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. USA 95: 4126-4133).
Furthermore, AcTPS1a, AcTPS1b, AcTPS1c, AcTPS1d and AcTPS2 have a motif called RxR, DDxxD, (N / D) Dxx (S / T) xxxE {called NSE / DTE motif} highly conserved in sesquiterpene synthases. (Figure 21).
On the other hand, it was predicted that AcTPS1a, AcTPS1b, AcTPS1c, AcTPS1d, and AcTPS2 have a plastid transit peptide, unlike ordinary known sesquiterpene synthases. FIG. 21 shows a comparison (alignment) of amino acid sequences of these five proteins and known enzymes having high homology with them.

(Functional analysis of five sesquiterpene synthase genes derived from Udo)
AcTps1a , AcTps1c , AcTps1d , AcTps1d and AcTps2 full-length cDNA sequences, which are five kinds of sesquiterpene synthase genes derived from Udo, were excised from the plasmid into which these were inserted with the restriction enzyme Fse I- Xho I, and the E. coli vector pETDuet- PET-AcTps1a, pET-AcTps1b, pET-AcTps1c, pET-AcTps1d and pET-AcTps2 were prepared by ligating to the Fse I- Xho I site of the multicloning site (MCS2) of 1 (Novagen) (FIG. 22). ). The trait of E. coli BL21 (DE3) using any one of plasmids pET-AcTps1a, pET-AcTps1b, pET-AcTps1c, pET-AcTps1d, pET-AcTps2 and plasmid pAC-Mev / Scidi (Non-patent Document 7) The resulting recombinant Escherichia coli was cultured, sesquiterpenes were extracted from the cultured Escherichia coli cells, and gas chromatography mass spectrometry (GC-MS) was further performed. These methods are as described in Non-Patent Documents 2, 3, 4, and 7.

The analysis by the GC-MS, the chromatogram of the E. coli strain ethyl acetate extracts which holds the vector pET21a and plasmids pAC-Mev / Scidi containing no AcTps1 (control), also not confirmed formation of any sesquiterpene In contrast, in an experimental group using E. coli containing one of the plasmids pET-AcTps1a, pET-AcTps1b, pET-AcTps1c, pET-AcTps1d and the plasmid pAC-Mev / Scidi, ethyl acetate from the cells was used. A new peak was observed in the extract. Comparison of mass spectra confirmed that these peaks (peaks 12, 13, 14 and 15 in FIG. 23) were β-elemene (FIG. 23).
Therefore, when overprinted with a preparation of β-elemene (manufactured by AvaChem Scientific), the retention time by GC analysis was completely consistent.
From the above results, it was confirmed that AcTps1a , AcTps1b , AcTps1c and AcTps1d are genes encoding β-elemene synthase.
Similarly, the chromatogram of ethyl acetate extracts of E. coli strains harboring the vector pET21a plasmid pAC-Mev / Scidi containing no AcTps2 (control), the generation of any sesquiterpene was not confirmed to, In the experimental group using E. coli containing plasmid pET-AcTps2 and plasmid pAC-Mev / Scidi, a new peak was observed in the ethyl acetate extract from the cells. By comparison of mass spectra, these peaks (peaks 16 and 17 in FIG. 24) were considered to be α-neoclovene and α-humulene (FIG. 24). Therefore, when the sample was overlaid with the α-humulene sample, the retention time of peak 17 by GC analysis completely matched.
Since the agreement between the mass spectrum of peak 16 in GC analysis and the α-neocloben mass spectrum in the database was very high at 99%, it was estimated that the peak was α-neocloben.
Thus, AcTps2 was considered to be a gene encoding α-neoclovene / α-humulene synthase. The chemical structures of α-neocloben and α-humulene are shown in FIG.
Based on the above results, selective production of β-elemen using one of the plasmids pET-AcTps1a, pET-AcTps1b, pET-AcTps1c, pET-AcTps1d, and Escherichia coli introduced with the plasmid pAC-Mev / Scidi. However, it was confirmed that α-neocloben and α-humulene can be produced using Escherichia coli introduced with plasmid pET-AcTps2 and plasmid pAC-Mev / Scidi.

(Determining cDNA sequence of monoterpene / sesquiterpene synthase gene derived from kosiabra)
Total RNA was extracted from shoots of Cosciabra ( Acanthopanax sciadophylloides ) (FIG. 1a) using RNeasy Plant Mini Kit (Qiagen). Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Degenerate oligonucleotide primer pair [Forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 19) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 4 -3 ′ (SEQ ID NO: 20)] was performed using cDNA as a template under the conditions described in Non-Patent Document 4, and an amplified fragment of 598 bp was obtained. The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer [sciad.lina.5race: 5′-GCCATTAATCCACCGATGTCTTCTCC-3 ′ (SEQ ID NO: 56) and sciad.lina. 3race: 5'-TTTCAGAGGGGGTGGAAGGACTTAGGC-3 '(SEQ ID NO: 57)] and Advantage2 Polymerase Mix (Clontech) were used to extend the fragment toward the 5' and 3 'ends according to the manufacturer's instructions, Were determined. Based on the obtained 5 'terminal sequence information, a newly synthesized oligonucleotide primer [linalool.5-RACE.332: 5'-GATGCCTAGCCGTTGCATGGTATCAA-3' (SEQ ID NO: 58)] was used to further add to the 5 'terminal. The full length cDNA sequence was determined.

(Isolation and structural analysis of one full-length monoterpene / sesquiterpene synthase gene cDNA from Kosiabra)
Oligonucleotide primer pair KS4.Nde-F (5′-AGACAGA CATATG GTTTCATATCATAATCCTTTAGAAG-3 ′ (SEQ ID NO: 59), underline indicates Nde I recognition site) and KS4 specific for the full-length cDNA sequence obtained in Example 16 PCR using Ksiabra cDNA as a template using .Kpn-R (5'-GAGGTAC GGTAC CTTAATACAGGGAACCCTCAAGG-3 '(SEQ ID NO: 60), underlined indicates Kpn I recognition site) and Advantage2 Polymerase Mix (Clontech) Reaction composition: according to manufacturer's instructions; reaction conditions: Denaturation 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes, 5 cycles, then 94 ° C for 30 seconds 25 cycles of 60 ° C. for 30 seconds and 72 ° C. for 2 minutes). The obtained cDNA was cloned using the plasmid pGEM-T Easy Vector System (Promega), and the nucleotide sequence was determined.
The cDNA nucleotide sequence is shown in SEQ ID NO: 61, and the amino acid sequence of the polypeptide encoded by the cDNA is shown in SEQ ID NO: 62. Further, the gene (cDNA) specified by SEQ ID NO: 61 was named AscTps3, and the protein (polypeptide) specified by SEQ ID NO: 62 encoded by AscTps3 was named AscTPS3.
AscTps3 contained a 1,749 bp open reading frame (ORF) and encoded a protein of putative 582 amino acid residues, molecular weight 67.0 kDa, and pI 5.67 (AscTPS3). By homology search against GenBank / EMBL / DDBJ databases, AscTPS3 is a known monoterpene / sesquiterpene synthase, linalool / nerolidol synthase from European grapevine ( Vitis vinifera ), 58%, and actinidia ( Actinidia arguta ) and linalool synthase (58%), chloroplast nerolidol synthase 1 (nerolidol synthase 1, chloroplastic-like) from European grape ( Vitis vinifera ) and 59% amino acid sequence identity (identity) Indicated.
The homology with known terpene synthases confirmed that AscTPS3 belongs to the TPS-g subfamily, a group of monoterpene synthases lacking the RRx8W motif (see N. Dudareva, D. Martin, CM Kish, N. Kolosova, N. Gorenstein, J. F? Ldt, B. Miller, J. Bohlmann (2003) (E) -β-Ocimene and Myrcene Synthase Genes of Floral Scent Biosynthesis in Snapdragon: Function and Expression of Three Terpene Synthase Genes of a New Terpene Synthase Subfamily. Plant Cell 15: 1227-1241). In addition, AscTPS3 contained the motifs RxR, DDxxD, (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in monoterpene / sesquiterpene synthases (Fig. twenty five).
On the other hand, AscTPS3 was predicted to have a plastid transit peptide, similar to the usual known monoterpene synthase. FIG. 25 shows a comparison (alignment) of amino acid sequences of this protein and known enzymes having high homology with these proteins.

(Functional analysis of one monoterpene / sesquiterpene synthase gene derived from Kosiabra)
A single monoterpene / sesquiterpene synthase gene AscTps3 full-length cDNA sequence derived from Cosiabra was excised from the plasmid into which it was inserted with the restriction enzyme Nde I- Kpn I, and the E. coli vector pETDuet-1 (manufactured by Novagen) The pET-AscTps3 plasmid was prepared by ligating to the Nde I- Kpn I site of the multiple cloning site (MCS2) (FIG. 26).
Escherichia coli BL21 (DE3) is transformed with plasmid pET-AscTps3 and plasmid pAC-Mev / Scidi (Non-patent Document 7), and the resulting recombinant Escherichia coli is cultured. The monoterpene / sesquiterpene was extracted and subjected to gas chromatography-mass spectrometry (GC-MS). These methods are as described in Non-Patent Documents 2, 3, 4, and 7.
The analysis by the GC-MS, the chromatogram of the ethyl acetate extracts of E. coli strains harboring the vector pET21a and plasmids pAC-Mev / Scidi containing no AscTps3 (control), the generation of any monoterpene / sesquiterpene However, in the experimental section using E. coli containing plasmid pET-AscTps3 and plasmid pAC-Mev / Scidi during culture, a new peak was observed in the ethyl acetate extract from the cells. . By comparison of mass spectra, these peaks (peaks 18 and 19 in FIG. 27) were considered to be linalool and nerolidol (FIG. 27).
Thus, when overprinted with a preparation of linalool and nerolidol (manufactured by Sigma-Aldrich), the retention time by GC analysis was completely consistent.
As described above, AscTps3 was found to be a gene encoding linalool / nerolidol synthase. The chemical structure of linalool is shown in FIG.
Moreover, from the above results, it was shown that selective production of linalool and nerolidol is possible using Escherichia coli introduced with plasmid pET-AscTps3 and plasmid pAC-Mev / Scidi.

(General)
In the present invention, β-caryophyllene synthase, germacrene D synthase, β-eudesmol synthase, β-cubeben synthase, β-elemene synthase, α-neocloben / α-humulene synthase, and linalool / nerolidol synthesis A new gene sequence of the enzyme was obtained. Furthermore, the novel synthase was confirmed to have low homology with other synthases.

  According to the present invention, β-caryophyllene, Germaclene D, β-eudesmol, β-cubene, β-elemene, α-neocloben, α-humulene, which are used for pharmaceuticals, agricultural chemicals, functional foods, fragrances, etc., or their raw materials, It has become possible to produce linalool or nerolidol efficiently and easily.

Claims (19)

The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 16 or 18;
(2) In the amino acid sequence described in SEQ ID NO: 16 or 18, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 16 or 18 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate of β-eudesmol,
(3) Farnesyl diphosphate having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 16 or 18 and substantially the same quality as the amino acid sequence shown in SEQ ID NO: 16 or 18 is converted into β-eudesmol. A gene encoding a polypeptide having an activity to convert,
(4) a gene comprising a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 or 17,
(5) a gene comprising a DNA comprising 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added in the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 or 17;
(6) A gene comprising 90% or more homology with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 or 17. Β-eudesmol synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 16 or 18,
(2) In the amino acid sequence described in SEQ ID NO: 16 or 18, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 16 or 18 An amino acid sequence having the activity of converting farnesyl diphosphate of β-eudesmol,
(3) Farnesyl diphosphate having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 16 or 18 and substantially the same quality as the amino acid sequence shown in SEQ ID NO: 16 or 18 is converted into β-eudesmol. Amino acid sequence having activity to convert. The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53,
(2) In the amino acid sequence of SEQ ID NO: 47, 49, 51 or 53, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 47, 49, 51 or 53 A gene encoding a polypeptide having an activity to convert farnesyl diphosphate substantially identical to the amino acid sequence described above into β-elemene,
(3) Farnesyl dinucleotide having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 and having substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 A gene encoding a polypeptide having an activity of converting phosphoric acid into β-elemente,
(4) a gene comprising a DNA comprising the base sequence set forth in SEQ ID NO: 46, 48, 50 or 52,
(5) a gene comprising a DNA comprising the base sequence set forth in SEQ ID NO: 46, 48, 50 or 52, wherein 1 to 50 base sequences are substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 46, 48, 50 or 52 and a DNA having a homology of 90% or more. Β-elemene synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53,
(2) In the amino acid sequence of SEQ ID NO: 47, 49, 51 or 53, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 47, 49, 51 or 53 An amino acid sequence having the activity of converting farnesyl diphosphate substantially the same amino acid sequence as described to β-elemene,
(3) Farnesyl dinucleotide having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 and substantially the same as the amino acid sequence set forth in SEQ ID NO: 47, 49, 51 or 53 An amino acid sequence having an activity of converting phosphoric acid into β-elemene. The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 ;
(2) In the amino acid sequence shown in SEQ ID NO: 6 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and farnesyl dilin is substantially the same as the amino acid sequence shown in SEQ ID NO: 6. A gene encoding a polypeptide having an activity of converting an acid into β-caryophyllene,
(3) having an amino acid sequence at least 90% homology to SEQ ID NO: 6, and having an activity of converting farnesyl diphosphate amino acid sequence substantially equivalent according to β- caryophyllene in SEQ ID NO: 6 A gene encoding a polypeptide,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 5 ,
(5) a gene comprising a DNA having a base sequence of SEQ ID NO: 5 wherein 1 to 50 base sequences are substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having the nucleotide sequence of SEQ ID NO: 5 and a DNA having 90% or more homology. Β-caryophyllene synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 6 ,
(2) In the amino acid sequence shown in SEQ ID NO: 6 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and farnesyl dilin is substantially the same as the amino acid sequence shown in SEQ ID NO: 6. An amino acid sequence having the activity of converting an acid to β-caryophyllene,
(3) having an amino acid sequence at least 90% homology to SEQ ID NO: 6, and having an activity of converting farnesyl diphosphate amino acid sequence substantially equivalent according to β- caryophyllene in SEQ ID NO: 6 Amino acid sequence. The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 or 10,
(2) In the amino acid sequence described in SEQ ID NO: 8 or 10, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 8 or 10 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate to germacrene D,
(3) Activity to convert farnesyl diphosphate having the homology of 90% or more with the amino acid sequence described in SEQ ID NO: 8 or 10 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 8 or 10 into Germacrene D A gene encoding a polypeptide having
(4) a gene comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 7 or 9,
(5) a DNA comprising a nucleotide sequence set forth in SEQ ID NO: 7 or 9, wherein the gene comprises a DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 7 or 9 and a DNA having 90% or more homology. Germacrene D synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence of SEQ ID NO: 8 or 10,
(2) In the amino acid sequence described in SEQ ID NO: 8 or 10, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and substantially the same as the amino acid sequence described in SEQ ID NO: 8 or 10 An amino acid sequence having the activity of converting farnesyl diphosphate to germane D
(3) Activity to convert farnesyl diphosphate having the homology of 90% or more with the amino acid sequence described in SEQ ID NO: 8 or 10 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 8 or 10 into Germacrene D Amino acid sequence having The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 or 41,
(2) In the amino acid sequence described in SEQ ID NO: 39 or 41, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence described in SEQ ID NO: 39 or 41 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate to β-cubeben,
(3) Convert farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 39 or 41 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 39 or 41 into β-cubeben A gene encoding a polypeptide having activity,
(4) a gene comprising a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 38 or 40,
(5) a DNA comprising a nucleotide sequence set forth in SEQ ID NO: 38 or 40, a gene comprising a DNA having 1 to 50 nucleotide sequences substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 38 or 40 and a DNA having 90% or more homology. Β-cubeben synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 39 or 41,
(2) In the amino acid sequence described in SEQ ID NO: 39 or 41, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and are substantially the same as the amino acid sequence described in SEQ ID NO: 39 or 41 An amino acid sequence having an activity of converting farnesyl diphosphate to β-cubeben;
(3) Convert farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 39 or 41 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 39 or 41 into β-cubeben An amino acid sequence having activity. The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 55,
(2) Farnesyl diphosphate having 1 to 20 amino acids substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 55, and substantially the same as the amino acid sequence described in SEQ ID NO: 55 A gene that encodes a polypeptide having an activity of converting to α-neocloben and α-humulene,
(3) Convert farnesyl diphosphate having homology of 90% or more with the amino acid sequence described in SEQ ID NO: 55 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 55 into α-neocloben and α-humulene A gene encoding a polypeptide having activity,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 54,
(5) a DNA comprising a base sequence set forth in SEQ ID NO: 54, a gene comprising DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 54 and having a homology of 90% or more. Α-neocloben / α-humulene synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 55,
(2) Farnesyl diphosphate having 1 to 20 amino acids substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 55, and substantially the same as the amino acid sequence described in SEQ ID NO: 55 An amino acid sequence having an activity to convert saponin to α-neocloben and α-humulene,
(3) Convert farnesyl diphosphate having homology of 90% or more with the amino acid sequence described in SEQ ID NO: 55 and substantially the same quality as the amino acid sequence described in SEQ ID NO: 55 into α-neocloben and α-humulene An amino acid sequence having activity. The gene shown in any one of the following (1) to (6) ;
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 62;
(2) In the amino acid sequence described in SEQ ID NO: 62, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl diphosphate is substantially the same as the amino acid sequence described in SEQ ID NO: 62 And a gene encoding a polypeptide having an activity to convert farnesyl diphosphate into linalool and nerolidol, respectively
(3) geranyl diphosphate and farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 62 and substantially the same as the amino acid sequence described in SEQ ID NO: 62, respectively, linalool and neroli A gene encoding a polypeptide having an activity of converting into a doll,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 61,
(5) a DNA comprising a base sequence set forth in SEQ ID NO: 61, a gene comprising DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added;
(6) A gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 61 and having a homology of 90% or more. Linalool / nerolidol synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 62,
(2) In the amino acid sequence described in SEQ ID NO: 62, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl diphosphate is substantially the same as the amino acid sequence described in SEQ ID NO: 62 And an amino acid sequence having an activity to convert farnesyl diphosphate into linalool and nerolidol, respectively.
(3) geranyl diphosphate and farnesyl diphosphate having a homology of 90% or more with the amino acid sequence described in SEQ ID NO: 62 and substantially the same as the amino acid sequence described in SEQ ID NO: 55, respectively, linalool and neroli An amino acid sequence having the activity of converting into a doll. A β-caryophyllene synthesizing agent comprising β-caryophyllene synthase having the amino acid sequence of any one of the following (1) to (3);
(1) the amino acid sequence set forth in SEQ ID NO: 6, 27, 29, 31 or 33,
(2) In the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 6, 27, 29, An amino acid sequence having an activity of converting farnesyl diphosphate substantially identical to the amino acid sequence of 31 or 33 to β-caryophyllene,
(3) It has 90% or more homology with the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33 and is substantially identical to the amino acid sequence described in SEQ ID NO: 6, 27, 29, 31 or 33 An amino acid sequence having the activity of converting homogeneous farnesyl diphosphate into β-caryophyllene.   Recombinant E. coli introduced with the gene according to any one of claims 1, 3, 5, 7, 9, 11, and 13. Recombinant Escherichia coli into which the following genes (1) and (2) are introduced in addition to the gene according to any one of claims 1, 3, 5, 7, 9, 11, and 13;
(1) Mevalonate pathway gene group that synthesizes from mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(2) Isopentenyl diphosphate isomerase gene. The mevalonate pathway gene group that synthesizes mevalonic acid or mevalonolactone to isopentenyl diphosphate and the isopentenyl diphosphate isomerase gene are genes derived from Streptomyces genus CL190 and / or genes derived from Saccharomyces cerevisiae Item 18. The recombinant Escherichia coli according to Item 17 . The recombinant E. coli according to any one of claims 16 to 18 is cultured in a medium containing mevalonic acid and / or mevalonolactone, or β-caryophyllene, gelmacrene D, β-eudesmol from a culture or cells obtained by culturing the recombinant Escherichia coli. , Β-cubeben, β-elemene, α-neocloben and α-humulene, or linalool and nerolidol, a method for producing sesquiterpenes