JP6399315B2 - Terpene synthase gene, acetoacetate hydrolase gene, and method for producing terpene - Google Patents

Description

The present invention relates to valeranol, hedicarol, α-selinen, α-copaene, β-osimene and α-farnesene, which are types of physiologically active sesquiterpenes useful for humans, and linalool, which is a type of monoterpene. And a protein synthesized from farnesyl diphosphate (geranyl diphosphate in the case of monoterpene), a gene encoding the protein, and a method for producing sesquiterpene (or monoterpene) using the gene.
The present invention further provides the production of a terpene by effectively culturing recombinant (genetic recombination) E. coli that biosynthesizes terpenes {sesquiterpenes, monoterpenes, tetraterpenes (carotenoids, etc.)}, and efficiently recovering the terpenes. Regarding the method.
The present invention further relates to an acetoacetate ester (acetoacetate fatty acid ester) hydrolase (esterase) gene and an efficient method for producing terpenes (isoprenoids, terpenoids) using the gene.

Terpenes (also called isoprenoids, terpenoids) are the most diverse group of compounds in nature, with over 23,000 species. The terpene contains 3,000 or more sesquiterpenes and 750 or more tetraterpenes (carotenoids). Terpenes contain many industrially useful products such as pharmaceuticals, agricultural chemicals, functional foods, fragrances, or the raw materials thereof.
However, terpenes have a small accumulation amount 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 technologies, materials, and genetic information for gene recombination (hereinafter referred to as “recombination”). Therefore, in recent years, research on technological development for mass production of terpenes 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 isopentenyl diphosphate (also referred to as isopentenyl pyrophosphate; hereinafter sometimes referred to as IPP), the first substrate of terpene biosynthesis.

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). In some cases, by sequential condensation with IPP using a synthetic enzyme (synthase), etc., it is converted to geranyl diphosphate having 10 carbon atoms (hereinafter sometimes referred to as GPP) and FPP having 15 carbon atoms. . 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.
Since E. coli does not biosynthesize the above terpenes, in order to biosynthesize these terpenes into E. coli, a biosynthetic enzyme gene (s) responsible for synthesis from FPP (or GPP) to the terpene is introduced into E. coli. Need to be expressed.

To date, the present inventors have mainly isolated sesquiterpene synthase genes from plants belonging to Zingiberaceae and Araliaceae and analyzed their functions (see Patent Documents 1 and 2). For example, 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).

Although Escherichia coli does not have a mevalonate pathway, studies have been actively conducted to introduce a gene group of enzymes of the mevalonate pathway (mevalonate pathway gene group; for example, Non-Patent Document 5) into E. coli. The first pioneering study was conducted by Kakinuma et al. (See Non-Patent Document 6). Kakinuma et al., A group of mevalonate pathway genes that synthesize from acetoacetyl-CoA (acetoacetyl-CoA; hereinafter referred to as acetoacetyl-CoA) to IPP, namely 3-hydroxy-3-methylglutaryl-CoA HMG-CoA) Synthetic enzyme (HMG-CoA synthase), HMG-CoA reductase (HMG-CoA reductase), mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), diphosphomevalonate 5 genes encoding decarboxylase (diphosphomevalonate decarboxylase; DPMVA decarboxylase), and a gene group encoding type 2 IPP isomerase (derived from Streptomyces genus CL190 strain; Non-patent document 5; Accession no AB037666; Hereinafter, it may be simply referred to as “mevalonate pathway gene group”}, Pantoea Ananate Introduced into Escherichia coli together with carotenoid (tetraterpene) biosynthetic genes derived from Pantoea ananatis { crtE , crtB , crtI , crtY , crtZ ; biosynthetic genes necessary to produce zeaxanthin from FPP} And expressed. This recombinant Escherichia coli can synthesize zeaxanthin efficiently by adding D-mevalonate lactone (D-mevalonate lactone; sometimes referred to as MVL) as a substrate to the medium and culturing. (Reference: Non-Patent Document 6).

The present inventors also used the same mevalonate pathway gene group derived from Streptomyces genus CL190 strain (Non-patent Document 5; Accession no AB037666) (including type 2 IPP isomerase gene) as in the case of Suganuma et al. The plasmid pACCYM184 was inserted into the E. coli vector pACYC184 {chloramphenicol (Cm) resistance} in a form (see Non-patent Document 7). Recombinant Escherichia coli introduced with plasmid pAC -Mev together with a plasmid for expression of the α-humulene synthase gene (α-humulene synthase; ZzZSS1 ) of 0.5 g / mL was added to the medium as a substrate. It was shown that 1 mg of α-humulene per 1 mL was produced by culturing (Reference: Patent Document 3, Non-Patent Document 7). In addition, when the ZzZSS1 gene was introduced into E. coli and expressed without introducing the plasmid pAC-Mev, the production amount of α-humulene remained at 1/11 that of the plasmid pAC-Mev. In addition, regarding the source of the mevalonate pathway gene group, the present inventors used the one derived from Streptomyces genus CL190 strain in the examples of the present specification, but use the corresponding gene group derived from yeast or other bacteria. (Reference: Non-Patent Document 8). In addition, when the β- eudesmol synthase gene ( ZzZSS2 ) of Hana ginger is introduced and expressed together with the group of mevalonate pathway genes, the recombinant Escherichia coli is cultured by adding MVL as a substrate to the culture medium to 100 per mL. It was shown to produce μg of β-eudesmol (see Non-Patent Document 4). In addition, it was shown that not only these sesquiterpenes but also the monoterpene linalool was produced in the above system (see: Patent Document 2).

Also, the end of the foreign genes in the plasmid pAC-Mev, prepare plasmid pAC-Mev / Scidi inserting the budding yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) 1 type of IPP isomerase from (IPP isomerase) gene (Scidi) (Reference: Patent Document 3, Non-Patent Document 7). pAC-Mev / Scidi terpene biosynthetic gene recombinant E. coli with (e.g., required lycopene biosynthesis of carotenoids crtE, crtB, crtI) when to introduce the expression, the amount of the terpene (e.g. lycopene) is, Scidi It was shown that the level was higher than when no gene was present (in the case of plasmid pAC-Mev) (see: Patent Document 3, Non-Patent Document 7). Incidentally, type 1 as the the IPP isomerase gene, in addition to baker's yeast Saccharomyces cerevisiae, astaxanthin-producing yeast xanthate Philo My Seth Dendororasu (Xanthophyllomyces dendrorhous) and astaxanthin production green alga Haematococcus pluvialis (Haematococcus pluvialis) eukaryotic, such as Those derived from organisms and some prokaryotes are known (eg Kajiwara, S., Fraser, PD, Kondo, K., and Misawa, N., Biochem. J. , 324: 421-426). , 1997), these genes can also be used in the present invention. Also in type 2 IPP isomerase gene, in addition to the genus Streptomyces CL190 strain, gene marine bacterium Brevundimonas (Brevundimonas) genus SD212 strain and marine bacteria Paracoccus (Paracoccus) genus prokaryotes such as N81106 strain has the knowledge (Misawa, N., Marine Drugs , 9 (5): 757-771, 2011), and these genes can also be used in the present invention.

As described above, the mevalonate pathway gene group (including type 2 IPP isomerase gene) is added to type 1 IPP isomerase (for example, Scidi gene) and introduced into E. coli, and MVL is added to the medium. As a result, it was shown that the amount of FPP (also GPP) produced in the recombinant Escherichia coli was increased by culturing. That is, sesquiterpene synthase (sesquiterpene synthase) gene using FPP as substrate, monoterpene synthase gene using GPP as substrate, or carotenoid (tetraterpene) starting from crtE (GGPP synthase gene) It was revealed that these products, such as sesqui (mono) terpenes or carotenoids, can be efficiently produced by introducing and expressing the biosynthetic genes together with the mevalonate pathway genes. Reference: Non-Patent Documents 7 and 8).

In addition to the mevalonate pathway gene group derived from the Streptomyces genus CL190 strain and the type 1 IPP isomerase gene ( Scidi gene), the present inventors further provided an acetoacetate-coenzyme A ligase derived from rat ( Rattus norvegicus ). A gene encoding (acetoacetate-CoA ligase; Aac1; acetoacetyl-CoA synthetase) was added, and the gene was introduced into E. coli and expressed (Reference: Patent Document 3, Non-Patent Documents 7 and 8). By culturing the recombinant Escherichia coli and adding acetoacetate (lithium acetoacetate; Li acetoacetate; hereinafter sometimes referred to as LAA) as a substrate to the medium, terpene can be efficiently treated as in the case of MVL addition. (Reference: Patent Document 3, Non-Patent Documents 7 and 8). In other words, it is 3.5 to 9.3 times higher (in the case of enzyme genes that synthesize carotenoids from FPP) than in the case of recombinant Escherichia coli in which only the enzyme gene that synthesizes terpenes from FPP is expressed. (Reference: Patent Document 3, Non-Patent Document 7).

  Furthermore, by using the system described above, it is possible to analyze the function of a newly obtained sesquiterpene (or monoterpene) synthase gene sequence whose function is unknown (see Non-Patent Document 8). That is, it is possible to examine whether recombinant E. coli having MVL or LAA assimilation ability introduced so as to express this gene sequence is producing sesquiterpenes (or monoterpenes). Does not produce terpenes). When a terpene is produced, by analyzing the chemical structure of the terpene, the terpene synthase gene sequence is an enzyme that synthesizes the product of a sesquiterpene having the chemical structure from FPP (or GPP monoterpene) This is because it is identified as a gene that encodes. More than 7,000 sesquiterpenes have already been isolated from plants, microorganisms, etc. (mainly plants), but many sesquiterpene synthase genes are still unknown. In the future, functional analysis of terpene synthase genes is expected to proceed with this system (see: Patent Documents 2 and 3, Non-Patent Document 8).

  Since MVL was an expensive reagent having an asymmetric carbon in the molecule (for example, 30,200 yen / g; Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), LAA, which is a relatively inexpensive reagent (for example, 11 , 100 yen / 1 g, 35,800 yen / 5 g; Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), the development of the utilization technology was significant. However, LAA is much more expensive than glucose (D-(+)-glucose; for example, 1,900 yen / 500 g; Tokyo Chemical Industry 2012-2013 catalog) as a medium sugar source. Development of a new substrate has been desired.

In general, sesquiterpenes (or monoterpenes), especially those that do not contain oxygen atoms in the molecule, are highly volatile, so they were produced during the cultivation of recombinant E. coli that biosynthesizes sesquiterpenes (or monoterpenes). There is a problem in that part of the sesquiterpene (or monoterpene) volatilizes, resulting in a reduction in product recovery. Therefore, a method in which about 20% of the medium amount of dodecane ( n- dodecane) is added and cultivated in a multi-layer, and the produced sesquiterpene (or monoterpene) is collected in the dodecane layer and used for GC-MS analysis has been frequently used. (Reference: Patent Documents 2 and 3, Non-Patent Document 7). However, since dodecane is difficult to remove with an evaporator, it has a problem that it is difficult to purify sesquiterpene (or monoterpene) collected in the dodecane layer.

  In general, when carotenoid products are obtained by culturing recombinant Escherichia coli that biosynthesizes tetraterpenes (carotenoids), the amount of carotenoid production per culture solution is considerably lower than other terpene products. It was. For example, the production amount per 1 mL of α-humulene, which is a sesquiterpene, is 1 mg (Ref: Patent Document 3, Non-Patent Document 7), whereas per 1 g (≈1 mL) of carotenoid lycopene. Was no more than 20 μg (see Non-Patent Document 9).

Patent 5457159 JP2013-63063A Patent 5405030

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 H. Alper, G. Stephanopoulos, Appl. Microbiol. Biotechnol. 78: 801-810, 2008

The present invention seeks to solve the following three related problems regarding the production of terpenes.
An object of the present invention is to obtain a novel enzyme gene necessary for the synthesis of sesquiterpene or monoterpene from FPP or GPP, and to provide a method for producing terpene using the obtained novel synthetic enzyme gene. (Problem 1).
The present invention further examines an effective culture method for E. coli and a method for efficiently producing and purifying terpenes when cultivating recombinant E. coli producing terpenes in a medium, and a method for efficiently producing terpenes. (Problem 2).
Furthermore, the present invention uses an acetoacetate ester (acetoacetate fatty acid ester) such as acetoacetate methyl ester or acetoacetate ethyl ester, which is an inexpensive substrate, and therefore a new acetoacetate hydrolase (esterase) gene is found. Another object is to provide a method for efficiently producing a terpene by utilizing a newly found esterase gene (Problem 3).

  In order to solve the above three related problems, the present inventors have conducted intensive research as follows.

<Problem 1>
(I) Obtaining a novel enzyme gene necessary for the synthesis of sesquiterpene or monoterpene from FPP or GPP, and providing a method for producing terpene using the obtained novel synthetic enzyme gene Problem 1.
Mevalonic acid isolated from Streptomyces genus CL190 strain (Non-patent Document 5; Accession no AB037666) for producing a large amount of FPP (or GPP) which is a substrate of sesquiterpene (or monoterpene) from LAA Pathway genes, ie HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase) In addition to 5 genes encoding diphosphomevalonate decarboxylase (DPMVA decarboxylase) and a gene encoding IPP isomerase (IPP isomerase; type 2), an IPP isomerase derived from Saccharomyces cerevisiae (IPP isomerase) isomerase; 1 type) encoding gene (Scidi) and La Use; (Aac1 acetoacetate-CoA ligase) encoding gene plasmid for expressing (Aacl) pAC-Mev / Scidi / Aacl { E. coli vector pACYC184 neither coenzyme A ligase - DOO (Rattus norvegicus) derived from acetoacetic acid; Chloramphenicol (Cm) resistance; Patent Literature 3, Non-Patent Literature 7} was prepared.
(Ii) cDNA is synthesized from total RNA extracted from Camellia flower plants and flowers of Freesia x hybrida , and bulbous tissue of Allium chinense G. Don. The full-length (sesqui) terpene synthase gene sequence was isolated and inserted into the E. coli expression vector pRSFDuet-1 (Novagen) to prepare the desired expression plasmid {kanamycin (Km) resistance}.
Each of the above plasmids is introduced into E. coli together with the plasmid prepared in (i), cultured in a medium containing LAA, Km, and Cm, and the sesquiterpene produced is analyzed to convert FPP to convert valeranol and / or Alternatively, valeranol / hedicarol synthase (synthase) gene that synthesizes hedicarol, α-serinen synthase gene that synthesizes α-serinen by converting FPP, and α-copaene that synthesizes FPP α -Copaene synthase gene and β-oscymene / α-farnesene synthase gene that synthesizes β-oscymene and α-farnesene by converting FPP were newly obtained. Furthermore, a linalool synthase gene that synthesizes linalool by converting GPP was newly obtained.
(Iii) E. coli expression of valeranol / hedicarol synthase gene, α-selinene synthase gene, α-copaene synthase gene, β-oscymene / α-farnesene gene, and linalool synthase gene obtained in (iii) One of the plasmids for use in humans is introduced into E. coli together with the plasmid prepared in (i) and cultured in a medium containing LAA, Km and Cm, thereby providing a kind of physiologically active sesquiterpene useful for humans A method for producing valeranol, hedicarol, α-selinen, α-copaene, β-oscymene and α-farnesene, and linalool, a kind of physiologically active monoterpene useful for humans We were able to.

<Problem 2>
(I) When culturing recombinant E. coli that biosynthesizes terpenes {sesquiterpenes, monoterpenes, tetraterpenes (carotenoids, etc.)} in a medium, an effective culture method for the E. coli, and efficient recovery of the terpenes It is the subject 2 of the present invention to examine a purification method and to provide a method for efficiently producing a terpene.
In general, sesquiterpenes (or monoterpenes), particularly those that do not contain oxygen atoms in the molecule, are highly volatile, so that the sesquiterpenes produced during the culture of recombinant E. coli that biosynthesizes sesquiterpenes (or monoterpenes) There is a problem in that part of the terpene (or monoterpene) volatilizes, resulting in a reduction in product recovery. Therefore, a method in which about 20% of the medium amount of dodecane ( n- dodecane) is added and cultivated in a multi-layer, and the produced sesquiterpene (or monoterpene) is collected in the dodecane layer and used for GC-MS analysis has been frequently used. (Reference: Patent Documents 2 and 3, Non-Patent Document 7). However, the present inventors often find that it is difficult to specify the structure of the produced sesquiterpene (or monoterpene) product only by GC-MS analysis (there is a problem with authenticity). As a result, it was concluded that NMR analysis was indispensable especially when there was no standard. On the other hand, in order to perform NMR analysis, it is necessary to purify the produced sesquiterpene (or monoterpene) product. However, since it is difficult to remove dodecane with an evaporator, the sesquiterpene (or monoterpene recovered in the dodecane layer is difficult to remove. The problem was that it was difficult to purify terpenes. Therefore, n -alkane having a carbon number of 10 or less with a low volatility temperature is added to about 20% of the amount of the medium, and the sesquiterpene (or monoterpene) product is recovered in the n -alkane layer, and the solvent is evaporated. I wanted to proceed with the purification by removing it. However, n -alkanes having 7 or less carbon atoms ( n -heptane) inhibited the growth of host E. coli. On the other hand, carbon atoms 8 (n - octane: n -octane) ~ carbons 10: n in (decane n -decane) - alkane did not inhibit growth of a host E. coli, moreover, conventional diaphragm evaporator decompression pump the solvent It was found that the purification can proceed efficiently. Thus, it was possible to solve the problem of examining an efficient method for recovering and purifying sesquiterpenes (or monoterpenes). Incidentally, n - octane or decane, a sesquiterpene (or monoterpene) to host the layered culture of biosynthesis, n - methods such that the product octane or decane layer recovered and purified has not been disclosed so far It was.
(Ii) The present inventors further added various sugars (about 1%) during the cultivation of recombinant E. coli that biosynthesizes tetraterpenes (carotenoids), and whether or not the amount of carotenoids per culture broth increases. investigated. As a result, when recombinant E. coli that biosynthesizes carotenoids is cultured in a medium containing fructose, galactose, trehalose, sorbitol, mannitol, and / or mannose, which are sugars that are not normally used in microorganism culture media, We found that the amount of carotenoids increased. As described above, an effective culture method for recombinant Escherichia coli producing carotenoids has been studied, and the problem of providing a method for efficiently producing carotenoids has been solved.

<Problem 3>
Use acetoacetate (acetoacetate acid methyl ester; MAA) or acetoacetate (acetoacetate: EAA) such as acetoacetic acid ethyl ester (EAA), which is an inexpensive substrate Therefore, terpenes {including sesquiterpenes, monoterpenes, and tetraterpenes (carotenoids) using the newly found esterase genes, including acetoacetate hydrolase (esterase) genes; terpenes are isoprenoids It is the third object of the present invention to provide a method for efficiently producing a terpenoid.
The prices of methyl acetoacetate (MAA) and ethyl acetoacetate (EAA) as the reagents were 2,800 yen / 500 g and 3,400 yen / 500 g, respectively ( Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog). Since the price of lithium acetoacetate (LAA) as a reagent was 35,800 yen / 5 g (Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), MAA and / or EAA was used instead of LAA. As a result, the cost can be significantly reduced.
Until now, there have been no reports of enzymes (esterases) or enzyme genes that can hydrolyze MAA or EAA (efficiently). Therefore, the present inventors obtained a base sequence from a genome sequence database for an ester hydrolase (esterase) gene containing 18 putative genes composed of 12 bacterial families classified from the primary amino acid sequence, and obtained a full-length sequence. Was isolated. Among the crude enzyme extract prepared from recombinant Escherichia coli introduced and expressed with these genes, synthesis of a significant amount of enzyme protein was confirmed in the soluble fraction in samples derived from 13 strains. When screening was performed using the hydrolytic activity for MAA or LAA as an index, three compounds having high acetoacetate hydrolyzing activity were found. Next, a plasmid (eg, pAC-Mev / Scidi / Aacl / pnbA) in which one of the above three genes was inserted into the plasmid pAC-Mev / Scidi / Aacl was prepared. The plasmid was introduced into E. coli together with a gene or gene group required for terpene (isoprenoid, terpenoid) synthesis from FPP (or GPP), and an evaluation test was performed. For example, the plasmid pAC-Mev / Scidi / Aacl, whether the gene group necessary for the synthesis of tetraterpene (carotenoid) lycopene or the gene necessary for the synthesis of sesquiterpene α-humulene is introduced. When introduced into E. coli together with / pnbA, MAA or EAA is assimilated at the same level or higher compared to LAA addition (positive control) in E. coli coexisting with plasmid pAC-Mev / Scidi / Aacl It was proved that we were able to achieve this task.

  The present invention has been completed based on the above findings. That is, the present invention provides the following [1] to [22].

[1] Acetoacetate (acetoacetate fatty acid ester) hydrolase (esterase) gene shown in any one of the following (1) to (7):
(1) a gene encoding a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 85 to 87,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 85 to 87, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 85 to 87 A gene encoding a polypeptide having an activity to hydrolyze an acetoacetate substantially the same as the amino acid sequence described in 1.
(3) 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 85 to 87, and substantially homogenous with the amino acid sequence described in any one of SEQ ID NOs: 85 to 87 A gene encoding a polypeptide having an activity of hydrolyzing acetoacetate,
(4) a gene comprising a DNA comprising the base sequence set forth in any one of SEQ ID NOs: 82 to 84 and 90,
(5) a gene comprising a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 82 to 84 and 90, wherein 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added; A gene comprising a DNA encoding a polypeptide having an activity of hydrolyzing acetoacetate,
(6) A gene comprising a DNA having a base sequence of any one of SEQ ID NOs: 82 to 84 and 90 and a DNA having 85% or more homology, and an acetoacetate ester (acetoacetate fatty acid ester) A gene comprising DNA encoding a polypeptide having an activity of hydrolyzing,
(7) It hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 82 to 84, 90, and acetoacetate (acetoacetate A gene comprising DNA encoding a polypeptide having an activity of hydrolyzing (fatty acid ester).

[2] Acetoacetate ester (acetoacetate fatty acid ester) hydrolase (esterase) having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence according to any one of SEQ ID NOs: 85 to 87,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 85 to 87, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 85 to 87 An amino acid sequence having an activity to hydrolyze an acetoacetate substantially the same as the amino acid sequence described in 1.
(3) 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 85 to 87, and substantially homogenous with the amino acid sequence described in any one of SEQ ID NOs: 85 to 87 An amino acid sequence having an activity of hydrolyzing acetoacetate.

[3] Recombinant E. coli introduced with the following genes (1) to (5):
(1) Gene or gene group of an enzyme that synthesizes a terpene (isoprenoid, terpenoid) from geranyl diphosphate or farnesyl diphosphate,
(2) type 1 and / or type 2 isopentenyl diphosphate isomerase gene or gene group,
(3) Mevalonate pathway genes that synthesize acetoacetyl-coenzyme A to isopentenyl diphosphate,
(4) Acetoacetate-coenzyme A ligase gene,
(5) Acetoacetate (acetoacetate fatty acid ester) hydrolase (esterase) gene.

[4] The recombinant Escherichia coli according to [3] above, wherein the acetoacetate (acetoacetate fatty acid ester) hydrolase (esterase) gene is the gene according to [1] above. .

[5] Obtaining a terpene from a culture or cells obtained by culturing the recombinant Escherichia coli according to [3] or [4] above in a medium containing methyl acetoacetate and / or ethyl acetoacetate A process for producing a terpene, characterized in that

[6] Gene shown in any one of the following (1) to (7):
(1) a gene encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 21 to 37,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 21 to 37, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 21 to 37 A gene encoding a polypeptide having an activity to convert farnesyl diphosphate substantially identical to the amino acid sequence described in 1 to valeranol and / or hedicariol,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 21 to 37 and is substantially the same as the amino acid sequence described in any one of SEQ ID NOs: 21 to 37 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate into valerianol and / or hedicariol,
(4) a gene comprising DNA consisting of the base sequence described in any one of SEQ ID NOs: 4 to 20,
(5) In the DNA consisting of the base sequence described in any one of SEQ ID NOs: 4 to 20, a gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added, And a gene comprising a DNA encoding a polypeptide having an activity of converting farnesyl diphosphate into valerianol and / or hedicariol,
(6) A gene comprising a DNA having a base sequence of any one of SEQ ID NOS: 4 to 20 and a DNA having 85% or more homology, and farnesyl diphosphate is converted to valerianol and / or heavy potash A gene consisting of DNA encoding a polypeptide having an activity of converting to oar,
(7) It hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 4 to 20, and farnesyl diphosphate is converted to valerianol and A gene comprising a DNA encoding a polypeptide having an activity to convert to hedicariol.

[7] Valerenol / Hedicariol synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence according to any one of SEQ ID NOs: 21 to 37,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 21 to 37, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 21 to 37 An amino acid sequence having an activity to convert farnesyl diphosphate substantially the same as the amino acid sequence described in 1 to valerianol and / or hedicariol,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 21 to 37 and is substantially the same as the amino acid sequence described in any one of SEQ ID NOs: 21 to 37 An amino acid sequence having an activity of converting farnesyl diphosphate into valeranol and / or hedicariol.

[8] 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 any one of SEQ ID NOs: 49 to 51,
(2) In the amino acid sequence according to any one of SEQ ID NOs: 49 to 51, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 49 to 51 A gene encoding a polypeptide having an activity to convert farnesyl diphosphate having substantially the same amino acid sequence as described above into α-serinene,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 49 to 51 and is substantially identical to the amino acid sequence described in any one of SEQ ID NOs: 49 to 51 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate to α-serinene,
(4) a gene comprising a DNA comprising the base sequence described in any one of SEQ ID NOs: 46 to 48,
(5) In the DNA consisting of the base sequence described in any one of SEQ ID NOs: 46 to 48, a gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added, And a gene comprising a DNA encoding a polypeptide having an activity of converting farnesyl diphosphate into α-serinene,
(6) A gene comprising a DNA having a nucleotide sequence of any one of SEQ ID NOs: 46 to 48 and a DNA having a homology of 85% or more, and an activity for converting farnesyl diphosphate into α-selinen A gene comprising a DNA encoding a polypeptide having
(7) It hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 46 to 48, and farnesyl diphosphate is converted to α-serinen. A gene comprising a DNA encoding a polypeptide having an activity of converting to.

[9] α-Serene synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence according to any one of SEQ ID NOs: 49 to 51,
(2) In the amino acid sequence according to any one of SEQ ID NOs: 49 to 51, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 49 to 51 An amino acid sequence having the activity of converting farnesyl diphosphate substantially the same as the amino acid sequence described in claim 1 to α-serinene,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 49 to 51 and is substantially identical to the amino acid sequence described in any one of SEQ ID NOs: 49 to 51 An amino acid sequence having an activity of converting farnesyl diphosphate to α-serinene.

[10] Gene shown in any one of the following (1) to (7):
(1) a gene encoding a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 62 to 69,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 62 to 69, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 62 to 69 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate substantially identical to the amino acid sequence described in one to α-copaene,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 62-69 and is substantially the same as the amino acid sequence described in any one of SEQ ID NOs: 62-69 A gene encoding a polypeptide having an activity of converting farnesyl diphosphate into α-copaene,
(4) a gene comprising a DNA comprising the base sequence set forth in any one of SEQ ID NOs: 54 to 61,
(5) In the DNA consisting of the base sequence described in any one of SEQ ID NOs: 54 to 61, a gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added, And a gene comprising a DNA encoding a polypeptide having an activity of converting farnesyl diphosphate into α-copaene,
(6) A gene consisting of a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 54 to 61 and a DNA having a homology of 85% or more and an activity for converting farnesyl diphosphate into α-copaene A gene comprising a DNA encoding a polypeptide having
(7) It hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 54 to 61, and farnesyl diphosphate is converted to α-copaene. A gene comprising a DNA encoding a polypeptide having an activity of converting to.

[11] α-Copaene synthase having the amino acid sequence of any one of the following (1) to (3):
(1) the amino acid sequence according to any one of SEQ ID NOs: 62 to 69,
(2) In the amino acid sequence described in any one of SEQ ID NOs: 62 to 69, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and any one of SEQ ID NOs: 62 to 69 An amino acid sequence having an activity to convert farnesyl diphosphate substantially identical to the amino acid sequence described in 1 to α-copaene,
(3) It has 90% or more homology with the amino acid sequence described in any one of SEQ ID NOs: 62-69 and is substantially the same as the amino acid sequence described in any one of SEQ ID NOs: 62-69 An amino acid sequence having an activity of converting farnesyl diphosphate to α-copaene.

[12] The linalool synthase 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: 75;
(2) In the amino acid sequence set forth in SEQ ID NO: 75, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl dilin is substantially the same as the amino acid sequence set forth in SEQ ID NO: 75 A gene encoding a polypeptide having an activity of converting an acid into linalool,
(3) a polypeptide having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 75 and having an activity of converting geranyl diphosphate substantially the same as the amino acid sequence set forth in SEQ ID NO: 75 to linalool The gene encoding,
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 74,
(5) In the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 74, the gene is composed of DNA in which 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added, and geranyl diphosphate is converted to linalool. A gene comprising DNA encoding a polypeptide having an activity of converting to
(6) From a DNA encoding a polypeptide which is a gene consisting of a DNA having the nucleotide sequence of SEQ ID NO: 74 and a DNA having 85% or more homology and having an activity of converting geranyl diphosphate to linalool The gene,
(7) A polypeptide that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 74 and has an activity of converting geranyl diphosphate into linalool. A gene consisting of DNA that encodes.

[13] Linalool synthase having any one of the following amino acid sequences (1) to (3):
(1) the amino acid sequence set forth in SEQ ID NO: 75,
(2) In the amino acid sequence set forth in SEQ ID NO: 75, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and geranyl dilin is substantially the same as the amino acid sequence set forth in SEQ ID NO: 75 An amino acid sequence having the activity of converting an acid to linalool,
(3) An amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 75 and having an activity of converting geranyl diphosphate having substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 75 to linalool .

[14] 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: 77;
(2) Farnesyl dilin in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 77 and are substantially the same as the amino acid sequence set forth in SEQ ID NO: 77 A gene encoding a polypeptide having an activity of converting an acid into β-cymene and α-farnesene,
(3) Farnesyl diphosphate having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 77 and substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 77 is converted into β-oscymene and α-farnesene. A gene encoding a polypeptide having the activity of
(4) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 76;
(5) In the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 76, the gene is composed of DNA in which 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added, and farnesyl diphosphate is β -A gene comprising DNA encoding a polypeptide having an activity to convert to ocimene and α-farnesene,
(6) A gene comprising a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 76 and a DNA having 85% or more homology and having an activity of converting farnesyl diphosphate into β-oscymene and α-farnesene A gene consisting of DNA encoding a peptide.
(7) It hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 76, and converts farnesyl diphosphate into β-oscymene and α-farnesene. A gene comprising DNA encoding a polypeptide having activity.

[15] β-cymene / α-farnesene 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: 77,
(2) Farnesyl dilin in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 77 and are substantially the same as the amino acid sequence set forth in SEQ ID NO: 77 An amino acid sequence having an activity of converting an acid into β-cymene and / or α-farnesene,
(3) Farnesyl diphosphate having a homology of 90% or more with the amino acid sequence of SEQ ID NO: 77 and substantially the same as the amino acid sequence of SEQ ID NO: 77 is converted to β-oscymene and / or α-farnesene An amino acid sequence having the activity of converting to.

[16] A recombinant Escherichia coli into which the gene according to any one of [6], [8], [10], [12] and [14] is introduced.

[17] The recombinant Escherichia coli according to [16] above is cultured from a culture or cells obtained by culturing in a medium. Valeranol, hedicarol, α-selinen, α-copaene, linalool, β-oscymene, And a process for producing terpenes, wherein α-farnesene is obtained.

[18] A terpene synthase gene-bearing host is cultured in a medium overlaid with one or more of n -alkanes having n -octane to decane (8 to 10 carbon atoms), and terpenes are recovered from the alkane layer after culture. And then purifying the terpene.

[19] A method for producing a terpene, wherein the host having the terpene synthase gene according to [18] is a recombinant Escherichia coli into which the terpene synthase gene has been introduced.

[20] The terpene according to the above [18] or [19] is valerianol, hedicarol, α-selinen, α-copaene, linalool, β-osimene and α-farnesene, and γ-amorphene, germane D Terpene, characterized in that it is aromadendrene, cis -muurola-4 (15), 5-diene { cis- muurola-4 (15), 5-diene}, β-cubeben and / or δ-kadinene Manufacturing method.

[21] Recombinant Escherichia coli into which a tetraterpene (carotenoid) synthase gene group has been introduced is cultured in a medium containing fructose, galactose, trehalose, sorbitol, mannitol and / or mannose. (Carotenoid) is extracted, The manufacturing method of the terpene characterized by the above-mentioned.

[22] A method for producing a terpene, wherein the tetraterpene (carotenoid) according to [21] is lycopene, β-carotene and / or zeaxanthin.

  In the present specification, “stringent conditions” in a 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 using hybridization encodes an active polypeptide is determined by, for example, introducing the DNA into Escherichia coli or the like and expressing it, and the Escherichia coli or the like producing the target protein. You can find out if you can do it. DNA obtained by the hybridization is each gene (for example, SEQ ID NO: 4-20, SEQ ID NO: 46-48, SEQ ID NO: 54-61, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 82-84 and SEQ ID NO: 90). Usually has high identity. High identity refers to 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity.

  The present invention is a kind of physiologically active sesquiterpenes useful for humans, valeranol, hedicarol, α-selinen, α-copaene, β-osimene, α-farnesene, and a monoterpene A protein (enzyme) that synthesizes linalool using FPP (only linalool GPP) as a substrate and a gene encoding the protein could be obtained and provided. According to the present invention, it was possible to provide a method for producing valeranol, hedicarol, α-selinene, α-copaene, β-oscymene, α-farnesene, and linalool in Escherichia coli and the like.

Furthermore, the present invention further includes a method in which about 20% of an n -alkane having 8 to 10 carbon atoms is added to the cultivated layer and cultured from the time when the recombinant host (E. coli) that synthesizes sesquiterpene (or monoterpene) is cultured in a medium. An efficient recovery and purification method for terpenes was devised, and a method for efficiently producing terpenes could be provided. The present invention also provides an effective culture method for recombinant Escherichia coli for synthesizing tetraterpenes (carotenoids), wherein fructose, galactose, trehalose, sorbitol, mannitol, and / or mannose is added to the medium and cultured. Developed and provided a method for efficiently producing tetraterpenes.

  The present invention further uses acetoacetic acid ester (acetoacetic acid fatty acid ester) hydrolase (esterase) gene to terpene in order to use acetoacetic acid methyl ester or acetoacetic acid ethyl ester, which is an inexpensive substrate. It was possible to provide a method for efficiently producing (isoprenoid, terpenoid).

(A) Flower of Camellia x hiemalis , and (b) The figure showing the above-ground part. The figure showing the amino acid sequence alignment of ChTPS1-1 and the known ginger plant sesquiterpene synthase (synthase). ZoTPS1 represents Zingiber officinale Roscoe-derived β-bisaboren synthase (BAI67934). 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. RDR, DDxxD, (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pRSF-ChTps1-x. CDNA corresponding to the ORF of ChTps1-x (1,665 bp or 1,563 bp) is inserted between the vector pRSFDuet-1 of Bgl I- Xho I. The figure showing the GC-MS analysis result of the sesquiterpene product of recombinant Escherichia coli. (A) GC chromatogram of ethyl acetate extracts of E. coli strains harboring plasmid pRSF-ChTps1-1 plasmid pAC-Mev / Scidi / Aacl including ChTps1-1. New peaks 1 and 2 are indicated by arrows. (B) GC chromatogram of ethyl acetate extracts of E. coli strains harboring plasmid pRSF-ChTps1-10 plasmid pAC-Mev / Scidi / Aacl including ChTps1-10. New peaks 1 and 2 are indicated by arrows. (C) GC chromatogram (control) of an ethyl acetate extract of Escherichia coli strain carrying the vector pRSFDuet-1 not containing ChTps1-x and the plasmid pAC-Mev / Scidi / Aacl. The figure which shows the structural-change prediction figure (b) of ¹ H NMR spectrum (a) of hedicarol at room temperature. The figure which shows the biosynthesis from the chemical structure and farnesyl diphosphate (FPP) of valeranol (Valerianol) and Hedycaryol (Hedycaryol) which are the sesquiterpenes which can be produced by this invention. Hedicalol is converted into Elemol by heat such as GC analysis. The figure showing the flower (a) of freesia ( Freesia x hybrida ) Airy purple, and the flower (b) of Airy peach. The figure showing the amino acid sequence alignment of Fh1TPS-y (y is 1-3) and the known ginger plant sesquiterpene synthase (synthase). ZoTPS1 represents Zingiber officinale Roscoe-derived β-bisaboren synthase (BAI67934). 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. RDR, DDxxD, (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pRSF-Fh1Tps-y and pRSF-Fh6Tps-z. CDNA corresponding to each ORF of Fh1Tps-y and Fh6Tps-z (1,701 bp) and cDNA (1,713 bp) is inserted between the vector pRSFDuet-1 of Nde I- Kpn I. The figure showing the GC-MS analysis result of the sesquiterpene product of recombinant Escherichia coli. (A) GC chromatogram of ethyl acetate extracts of the plasmid containing Fh1Tps -1 pRSF-Fh1Tps-1 and plasmid pAC-Mev / Scidi / Aacl E. coli strains harboring. A new peak 1 is indicated by an arrow. (B) GC chromatogram of ethyl acetate extracts of the plasmid containing the Fh1Tps -2 pRSF-Fh1Tps-2 plasmid pAC-Mev / Scidi / Aacl E. coli strains harboring. A new peak 1 is indicated by an arrow. (C) plasmid containing Fh1Tps -3 pRSF-Fh1Tps-3 plasmid pAC-Mev / Scidi / Aacl GC chromatogram of ethyl acetate extracts of E. coli strains harboring. A new peak 1 is indicated by an arrow. (D) GC chromatogram (control) of an E. coli strain ethyl acetate extract containing the vector pRSFDuet-1 not containing Fh1Tps-y and the plasmid pAC-Mev / Scidi / Aacl. Shows the chemical structure of α-selinene and α-copaene, biosynthesis from farnesyl diphosphate (FPP), sesquiterpenes derived from freesia flowers that can be produced in the present invention. Figure. The figure showing the amino acid sequence alignment of Fh6TPS-z (z is 1-8) and the known ginger plant sesquiterpene synthase (synthase). ZoTPS1 represents Zingiber officinale Roscoe-derived β-bisaboren synthase (BAI67934). 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. RDR, DDxxD, (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks. The figure showing the GC-MS analysis result of the sesquiterpene product of recombinant Escherichia coli. Of the 8 Fh6Tps-z , the top 4 analysis results with the highest activity are shown. (A) GC chromatogram of ethyl acetate extracts of the plasmid containing Fh6Tps -1 pRSF-Fh6Tps-1 and plasmid pAC-Mev / Scidi / Aacl E. coli strains harboring. A new peak 1 is indicated by an arrow. (B) GC chromatogram of ethyl acetate extracts of the plasmid containing Fh6Tps -2 pRSF-Fh6Tps-2 plasmid pAC-Mev / Scidi / Aacl E. coli strains harboring. A new peak 1 is indicated by an arrow. (C) plasmid containing Fh6Tps -3 pRSF-Fh6Tps-3 plasmid pAC-Mev / Scidi / Aacl GC chromatogram of ethyl acetate extracts of E. coli strains harboring. A new peak 1 is indicated by an arrow. (D) GC chromatogram of ethyl acetate extracts of the plasmid containing Fh6Tps -4 pRSF-Fh6Tps-4 plasmid pAC-Mev / Scidi / Aacl E. coli strains harboring. A new peak 1 is indicated by an arrow. (E) GC chromatogram (control) of an ethyl acetate extract of an Escherichia coli strain carrying the vector pRSFDuet-1 not containing Fh6Tps-z and the plasmid pAC-Mev / Scidi / Aacl. The figure showing a camellia ( Camellia saluenensis variety name: Inochi fragrance) flower. The figure showing the amino acid sequence alignment of CsTPS1 and the known ginger family plant-derived sesquiterpene synthase (synthase). ZoTPS1 represents Zingiber officinale Roscoe-derived β-bisaboren synthase (BAI67934). 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. RDR, DDxxD, (N / D) Dxx (S / T) xxxE motifs highly conserved in monoterpene / sesquiterpene synthase are indicated by asterisks. The figure showing the structure of plasmid pRSF-CsTps1. CDNA corresponding to the ORF of CsTps1 (1,728 bp) is inserted between the vector pRSFDuet-1 of Nde I- Kpn I. The figure showing the analysis result of the monoterpene product of recombinant Escherichia coli. (A) GC chromatogram of ethyl acetate extracts of E. coli strains harboring plasmid pRSF-CsTps1 plasmid pAC-Mev / Scidi / Aacl including CsTps1. A new peak 1 is indicated by an arrow. (B) CsTps1 do not contain the vector pRSFDuet-1 and plasmid pAC-Mev / Scidi / Aacl GC chromatogram of E. coli strain ethyl acetate extracts to hold the (control). The figure which shows the biosynthesis from the chemical structure of linalool (linalool) which is a monoterpene which can be produced by this invention, and geranyl diphosphate (GPP). It is a figure which shows the process of the whole RNA of a rakkyo ( Allium chinense G.Don), and the total RNA extraction from a bulb. The figure showing the analysis result of the sesquiterpene product of recombinant Escherichia coli. GC chromatogram of dodecane extract of E. coli strain carrying (dodecane layer) Plasmid pAlcTPS1 plasmid pAC-Mev / Scidi / Aacl comprising (a) AlcTPS1. A new peak 1 is indicated by an arrow. GC chromatogram of (b) AlcTPS1 do not contain the vector pRSFDuet-1 and plasmid pAC-Mev / Scidi / Aacl dodecane extract of E. coli strains harboring (dodecane layer) (control). The figure showing the analysis result of the sesquiterpene product of recombinant Escherichia coli. GC chromatogram of dodecane extract of E. coli strain carrying (dodecane layer) Plasmid pAlcTPS1 plasmid pAC-Mev / Scidi / Aacl comprising (a) AlcTPS1. A new peak 2 is indicated by an arrow. GC chromatogram of (b) AlcTPS1 do not contain the vector pRSFDuet-1 and plasmid pAC-Mev / Scidi / Aacl dodecane extract of E. coli for holding (dodecane layer) (control). It is a figure which shows the MS spectrum (a) of peak 1 (FIG. 20), and the MS spectrum (b) of (beta) -ocimene. FIG. 22 shows an MS spectrum (a) of peak 2 (FIG. 21) and an MS spectrum (b) of α-farnesene. The PDA-HPLC analysis figure of the solvent extract of colon_bacillus | E._coli (it hold | maintains plasmid pAC-Mev / Scidi / Aacl) which expressed the gamma-amorphene synthase gene ( ZoTps5 ) of ginger. Structures of four types of sesquiterpenes produced by Escherichia coli (containing plasmid pAC-Mev / Scidi / Aacl ) expressing the gamma-amorphene synthase gene ( ZoTps5 ) of ginger. The TLC analysis figure of the solvent extract of colon_bacillus | E._coli (it hold | maintains plasmid pAC-Mev / Scidi / Aacl) which expressed the Kubeben synthase gene ( AeTps1 ). Structures of four types of sesquiterpenes produced by Escherichia coli (holding the plasmid pAC-Mev / Scidi / Aacl ) expressing the Kubanben synthase gene ( AeTps1 ) It is a figure which shows the esterase activity evaluation of the various hydrolase with respect to acetoacetic acid ethyl ester (EAA) and acetoacetic acid methyl ester (MAA). It is a figure which shows the structure of plasmid pAC-Mev / Scidi / Aacl / pnbA and pAC-Mev / Scidi / Aacl / pnbAopt. Lycopene produced by lycopene-producing Escherichia coli introduced with plasmid pAC-Mev / Scidi / Aacl / pnbA or pAC-Mev / Scidi / Aacl / pnbAopt using lithium acetoacetate (LAA), MAA, or EAA as a substrate It is the figure which compared the production amount. The plasmid pAC-Mev / Scidi / Aacl was used as a control having no esterase activity. The left figure shows the amount of lycopene produced per gram of dry cell weight (DCW), and the right figure shows the amount of lycopene produced per liter of medium. FIG. 3 is a diagram comparing the amount of α-humulene produced by α-humulene-producing Escherichia coli introduced with plasmid pAC-Mev / Scidi / Aacl / pnbA when LAA, MAA, or EAA is used as a substrate. The plasmid pAC-Mev / Scidi / Aacl was used as a control having no esterase activity. The figure shows the results of examining the titer when lycopene-producing Escherichia coli (containing plasmids pACCRT-EIB and pRK-idi) was cultured in baffled Erlenmeyer flasks by adding various sugars 1% to LB medium. is there. It is a figure which shows the result of the rat brain lipid peroxidation suppression test using (alpha) -copaene ((alpha) -copaene) and catechin (catechin; positive control).

The present invention is described in detail below.
(1. Effects of mevalonate pathway gene cluster and IPP isomerase gene transfer)
Although Escherichia coli does not have a mevalonate pathway, studies have been actively conducted to introduce an enzyme gene group (mevalonate pathway gene group) of the mevalonate pathway into Escherichia coli. The first pioneering study was conducted by Kakinuma et al. (See Non-Patent Document 6). Kakinuma et al., A group of mevalonate pathway genes that synthesize acetoacetyl-CoA to IPP, that is, HMG-CoA synthase, HMG-CoA reductase, HMG-CoA reductase, mevalonate kinase ( 5 genes encoding mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), diphosphomevalonate decarboxylase (DPMVA decarboxylase), and genes encoding 2 types of IPP isomerase (IPP isomerase) {Derived from Streptomyces genus CL190; Non-Patent Document 5; Accession no AB037666} from Pantoea ananatis carotenoid (also called tetraterpene) biosynthetic genes { crtE , crtB , crtI , crtY , crtZ; to make the zeaxanthin (zeaxanthin) from FPP It was introduced into E. coli with essential biosynthetic gene cluster}, and expressed. This recombinant Escherichia coli can synthesize zeaxanthin efficiently by adding D-mevalonate lactone (D-mevalonate lactone; sometimes referred to as MVL) as a substrate to the medium and culturing. (Reference: Non-Patent Document 6).
The present inventors also used the same mevalonate pathway gene group derived from Streptomyces genus CL190 strain (Non-patent Document 5; Accession no AB037666) (including type 2 IPP isomerase gene) as in the case of Suganuma et al. Was inserted into the E. coli vector pACYC184 {chloramphenicol (Cm) resistance} in the form of plasmid pAC-Mev (see Non-patent Document 7). Recombinant Escherichia coli introduced with plasmid pAC -Mev together with a plasmid for expression of the α-humulene synthase gene (α-humulene synthase; ZzZSS1 ) of 0.5 g / mL was added to the medium as a substrate. It was shown that 1 mg of α-humulene per 1 mL was produced by culturing (Reference: Patent Document 3, Non-Patent Document 7). In addition, when the ZzZSS1 gene was introduced into E. coli and expressed without introducing the plasmid pAC-Mev, the production amount of α-humulene remained at 1/11 that of the plasmid pAC-Mev. In addition, regarding the source of the mevalonate pathway gene group, the present inventors used the one derived from Streptomyces genus CL190 strain in the examples of the present specification, but use the corresponding gene group derived from yeast or other bacteria. (Reference: Non-Patent Document 8). In addition, when the β- eudesmol synthase gene ( ZzZSS2 ) of Hana ginger is introduced and expressed together with the group of mevalonate pathway genes, the recombinant Escherichia coli is cultured by adding MVL as a substrate to the culture medium to 100 per mL. It was shown to produce μg of β-eudesmol (see Non-Patent Document 4). In addition, it was shown that not only these sesquiterpenes but also the monoterpene linalool is produced in the above system (see: Patent Document 3).
Also, the end of the foreign genes in the plasmid pAC-Mev, prepare plasmid pAC-Mev / Scidi inserting the budding yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) 1 type of IPP isomerase from (IPP isomerase) gene (Scidi) (Reference: Patent Document 3, Non-Patent Document 7). pAC-Mev / Scidi terpene biosynthetic gene recombinant E. coli with (e.g., required lycopene biosynthesis of carotenoids crtE, crtB, crtI) when to introduce the expression, the amount of the terpene (e.g. lycopene) is, Scidi It was shown that the level was higher than when no gene was present (in the case of plasmid pAC-Mev) (see: Patent Document 3, Non-Patent Document 7). Examples of type 1 IPP isomerase genes include genes possessed by eukaryotic microorganisms such as baker's yeast Saccharomyces cerevisiae and green alga Haematococcus pluvialis and some prokaryotic organisms ( Kajiwara, S., Fraser, PD, Kondo, K., and Misawa, N., Biochem. J. , 324: 421-426, 1997), these genes can also be used in the present invention. Also, in the type 2 IPP isomerase gene, in addition to the type 2 IPP isomerase gene derived from E. coli (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), marine bacterium shake Bande O MONAS (Brevundimonas) prokaryotic genes from such type 2 IPP isomerase gene of the genus SD212 strain and Paracoccus (Paracoccus) genus N81106 strain is known (Misawa, N., marine Drugs, 9 (5): 757-771, 2011), these genes can also be used in the present invention.
As described above, the mevalonate pathway gene group (including type 2 IPP isomerase gene) is introduced into E. coli by adding type 1 IPP isomerase (eg, Scidi gene), and cultured with MVL added to the medium. As a result, it was shown that the amount of FPP produced in the recombinant E. coli was increased to a large amount. In other words, the carotenoid biosynthetic genes starting from sesquiterpene synthase (sesquiterpene synthase) gene, monoterpene synthase gene, or crtE (GGPP synthase gene) using FPP as a substrate are mevalonate pathway genes It was revealed that these products, such as sesqui (mono) terpenes or carotenoids, can be efficiently produced by introducing and expressing at the same time as the group (see Non-Patent Documents 7 and 8). .

(2. Effects of acetoacetate and acetoacetate ester utilization gene transfer)
Since D-mevalonolactone (MVL) contains asymmetric carbon in the molecule, it is expensive (for example, 1 g: 30,200 yen according to the catalog of Tokyo Chemical Industry Co., Ltd. 2012-2013) and difficult to put into practical use. Construction of a system for efficiently producing terpenes using a simple substrate has been desired. Therefore, the present inventors have added a rat ( Rattus norvegicus ) in addition to the mevalonate pathway gene group (including the type 2 IPP isomerase gene) derived from the Streptomyces genus CL190 strain and the type 1 IPP isomerase gene ( Scidi gene). ) -Derived acetoacetate-coenzyme A ligase gene (acetoacetate-CoA ligase; Aac1; acetoacetyl-CoA synthetase) was added and introduced into E. coli and expressed (Reference: Patent Document 3, Non-patent Document) 7, 8). By culturing the recombinant Escherichia coli and adding LAA as a substrate (final concentration 1.0 g / L) to the medium, it was shown that terpenes can be efficiently produced as in the case of adding MVL (see: Patent Document 3, Non-Patent Documents 7 and 8). In other words, it is 3.5 to 9.3 times higher (in the case of enzyme genes that synthesize carotenoids from FPP) than in the case of recombinant Escherichia coli in which only the enzyme gene that synthesizes terpenes from FPP is expressed. (Reference: Patent Document 3, Non-Patent Document 7). As the mevalonate pathway gene group for synthesizing up to isopentenyl diphosphate, the above-mentioned mevalonate pathway gene group derived from Streptomyces genus CL190 strain (Reference: Non-Patent Document 5) can be used. Mevalonate pathway genes derived from Saccharomyces cerevisiae (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), bacteria Streptococcus pneumonia ( Streptococcus pneumonia ) Mevalonate Pathway Gene Group (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) Etc. can also be used.
Furthermore, by using the system described above, it is possible to analyze the function of a newly obtained sesquiterpene (or monoterpene) synthase gene sequence whose function is unknown (see Non-Patent Document 8). That is, it is possible to examine whether recombinant E. coli having an ability to assimilate MVL or LAA introduced so as to express this gene sequence is producing sesquiterpenes (or monoterpenes). Does not produce terpenes). When terpenes are produced, by analyzing the chemical structure of the terpene, the terpene synthase gene sequence encodes an enzyme that synthesizes a sesquiterpene (or monoterpene) product with that chemical structure from FPP. It is because it is identified that it is a gene. More than 7,000 sesquiterpenes have already been isolated from plants and microorganisms (mainly plants), but many sesquiterpene synthase genes are still unknown. In the future, functional analysis of terpene synthase genes is expected to proceed with this system (see: Patent Documents 2 and 3, Non-Patent Document 8).
Since MVL was an expensive reagent having an asymmetric carbon in the molecule (for example, 30,200 yen / g; Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), LAA, which is a relatively inexpensive reagent (for example, 11 , 100 yen / g, 35,800 yen / 5 g; Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), the development of the utilization technology was significant. However, LAA is much more expensive than glucose (D-(+)-glucose; for example, 1,900 yen / 500 g; Tokyo Chemical Industry 2012-2013 catalog) as a medium sugar source. Development of a new substrate has been desired.
The prices of methyl acetoacetate (MAA) and ethyl acetoacetate (EAA) as the reagents were 2,800 yen / 500 g and 3,400 yen / 500 g, respectively ( Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog). In addition, since the price of lithium acetoacetate (LAA) as a reagent was 35,800 yen / 5 g (Tokyo Chemical Industry Co., Ltd. 2012-2013 catalog), MAA and / or EAA is used instead of LAA. As a result, the cost can be significantly reduced.
Until now, there have been no reports of enzymes (esterases) or enzyme genes that can hydrolyze MAA or EAA (efficiently). Therefore, the present inventors obtained a base sequence from a genome sequence database for an ester hydrolase (esterase) gene containing 18 putative genes composed of 12 bacterial families classified from the primary amino acid sequence, and obtained a full-length sequence. Was isolated. Among the crude enzyme extract prepared from recombinant Escherichia coli introduced and expressed with these genes, synthesis of a significant amount of enzyme protein was confirmed in the soluble fraction in samples derived from 13 strains. When screening was performed using the hydrolytic activity for MAA or LAA as an index, it was confirmed that PnbA, PA3859 and SGO0795 have high acetoacetate hydrolyzing activity in the three gene expression strains. Next, the plasmid pAC-Mev / Scidi / Aacl {both using the E. coli vector pACYC184; chloramphenicol (Cm) resistance; Patent Document 3, Non-patent Document 7} is inserted with one of the above three genes. Produced. Examples of the structures of plasmids pAC-Mev / Scidi / Aacl / pnbA and pAC-Mev / Scidi / Aacl / pnbAopt are shown in FIG. Plasmid pAC-Mev / Scidi / Aacl / pnbA (or a similar plasmid containing pAC-Mev / Scidi / Aacl / pnbAopt, or other acetoacetate hydrolase gene) from FPP (or GPP) to terpene (isoprenoid, Terpenoids) were introduced into Escherichia coli together with genes or gene groups necessary for synthesis, and an evaluation test was conducted. For example, the plasmid pAC-Mev / Scidi / Aacl, whether the gene group necessary for the synthesis of tetraterpene (carotenoid) lycopene or the gene necessary for the synthesis of sesquiterpene α-humulene is introduced. When introduced into E. coli with / pnbA (or pAC-Mev / Scidi / Aacl / pnbAopt), is the same level as when LAA was added in E. coli coexisting with plasmid pAC-Mev / Scidi / Aacl (positive control)? It became clear that the assimilation ability of MAA or EAA beyond it was shown (FIG. 30, FIG. 31). That is, according to the present invention, by using MAA or EAA which is an inexpensive substrate, terpenes {including sesquiterpenes, monoterpenes, and tetraterpenes (carotenoids); Can be manufactured.

{3. Terpene synthase (terpene synthase; function of terpene produced)
Enzyme proteins that synthesize monoterpenes, sesquiterpenes, and some diterpenes have homology to each other, and are collectively referred to as terpene synthase (TPS). Monoterpenes and sesquiterpenes are the main components contained in plant essential oils, and are plant fragrance and fragrance components. Monoterpenes include famous fragrances and fragrances such as limonene (for example citrus scents), linalool (for example scents for lavender and bergamot), menthol (scents for mint plants), camphor (camphor). Yes. To date, linalool synthase genes have been obtained from various plants such as sarunasi ( Actinidia arguta ), matatabi ( Actinidia polygama ), ginger ( Zingiber officinale ), turmeric (autumn turmeric; Curcuma longa ), All of the functionally analyzed linalool synthases are known to share the nerving activity of sesquiterpene (HJ Koo, DR Gang, Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues. PLoS One 7: e51481, 2012). Therefore, linalool synthase is often also referred to as linalool / nerolidol synthase. In addition, linalool has an unpleasant odor while linalool has a pleasant scent. On the other hand, linalool synthase (CsTPS1) derived from camellia ( Camellia saluenensis variety name: Inochi fragrance) included in the present invention has no nerolidol synthesizing activity. Therefore, CsTPS1 is a functionally novel enzyme.
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 (TPS) 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). In addition, in plants belonging to Zingiberaceae, from ginger (ginger; Zingiber officinale ), as a sesquiterpene synthase, germacrene D synthase gene ( ZoGeD ; non-patent document 1), β-bisabolen synthesis The enzyme (β-bisabolene synthase) gene ( ZoTPS1 ; Patent Document 1, Non-patent Document 2) and the γ-amorphene synthase gene ( ZoTPS5 ; Patent Document 1) were isolated, and their structure and function analysis Was done. Also, 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 the present invention, as an enzyme gene for synthesizing a sesquiterpene from FPP, a valerianol / hedycaryol synthase gene (for example, ChTps1-1 ), an α-selinene synthase gene ( For example, Fh1Tps-1 ), α-copaene synthase gene (eg Fh6Tps-1 ), and β-ocimene / α-farnesene synthase gene ( AlcTPS1 ) Newly found. In particular, the enzyme that synthesizes valerianol and the gene that encodes the enzyme have not been known so far and have been disclosed for the first time in the world by this specification. In addition, the enzyme that synthesizes α-selinen or α-copaene almost as a single product and the gene encoding it have not been known so far (at least both academic papers and sequence information have been disclosed). It was first disclosed in the world by this specification.
Furthermore, any one of the plasmids for expressing Escherichia coli of the sesquiterpene (and monoterpene) synthase gene included in the present invention is introduced into E. coli together with the Cm resistant plasmid (for example, pAC-Mev / Scidi / Aacl / pnbA). Introducing and culturing in a medium containing LAA, Km and Cm efficiently produces valerianol, hedicarol, α-selinen, α-copaene, β-oscymene and α-farnesene (and linalool) be able to. These sesquiterpenes (and monoterpenes) are expected to have physiological functions useful for human health. For example, it is thought that valeranol has a tonic effect on ovarian function, α-copaene has an anti-inflammatory effect, and β-ocimene has an antibacterial and antiviral effect. Also in the present invention, rat brain lipid peroxidation inhibition test ( in vitro antioxidant activity test) was performed on α-copaene, and α-copaene has a mild antioxidant activity (IC ₅₀ = 35.1 μM). Actually showed that.

(4. Escherichia coli strain and genetic recombination experiment method)
In the Examples of the present specification, E. coli B strain BL21 (DE3) and K12 strain JM101 or JM109 were used. However, various strains such as DH5, HB101, LE392, and JM109 (DE3) of Escherichia coli K12 strain exist in E. coli strains. Therefore, E. coli strains are not limited to BL21 (DE3), JM101, and JM109.
In the following examples, LB medium and TB medium were used as the culture medium for recombinant Escherichia coli, but there are many mediums such as 2 x YT medium and M9 medium as the culture medium for Escherichia coli. It is not limited to TB media.
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.

(5. Hosts other than recombinant E. coli)
In the method for producing a sesquiterpene of the present invention, insects, yeasts, plant cell systems, cell-free systems (such as wheat germ) and the like can be used as hosts other than recombinant E. coli.
Also in these hosts, sesquiterpenes (or monoterpenes) can be produced in the same manner as in the recombinant Escherichia coli system by introducing appropriate synthase genes. Synthetic enzyme genes necessary for each host are as follows.
Yeast system: These sesquiterpenes can be produced by introducing and expressing the sesquiterpene (or monoterpene) synthase gene of the present invention in yeast using a yeast expression vector. 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: These sesquiterpenes can be produced by introducing the sesquiterpene (or monoterpene) synthase gene of the present invention into insects using an insect expression vector and expressing them. In addition, it is thought that the target sesquiterpene production can be improved by co-expressing the HMG-CoA reductase gene.
Plant cell system: The sesquiterpene (or monoterpene) synthase gene of the present invention can be produced by introducing it into a plant using a plant expression vector and expressing it. Become. In addition, it is considered that the target terpene production can be improved by co-expressing the IPP isomerase ( idi ) or HMG-CoA reductase gene. In addition, when a foreign gene is introduced and expressed directly into chloroplasts 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.

(6. Culture of recombinant Escherichia coli for biosynthesis of sesquiterpenes, monoterpenes, etc., and methods for recovering and purifying the produced sesquiterpenes, monoterpenes, etc.)
In the present invention, n -alkane having 8 to 10 carbon atoms of n -octane or decane is overlaid on a culture medium, cultivating recombinant Escherichia coli for biosynthesis of terpenes (sesquiterpenes, monoterpenes, etc.), and the produced terpenes A manufacturing method has been invented for efficiently recovering the product. It should be noted that there has never been a production method in which n -octane or decane is overlaid and the host is cultured and the produced terpene is recovered.
The purification method of the present invention, which begins with the layer culture of n -octane or decane, is a method for efficiently isolating and purifying terpenes from recombinant Escherichia coli that biosynthesizes sesquiterpenes, monoterpenes, etc. Includes the following steps (I), (II), (III) and (IV) or (I), (II) and (V) in this order.
(I) A step of extracting terpenes produced by overlaying and culturing an n-alkane having 8 to 10 carbon atoms in the medium during recombinant E. coli culture (liquid shaking culture) to the alkane phase (extraction step) .
(II) A step of carrying out two-phase partitioning between the alkane phase obtained in step (I) and a suitable aqueous solvent, recovering the alkane phase containing terpenes and removing impurities (separation step).
(III) A step in which the alkane phase obtained in step (II) is purified by preparative HPLC using a reversed phase system (ODS or the like) column (preparation step).
(IV) Pure terpenes with a good recovery rate are obtained by recovering terpenes in the alkane phase in the alkane / aqueous solvent system from the solution containing pure terpenes obtained in step (III) and concentrating to dryness. Step for removing (solvent removal step).
(V) A step in which the alkane phase obtained in step (II) is purified by an open column using silica gel or by preparative HPLC (preparation step).
In the following [1] to [5], the steps (I) to (V) will be described in detail.

[1] The medium for culturing recombinant Escherichia coli in step (I) may be any medium as long as Escherichia coli grows smoothly and produces the desired terpenes in the presence of an appropriate gene expression inducer. Cultivation of E. coli is carried out with 10% -50% (V / V) n -alkane layered on E. coli growth medium whose solvent is water (10-50 mL n -alkane in 100 mL medium). . The n -alkane used at this time should have a longer carbon chain than n -octane (C8) (alkanes with a shorter carbon chain than n -octane are highly soluble in water, and thus inhibit the growth of recombinant E. coli. To end up). On the other hand, the longer the carbon, the higher the boiling point of the alkane and the more difficult it is to remove the solvent in a vacuum concentrator such as an evaporator. Specifically, alkanes from n -octane (C8) to decane (C10) are used. It is simple (if it is an n -alkane from C8 to C10, it can be concentrated with a normal diaphragm pump under heating at 40 to 50 ° C.). As for terpenes consisting of C and H (hydrocarbon), or terpenes having one hydroxyl group introduced therein, 95% or more of the terpenes produced in Escherichia coli are layered C8-C10 n -alkane phase. To be distributed.

[2] The water-containing solvent used in step (II) is preferably one that does not dissolve in the C8-C10 n -alkane used in step (I) and has alkaline properties. Specifically, a mixture of about 0.1 to 1 N KOH or NaOH solution with methanol or acetonitrile is used in the same volume as the alkane phase. However, when the volume of the alkane phase used in the step (I) is small, it is better to further add alkane in order to facilitate the subsequent two-phase distribution. However, where added alkane is n of C8 to C10 - need not be limited to alkanes, n of C8 to C10 - can be used alkane having an affinity for alkane, preferably, C₅-C₈ the n - Alkanes can be mentioned. We used C6 n -hexane. Since most of the terpenes are neutral substances with low polarity, by carrying out a two-phase partition with an alkaline hydrous solvent and an alkane (consisting of the original C8-C10 n -alkane and the alkane added later), Acidic substances containing free fatty acids produced in large quantities by recombinant Escherichia coli and neutral substances with relatively high polarity are distributed to the hydrous solvent side, while terpenes are distributed to the alkane phase, so impurities can be efficiently removed. it can. However, the mixing ratio of methanol or acetonitrile and 0.1 to 1 N KOH or NaOH needs to be adjusted depending on the type of terpene. For example, methanol or acetonitrile: alkaline water = 90: 10 is used for terpenes (hydrocarbons) composed of only C and H, and acetonitrile: alkali water = 50: 50 is used for terpenes into which one hydroxyl group is introduced. There is a need. This is a device for distributing almost all of the terpenes to the alkane phase.

  [3] Step (III) is a technique for completely purifying and recovering the terpenes roughly purified in step (II). Many terpenes are weakly adsorbed on silica gel because of their low polarity, and it is often difficult to use a silica gel resin for purification to a pure product. As for the purification of terpenes, a method using a difference in boiling point such as fractional distillation has been known, but the conventional method is suitable for mass processing (g scale) but difficult to apply on the mg scale ( In many cases, clear separation is difficult and recovery rate is poor. On the other hand, by performing HPLC fractionation using reverse-phase resin (C18 or C30 HPLC column) and 90-100% acetonitrile or methanol as solvent, terpenes can be completely cleared in many cases with high recovery rate. It can be purified. For detection by HPLC, UV end absorption (200-220 nm) or differential refraction (RI) can be used. When UV end absorption is used for detection, a solvent composed of acetonitrile and water that does not absorb in the same region. Is preferably selected.

[4] Step (IV) is a method for obtaining a pure product by removing the solvent from the terpenes completely purified in step (III). Since many terpenes have a low boiling point and a property of azeotroping with water, most of the terpenes can be obtained by subjecting the fraction collected in step (III) to vacuum concentration in an apparatus such as an evaporator. Vaporizes with the solvent. To prevent this, the same amount of water is added to the HPLC-prepared solvent (90-100% acetonitrile or methanol) to make an acetonitrile or methanol solution of about 50%, and this solution has a low boiling point and is liquid at room temperature and normal pressure. Alkanes (specifically n -pentane (C5) or n -hexane (C6)) are distributed in two phases in equal volumes to distribute terpenes into the alkane phase, and then sodium sulfate (anhydrous) is added to the alkane phase. By thoroughly dehydrating and removing the solvent with an evaporator at a low vacuum of about 150 mmHg, terpenes can be made pure without loss. Moreover, spraying of dry nitrogen gas can be used as a solvent removal (alkane) without using a vacuum concentrator such as an evaporator.

  [5] Step (V) is a method different from step (III) for completely purifying and recovering the terpenes roughly purified in step (II). As described in [3], many terpenes have low polarity, and silica gel is often difficult to purify to a pure product. However, when there are not many impurities, or when the polarities are separated between existing terpenes, terpenes that do not contain OH groups use hexane as a developing solvent, and terpenes that contain OH groups develop. Purification to a pure product is possible using a silica gel open column or preparative HPLC with a solvent of hexane: ethyl acetate mixed solvent. Since water is not contained in the developing solvent when this method is used, the step (IV) is not necessary, and the fraction solution can be concentrated as it is with a vacuum concentrator such as a low vacuum evaporator of about 150 mmHg.

(7. Method for producing terpene using acetoacetate)
Furthermore, the method for producing terpenes (isoprenoids) according to the present invention is characterized in that recombinant Escherichia coli is cultured in a medium containing acetoacetate (acetoacetate fatty acid ester) to obtain terpenes from the culture or the cells. It is.
Examples of the acetoacetate ester include acetoacetate methyl ester (MAA), acetoacetate ethyl ester (EAA), acetoacetate propyl ester, acetoacetate butyl ester, acetoacetate tert -butyl ester, etc. Among these, inexpensive raw materials It is preferred to use acetoacetic acid methyl ester (MAA) and / or acetoacetic acid ethyl ester (EAA).
The concentration of acetoacetate in the medium is not particularly limited as long as Escherichia coli can produce terpene, but it is preferably 1 to 25 mM, and more preferably 1 to 10 mM. Medium components other than acetoacetate may be the same as those contained in a general E. coli culture medium.
The temperature at the time of MAA and / or EAA addition culture is not particularly limited as long as recombinant E. coli can grow, but it is preferably 15 to 30 ° C, more preferably 18 to 22 ° C.
Although the culture time is not particularly limited, the culture is preferably performed for 12 to 72 hours, more preferably for 24 to 48 hours after the transgene expression.
The collection / purification of sesquiterpenes, monoterpenes, etc. from the culture or the cells is as described in 6. above. Can be carried out according to the method disclosed in.

{8. Recombinant Escherichia coli Culture Method for Biosynthesis of Tetraterpenes (Carotenoids)}
When culturing recombinant E. coli that biosynthesizes tetraterpenes (carotenoids), the above-mentioned 7. In addition, fructose, galactose, trehalose, sorbitol, mannitol, and / or mannose may be added to the medium under the conditions shown in 1) or normal culture conditions and cultured.
Fructose, galactose, trehalose, and sorbitol are carbon sources that significantly increase the amount of carotenoid production per culture solution, and mannitol, a carbon source that increases the carotenoid production amount per culture solution, although not as much as the previous four. Mannose is mentioned. In addition, the effect on glucose, sucrose, lactose, maltose, and starch was not recognized by the increase in the amount of microbial cells per culture solution. As described above, according to the contents disclosed in this example, an efficient culture method was devised in which fructose, galactose, trehalose, sorbitol, mannitol, and / or mannose was added to a medium to culture carotenoid-producing Escherichia coli.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

{Determination of sesquiterpene synthase (sesquiterpene synthase) gene cDNA sequence from citrus camellia}
Total RNA was extracted from flowers of Camellia x hiemalis (Fig. 1) 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 based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 3 {Forward: 5′-TTYCGAYTIYTIMGRMARCAIGG-3 ′ (SEQ ID NO: 38) and reverse: 5′-TAIGHRTCAWAIRTRTCRTC- 3 ′ (SEQ ID NO: 39)} was used, and PCR was performed using cDNA as a template under the conditions as described in Non-Patent Document 3, and an amplified fragment of 938 bp was obtained (in SEQ ID NO: 38 and SEQ ID NO: 39, “ “Y” is pyrimidine (C or T), “R” is purine (A or G), “I” is inosine, “M” is A or C, “H” is A or C or T, “W” is A Or T). The obtained amplified fragment was cloned using pGEM-T Easy Vector System (Promega) and the nucleotide sequence was determined. It was found that the obtained clone had the reverse primer sequence (SEQ ID NO: 39) at both ends and was amplified only with the reverse primer. Since 5 ′ side contained the starting methionine and 5 ′ UTR sequence, in order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer (White.3 race-2) : Using 5′-TTTAGACAAACAGCTATGGGACAA-3 ′ (SEQ ID NO: 1) and Advantage2 Polymerase Mix (Clontech), the fragment was extended toward the 3 ′ end according to the manufacturer's instructions to determine the full-length cDNA sequence.

{Acquisition of Valerenanol / Hedicariol Synthase ( Valeranol / Hedicariol Synthase) Gene ( ChTps1 ) cDNA}
Oligonucleotide primer pair Wh.Bgl-F specific to the full-length cDNA sequence obtained in Example 1 (5′-AGACAGA AGATCT CATGGCTTCATCTCAAGTTGGTGA-3 ′ (SEQ ID NO: 2), underline indicates Bgl II recognition site) and Wh PCR using .Xho-R (5'-GAGGTAC CTCGAG TCACATGGGAATTGGATCTTCGA-3 '(SEQ ID NO: 3), 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 4 minutes). The obtained cDNA was cloned using the plasmid pGEM-T Easy Vector System (Promega), and the nucleotide sequence was determined. The cDNA nucleotide sequences are shown in SEQ ID NOs: 4 to 20, and the amino acid sequences of the respective polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 21 to 37. Further, the cDNA specified by SEQ ID NOs: 4 to 20 is named ChTps1-x (x is 1 to 17), and the respective proteins specified by SEQ ID NOs: 21 to 37 encoded by ChTps1-x are ChTPS1-x ( x was named 1-17).
ChTps1-1 contained a 1665 bp open reading frame (ORF) and encoded a protein (ChTPS1-1) with a putative 554 amino acid residue, a molecular weight of 64.0 kDa, and a pI of 5.15. Based on homology search against GenBank / EMBL / DDBJ database, ChTPS1-1 is a known sesquiterpene synthase, Lavandula pedunculata- derived germacrene A synthase and 51%, Doremi from Valeriana officinalis L. It showed 50% amino acid sequence identity with narpene synthase and 50%, and tomato ( Solanum lycopersicum ) terpene synthase. Because of its homology with known sesquiterpene synthases, ChTPS1-1 is a group of angiosperm sesquiterpenes and diterpene synthases, TPS-a subfamily (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, ChTPS1-1 contained the motifs DDxxD, RDR and (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 2). . On the other hand, like the known sesquiterpene synthase, ChTPS1-1 was predicted not to contain a plastid transit peptide.
ChTps1-10 contained a 1665 bp ORF and encoded a protein (ChTPS1-10) with a putative 554 amino acid residue, a molecular weight of 64.0 kDa, and a pI of 5.20. Based on homology searches against the GenBank / EMBL / DDBJ database, ChTPS1-10 is a known sesquiterpene synthase, Lavandula pedunculata- derived germacrene A synthase, 52%, Doremi from Valeriana officinalis L. The amino acid sequence identity was found to be 51% with nomin synthase and 50% with terpene synthase derived from tomato ( Solanum lycopersicum ). Because of its homology with known sesquiterpene synthases, ChTPS1-10 is a subfamily of angiosperm sesquiterpenes and diterpene synthases (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, ChTPS1-10 contained the motifs DDxxD, RDR and (N / D) Dxx (S / T) xxxE {called NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 2) . On the other hand, like the known sesquiterpene synthase, ChTPS1-10 was predicted not to contain a plastid transit peptide.
Most ChTps1-x contained a 1665 bp ORF, whereas ChTps1-13 contained a 1563 bp ORF due to the deletion of the 102 bp fragment, a protein of putative 520 amino acid residues, molecular weight 60.4 kDa, and pI 5.33 (ChTPS1-13) was encoded. By homology search against GenBank / EMBL / DDBJ database, ChTPS1-13 is a known sesquiterpene synthase, Lavandula pedunculata- derived Germacrene A synthase and 49%, tomato ( Solanum lycopersicum ) terpene synthase (terpene synthase) and 48%, and the amino acid sequence identity (Driminol synthase) derived from Valeriana officinalis L. was 48%. Because of its homology with known sesquiterpene synthases, ChTPS1-13 is a group of angiosperm sesquiterpenes and diterpene synthases, TPS-a subfamily (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, ChTPS1-13 contained the motifs DDxxD, RDR and (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 2). . On the other hand, like the known sesquiterpene synthase, ChTPS1-13 was predicted not to contain a plastid transit peptide.

(Valeria and hedicariol production by E. coli with plasmid pRSF-ChTps1-x and plasmid pAC-Mev / Scidi / Aacl)
The plasmid DNA into which the full - length ChTps1-x sequence has been inserted is digested with the restriction enzyme Bgl I- Xho I, and the resulting ChTps1-x sequence is pRSFDuet-1 vector {kanamycin (Km) resistance; Novagen} Bgl The plasmid pRSF-ChTps1-x was ligated to the II- Xho I site (FIG. 3).
Plasmid pAC-Mev / Scidi / Aacl {chloramphenicol (Cm) resistance; Patent Document 3, Non-patent Document 7} and plasmid pRSF-ChTps1-x were used to transform Escherichia coli BL21 (DE3). Transformed. In addition, E. coli transformation method, recombinant E. coli culture method, extraction of sesquiterpene from cultured E. coli cells, and analysis method using gas chromatograph mass spectrometer (GC-MS: GCMS-QP5050 manufactured by Shimadzu) It is as Patent Documents 2 and 3 or Non-Patent Document 7. However, LAA was used instead of MVL as a substrate for culturing recombinant E. coli, 50 mg / L Km and 30 mg / L Cm were used as drugs, and dodecane was not overlaid during the culture. As a result of analysis using GC-MS, in the control group in which IPTG was added to the E. coli culture containing the plasmid pRSFDuet-1 and the plasmid pAC-Mev / Scidi / Aacl, a new sesquioxide was added to the ethyl acetate extract from the cells. While terpene formation was not confirmed, in the experimental section where IPTG induction was carried out in E. coli culture containing plasmid pRSF-ChTps1-x and plasmid pAC-Mev / Scidi / Aacl, the ethyl acetate extract from the cells Two new peaks were observed (peaks 1 and 2; FIG. 4). Peak 1 was consistent with elemol in the MS database. On the other hand, peak 2 was not hit in comparison with the database.
Further, the present inventors cultured 1 L of E. coli BL21 (DE3) into which plasmids pAC-Mev / Scidi / Aacl and pRSF-ChTps1-x were introduced, purified peak 1 and peak 2 from recombinant E. coli, and NMR analysis. Went. As a result, peaks 1 and 2 were identified as hedycaryol and valerianol, respectively (Example 4). FIG. 5 shows the ¹ H NMR spectrum of hedicarol and the predicted structural change of hedicarol at room temperature. It can be seen that each signal in the ¹ H NMR spectrum is very broad because of the structural change of this hedicarol. Since hedicarol is expected to be converted to elemol by heat such as GC analysis, it is a reasonable result that GC-MS analysis shows that peak 1 is elemol.
From the above results, it was found that ChTps1-x is a gene encoding valerianol / hedycaryol synthase. The synthetic activity of valerianol and hedicariol was highest in ChTps1-1 , and gradually decreased as the clone number (x) progressed from ChTps1-2 to ChTps1-9 . The main product in most clones was valeranol , but in ChTps 1-10 , hedicariol was more than valeranol . Clones from ChTps1-11 to ChTps1-16 synthesized only valeranol . In ChTps 1-17 , only hedicariol was detected. Table 1 shows the GC-MS peak intensities of valerianol and hedicariol synthesized by each ChTps1-x clone and recombinant Escherichia coli containing the clone. The chemical structure of valeranol and hedicariol is shown in FIG.
From the above results, it is shown that selective production of valeranol and hedicariol (Elemol) is possible using E. coli introduced with plasmid pRSF-ChTps1-x and plasmid pAC-Mev / Scidi / Aacl. It was. In addition, since the enzyme which synthesize | combines a valerianol and the gene which codes this were not known until now, it was disclosed for the first time in the world by this specification. Valeranol is considered to have a tonic effect on ovarian function, but in the future, valeranol could be easily provided by the production method of the present invention. It became possible to do. Discovery of new functionality and deepening of knowledge of functionality are expected for valeranol.

Peak intensity of terpenes detected from ChTps1-x recombinant E. coli (darker shade indicates higher peak intensity. TIC: total ion chromatogram, ND: not determined).

(Purification and identification of valericanol and hedicariol)
Escherichia coli BL21 (DE3) introduced with plasmid pRSF-ChTps1-1 and plasmid pAC-Mev / Scidi / Aacl was transferred to TB containing 50 mg / L Km and 30 mg / L Cm (Terific Broth; J. Sambrook, DW Russel, Molecular Cloning, 3rd edition, Cold Spring Harbor, NY, 2001) 1 L. At this time, 200 mL of n -octane was added and cultured. After completion of the culture, first culture (broth 1 L + n - octane 200 mL) was stirred well and put in 2 L capacity separating funnel, clear the lower (aqueous layer) was discarded {upper layer at this point (n -Octane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of 50% methanol (0.1 N NaOH 250 mL + methanol 250 mL) were added to the separatory funnel and stirred well. In this operation, the emulsion almost disappeared and separated into two layers, so the lower layer (alkali 50% methanol layer) was discarded. Again, 500 mL of alkali 50% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting).
Next, 20 L of this concentrate was analyzed and fractionated by the following PDA-HPLC (high performance liquid chromatography with photodiode array detector) under the following conditions.
Column: Developsil C30-UG-520 mm x 250 mm (Nomura Chemical)
Solvent: 90% CH ₃ CN
Flow rate: 8.0 mL / min
Detection: 200-500 nm (PDA)
The two main peaks, that is, the peak with a retention time of 16.2 minutes (peak 1) and the peak with a retention time of 18.0 minutes (peak 2) were divided into about 10 times. Inject times). After completing 10 peak fractions, add the same amount of water as the collected total eluate (80-150 mL) and place in a 500 mL separatory funnel, where the same amount (elution solvent x 2 (160 -300 mL)) n -pentane was added and stirred well to recover the n -pentane layer. The n -pentane layer collected by repeating the same operation once again was transferred to a 1 L Erlenmeyer flask, dried over anhydrous sodium sulfate, and concentrated under reduced pressure (150 mmHg setting) with an evaporator. When the concentrate reaches about 5 mL, transfer the remaining solvent to the 25 mL eggplant flask. Concentration with the evaporator is stopped as soon as n -pentane disappears. Finally, dry nitrogen gas is blown for about 5 minutes to remove the solvent. Distilled completely.
As a result of the above operations, 13.1 mg of the compound of peak 1 {identified as valerianol represented by the following formula (1) by various analyzes} was obtained as a pure product. The compound of peak 2 was obtained as a mixture in an amount of 4.2 mg [{hedycaryol represented by the following formula (2) is present in this mixture from GC-MS analysis. In GC-MS, hedycaryol is detected as elemol represented by the following formula (3) generated by heating change}]. The compound of peak 1 was identified as valerianol by NMR analysis and GC-MS analysis.

Spectroscopic analysis data of valerianol:
GC-MS retention time 38.8 min, m / z 222 (M ⁺ ). ¹ H-NMR (CDCl ₃ ): 0.91 (d, J = 7.0 Hz, 3H, H-11), 0.8 95 (m, 1H, H-6), 1.11 (s, 3H, H-15), 1.15 (s, 3H, H-13), 1.15 (m, 1H, H-9), 1.16 (s, 3H, H-14), 1.36 (m, 1H, H-2), 1.40 (m, 1H, H-9), 1.56 (m, 1H, H-1) ), 1.68 (m, 1H, H-2), 1.70 (m, 1H, H-8), 1.80-1.95 (2H, H-3), 1.88 (m, 1H , H-6), 2.04 (m, 1H, H-6), 2.26 (m, 1H, H-6), 5.33 (m, 1H, H-4). ¹³ C-NMR ( CDCl ₃ ): 15.5 (C-11), 23.7 (C-3), 25.3 (C-15), 26.9 (C-2), 27.0 (C-13), 27 .1 (C-14), 29.6 (C-7), 32.5 (C-6), 35.2 (C-9), 37.7 (C-10), 39.3 (C- 1), 44.2 (C-8), 72.9 C-12), 119.0 (C-4), 143.1 (C-5),

Elemol spectroscopic data:
GC-MS retention time 36.9 min, m / z 222 (M ⁺ ). ¹ H-NMR (CDCl ₃ ): 0.98 (s, 3H, H-13), 1.20 (s, 6H, H -8 and H-9), 1.38 (m, 1H, H-1), 1.40-1.50 (2H, H-6), 1.44 (m, 1H, H-2), 1 .59 (m, 1H, H-2), 1.60-1.70 (2H, H-5), 1.71 (s, 3H, H-12), 1.96 (dd, J = 3. 3. 12.4 Hz, H-3), 4.59 (s, 1H, H-11), 4.82 (s, 1H, H-11), 4.89 (d, J = 11.8 Hz , 1H, H-15), 4.90 (d, J = 16.5 Hz, 1H, H-15), 5.80 (dd, J = 11.8, 16.5 Hz, 1H, H-14 ¹³ C-NMR (CDCl ₃ ): 16.5 (C-13), 22.5 (C-6), 24.8 (C-12), 27.1 (C-8 and C-9) , 28.4 (C-2), 39.7 (C-4), 39.8 (C-5), 49.3 (C-1), 52.6 (C-3), 72.4 ( C-7), 109.9 (C-15) , 112.0 (C-11), 147.9 (C-10), 150.2 (C-14).

{Determining cDNA sequence of sesquiterpene synthase (sesquiterpene synthase) gene from freesia}
Total RNA was extracted from Freesia x hybrida Airy Purple and Airy Peach flowers (FIG. 7) using RNeasy Plant Mini Kit (Qiagen). CDNA was prepared from 1.0 μg of total RNA using SMARTer RACE cDNA amplification kit (Takara Bio) according to the manufacturer's instructions. Degenerate oligonucleotide primer pairs (forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 38) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC- based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 3 Using 3 ′ (SEQ ID NO: 39), PCR was performed using cDNA as a template under the conditions described in Non-Patent Document 3, and amplified fragments of 611 bp (Airy Purple) and 359 bp (Airy Peach) were obtained. The amplified fragment was cloned using pGEM-T Easy Vector System (Promega), and the nucleotide sequence was determined.The clone obtained from Airy Purple was amplified with a pair of forward and reverse degenerate oligonucleotide primers. On the other hand, the clone obtained from Airy Peach has the sequence of the reverse primer (SEQ ID NO: 39) at both ends, In order to isolate each full-length cDNA, SMARTer RACE cDNA Amplification Kit (Takara Bio), oligonucleotide primer and Advantage2 Polymerase Mix (Clontech) were used. The fragment was extended toward the 5 ′ and 3 ′ ends according to the manufacturer's instructions and the full-length cDNA sequence was determined.Note that the oligonucleotide primer was FR.purple.5-R (5′− for Airy Purple). TCAAGTATATCCTCACCAGGGATGCT-3 ′) (SEQ ID NO: 40) and FR.purple.3-F (5′-AGCAAGGAAGATGCTGACAAAGGT-3 ′) (SEQ ID NO: 41), while for Airy Peach, Fpe1.5race (5′-GTTGTTTGATAAAATAATCACCCCAAAC-3 ′) (SEQ ID NO: 42) and FPe 1.3 race (5′-TCATTTCTCTTCGGTTCCGATTGTTGAG-3 ′) (SEQ ID NO: 43) were used.

{Acquisition of α-selinene synthase (α-selinene synthase) gene ( Fh1Tps ) cDNA}
Oligonucleotide primer pair FPu1.Nde-F specific to the full-length cDNA sequence obtained in Example 5 (5′-AGACAGA CATATG GAGTCAG-CTGCTGG-3 ′ (SEQ ID NO: 44), underline indicates Nde I recognition site) And FPu1.Kpn-R (5′-GTAC GGTACC CTAGAAATAGTCATCTTCGAAGGAAATTGG-3 ′ (SEQ ID NO: 45), underlined indicates Kpn I recognition site) and Advantage2 Polymerase Mix (manufactured by Clontech) using Airy purple cDNA as a template PCR (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, 60 ° C. for 30 seconds and 72 ° C. for 4 minutes for 25 cycles). 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 NOs: 46 to 48, and the amino acid sequences of the respective polypeptides encoded by the cDNA are shown in SEQ ID NOs: 49 to 51. Further, the cDNA specified by SEQ ID NOs: 46 to 48 is named Fh1Tps-y (y is 1 to 3), and the proteins (polypeptides) specified by SEQ ID NOs: 49 to 51 encoded by Fh1Tps-y are respectively Fh1TPS. -y (y is 1-3).
Each of Fh1Tps-y contains a 1701 bp open reading frame (ORF) and encodes a protein (Fh1TPS-y) with a putative 566 amino acid residue, a molecular weight of 66.4 kDa, and a pI of 4.98 to 4.99. (Table 2). Based on a homology search against the GenBank / EMBL / DDBJ database, Fh1TPS-1 is a known sesquiterpene synthase, sesquiterpene synthase from American oil palm ( Elaeis oleifera ), 52%, β-caryophyllene synthase from grape ( Vitis vinifera ) (Beta-caryophyllene synthase) and 47%, sesquiterpene synthase 3 (Sesquiterpene synthase 3) derived from Zingiber zerumbet and 48-49% amino acid sequence identity (Table 2). Due to its homology with known sesquiterpene synthases, Fh1TPS-y is a subfamily of angiosperm sesquiterpenes and diterpene synthases (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, Fh1TPS-y contained the motifs DDxxD, RDR, and (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 8). . On the other hand, unlike the known sesquiterpene synthase, Fh1TPS-y was predicted to contain a plastid transit peptide.

Fh1TPS-y molecular weight, pI, and similarity to other terpene synthases by homology search.

(Α-Selene production by E. coli with plasmid pRSF-Fh1Tps and plasmid pAC-Mev / Scidi / Aacl)
The plasmid DNA into which the full - length Fh1Tps1-y sequence was inserted was digested with the restriction enzyme Nde I- Kpn I, and the resulting Fh1Tps1-y sequence was converted to the Nde I- Kpn I site of the pRSFDuet-1 vector (Km resistant; Novagen). To construct a pRSF-Fh1Tps-y plasmid (FIG. 9).
Plasmid pRSF-Fh1Tps-y and plasmid pAC-Mev / Scidi / Aacl (Cm resistance; Patent Document 3, Non-Patent Document 7) were used to transform E. coli BL21 (DE3). In addition, E. coli transformation method, recombinant E. coli culture method, extraction of sesquiterpene from cultured E. coli cells, and analysis method using gas chromatograph mass spectrometer (GC-MS: GCMS-QP5050 manufactured by Shimadzu) It is as Patent Documents 2 and 3 or Non-Patent Document 7. However, LAA was used instead of MVL as a substrate for culturing recombinant E. coli, 50 mg / L Km and 30 mg / L Cm were used as drugs, and dodecane was not overlaid during the culture. As a result of analysis using GC-MS, in the control group in which IPTG was added to the E. coli culture containing the plasmid pRSFDuet-1 and the plasmid pAC-Mev / Scidi / Aacl, a new sesquioxide was added to the ethyl acetate extract from the cells. While terpene formation was not confirmed, in the experimental group in which IPTG induction was performed in E. coli culture containing plasmid pRSF-Fh1Tps-y and plasmid pAC-Mev / Scidi / Aacl, the ethyl acetate extract from the cells One new peak was observed inside (FIG. 10). Peak 1 was analyzed by NMR and identified as α-selinene (Example 8). From this result, it was found that Fh1Tps-y is a gene encoding α-selinene synthase. Synthetic activity Fh1Tps-1 of alpha-selinene is 1.67 × 10 ⁶ TIC (α- Serinenpiku height, hereinafter the same) as the highest, Fh1Tps-2 is ^{1.13 × 10 6 TIC, Fh1Tps-} 3 is 0 It gradually decreased to 0.94 × 10 ⁶ TIC. The chemical structure of α-selinene is shown in FIG.
In addition, since the enzyme which synthesize | combines (alpha)-serinen as a substantially single product, and the gene which codes this were not known until now, it is (alpha) -derived from Freesia ( Freesia x hybrida ) Airy purple disclosed here. Linen synthase (Fh1TPS-y) and the same gene ( Fh1Tps-y ) are the first reports. In addition, a known enzyme protein that synthesizes α-selinen as a by-product {for example, SES derived from sweet basil ( Ocimum basilicum ) (Y. Iijima et al., Plant Physiol. 136: 3724-3736, 2004)} Are also phylogenetically distant (all have a low homology of less than 50%). In the future, since α-selinen can be easily provided by the production method of the present invention, it has become possible to conduct various physiological function tests on α-serinen. It is expected to discover new functionality and deepen knowledge of functionality for α-selinen.

(Identification of α-selinen)
E. coli BL21 (DE3) introduced with plasmid pRSF-Fh1Tps-1 and plasmid pAC-Mev / Scidi / Aacl was cultured in 1 L of TB (Terific Broth) containing 50 mg / L Km and 30 mg / L Cm. . At this time, 200 mL of n -octane was added and cultured. After completion of the culture, first culture (broth 1 L + n - octane 200 mL) was stirred well and put in 2 L capacity separating funnel, clear the lower (aqueous layer) was discarded {upper layer at this point (n -Octane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of 50% methanol (0.1 N NaOH 250 mL + methanol 250 mL) were added to the separatory funnel and stirred well. In this operation, the emulsion almost disappeared and separated into two layers, so the lower layer (alkali 50% methanol layer) was discarded. Again, 500 mL of alkali 50% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting).
Next, 20 L of this concentrate was analyzed by PDA-HPLC (high performance liquid chromatography with photodiode array detector) shown below.
Column: Developsil C30-UG-520 mm x 250 mm (Nomura Chemical)
Solvent: 90% CH ₃ CN
Flow rate: 8.0 mL / min
Detection: 200-500 nm (PDA)
As a result, it was estimated that the peak with a retention time of 55.0 min was a terpene compound (by preliminary GC-MS analysis), so that each peak was divided into about 10 times under the same conditions (the concentrate 2). 10 mL each of 200 L was injected). After completing 10 peak fractions, add the same amount of water as the collected total eluate (80-150 mL) and place in a 500 mL separatory funnel, where the same amount (elution solvent x 2 (160 -300 mL)) n -pentane was added and stirred well to recover the n -pentane layer. The n -pentane layer collected by repeating the same operation once again was transferred to a 1 L Erlenmeyer flask, dried over anhydrous sodium sulfate, and concentrated under reduced pressure (150 mmHg setting) with an evaporator. When the concentrate reaches about 5 mL, transfer the remaining solvent to the 25 mL eggplant flask. Concentration with the evaporator is stopped as soon as n -pentane disappears. Finally, dry nitrogen gas is blown for about 5 minutes to remove the solvent. Distilled completely.
With the above operation, 5.6 mg of a pure compound was obtained. This compound was identified as α-selinene represented by the following formula (4) by NMR analysis and GC-MS analysis.

Spectroscopic analysis data for α-selinen:
GC-MS retention time 28.7 min, m / z 204 (M ⁺ ). ¹ H-NMR (CDCl ₃ ): 0.80 (s, 3H, H-15), 1.18 (m, 1H, H -6), 1.20 (m, 1H, H-9), 1.30-1.40 (2H, H-1), 1.46 (m, 1H, H-9), 1.50-1 .60 (2H, H-8), 1.61 (s, 3H, H-11), 1.73 (s, 3H, H-14), 1.75 (m, 1H, H-6), 1 .90 (m, 1H, H-2), 1.92 (m, 1H, H-7), 2.15 (m, 1H, H-2), 4.70 (s, 1H, H-13) , 4.72 (s, 1H, H-13), 5.32 (m, 1H, H-3). ¹³ C-NMR (CDCl ₃ ): 15.6 (C-15), 20.9 (C -14), 21.2 (C-11), 23.0 (C-2), 26.8 (C-8), 28.9 (C-6), 32.3 (C-10), 37 .9 (C-1), 40.2 (C-9), 46.8 (C-5), 46.8 (C-7), 108.2 (C-13), 120.9 (C- 3), 135.1 (C- 4), 151.0 (C-12).

{Acquisition of α-copaene synthase (α-copaene synthase) gene ( Fh6Tps ) cDNA}
A pair of oligonucleotide primers specific for the full-length cDNA sequence obtained in Example 5 FPe1.Nde-F (5′-AGACAGA CATATG GAGTCAGTACTGCTGAGCT-3 ′ (SEQ ID NO: 52), underlined indicates Nde I recognition site) and FPe1 PCR using Airy Peach cDNA as a template using Kpn -R (5'-GTAC GGTACC CTAGTAGTAGTCCCCCGGGAAG-3 '(SEQ ID NO: 53), 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 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. The cDNA nucleotide sequence is shown in SEQ ID NOs: 54 to 61, and the amino acid sequences of the respective polypeptides encoded by the cDNA are shown in SEQ ID NOs: 62 to 69. Further, the cDNA specified by SEQ ID NOs: 54 to 61 is named Fh6Tps-z (z is 1 to 8), and the proteins (polypeptides) specified by SEQ ID NOs: 62 to 69 encoded by Fh6Tps-z are respectively Fh6TPS. -z (z is 1-8).
All of Fh6Tps-z contain a 1713 bp open reading frame (ORF), a protein (Fh6TPS-z) with a putative 570 amino acid residue, a molecular weight of 66.7-66.8 kDa, and a pI of 4.85 to 4.88. (Table 3). Based on homology searches against GenBank / EMBL / DDBJ databases, Fh6TPS-z is a known sesquiterpene synthase, sesquiterpene synthase from American oil palm ( Elaeis oleifera ) and 51%, β-caryophyllene synthase from grape ( Vitis vinifera ) (Beta-caryophyllene synthase) and 46%, and sesquiterpene synthase 3 (Sesquiterpene synthase 3) derived from Zingiber zerumbet showed 48-49% amino acid sequence identity (Table 3). Because of homology with known sesquiterpene synthases, Fh6TPS-z is a subfamily of angiosperm sesquiterpenes and diterpene synthases (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, FH6TPS-z contained the motifs DDxxD, RDR and (N / D) Dxx (S / T) xxxE {referred to as NSE / DTE motif} highly conserved in sesquiterpene synthase (Fig. 12). . On the other hand, unlike known sesquiterpene synthases, Fh6TPS-z was predicted to contain a plastid transit peptide.

Molecular weight of Fh6TPS-z, pI, and similarity to other terpene synthases by homology search.

(Α-Copaene production by E. coli with plasmid pRSF-Fh6Tps-z and plasmid pAC-Mev / Scidi / Aacl)
Plasmid DNA with the full - length Fh6Tps-z sequence inserted was digested with the restriction enzyme Nde I- Kpn I, and the resulting Fh6Tps-z sequence was converted into the Nde I- Kpn I site of the pRSFDuet-1 vector (Km resistant; manufactured by Novagen). To produce a pRSF-Fh6Tps-z plasmid (FIG. 9).
Escherichia coli BL21 (DE3) was transformed with plasmid pRSF-Fh6Tps-z and plasmid pAC-Mev / Scidi / Aacl (Cm resistance; Patent Document 3, Non-Patent Document 7). In addition, E. coli transformation method, recombinant E. coli culture method, extraction of sesquiterpene from cultured E. coli cells, and analysis method using gas chromatograph mass spectrometer (GC-MS: GCMS-QP5050 manufactured by Shimadzu) It is as Patent Documents 2 and 3 or Non-Patent Document 7. However, LAA was used instead of MVL as a substrate for culturing recombinant E. coli, 50 mg / L Km and 30 mg / L Cm were used as drugs, and dodecane was not overlaid during the culture. As a result of analysis using GC-MS, in the control group to which IPTG was added during the cultivation of E. coli containing plasmid pRSFDuet-1 and plasmid pAC-Mev / Scidi / Aacl, a novel substance was extracted into the ethyl acetate extract from the cells. In the experimental section where IPTG induction was performed in E. coli culture containing the plasmid pRSF-Fh6Tps-z and the plasmid pAC-Mev / Scidi / Aacl, ethyl acetate was extracted from the cells. A new peak 1 was observed in the liquid (FIG. 13). Peak 1 was analyzed by NMR and identified as α-copaene (Example 11). From this result, it was found that Fh6Tps-z is a gene encoding α-copaene synthase. The synthetic activity of α-copaene was highest in Fh6Tps-1 , and decreased as the clone number (z) progressed from Fh6Tps- 2 to Fh6Tps-8 . Table 4 shows the GC-MS peak intensity of each Fh6Tps-z clone and α-copaene synthesized by recombinant E. coli containing the clone. The chemical structure of α-copaene is shown in FIG.
From the above results, it was shown that selective production of α-linenene was possible using E. coli into which plasmid pRSF-Fh1Tps-y and plasmid pAC-Mev / Scidi / Aacl were introduced. It was also shown that selective production of α-copaene was possible using E. coli introduced with plasmid pRSF-Fh6Tps-z and plasmid pAC-Mev / Scidi / Aacl.
In addition, about the enzyme which synthesize | combines (alpha) -copaene almost as a single product, and the gene which codes this, it was not known until now in what both the academic paper and the sequence information were disclosed. Therefore, the α-copaene synthase (Fh6TPS-z) derived from Freesia × hybrida Airy Peach and the same gene ( Fh6Tps-z ) disclosed here are the first reports. Also known enzyme proteins that synthesize α-copaene as a by-product {eg, RcSeTPS1 derived from castor bean; Ricinus communis (X. Xie, J. Kirby, JD Keasling, Phytochem. 78: 20-28, 2012) } Is also phylogenetically distant (all have a low homology of 50% or less). In the future, since α-copaene can be easily provided by the production method of the present invention, it has become possible to conduct various physiological function tests on α-copaene. Although α-copaene is considered to have anti-inflammatory effects, it is expected to discover new functionalities and deepen functional knowledge.

Peak intensity of terpenes detected from Fh6Tps-z recombinant E. coli (TIC: total ion chromatogram, tr: trace).

(Identification of α-copaene)
E. coli BL21 (DE3) introduced with plasmid pRSF-Fh6Tps-1 and plasmid pAC-Mev / Scidi / Aacl was cultured in TB (Terific Broth) 1 L containing 50 mg / L Km and 30 mg / L Cm. . At this time, 200 mL of n -octane was added and cultured. After completion of the culture, first culture (broth 1 L + n - octane 200 mL) was stirred well and put in 2 L capacity separating funnel, clear the lower (aqueous layer) was discarded {upper layer at this point (n -Octane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of alkali 90% methanol (50 mL of 0.5 N NaOH + 450 mL of methanol) were added to the separatory funnel and stirred well. In this operation, the emulsion disappeared and it was clearly divided into two layers, so the lower layer (alkali 90% methanol layer) was discarded. Again, 500 mL of alkaline 90% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting). The concentrate was applied to silica gel (Silica Gel 60, Kanto Chemical) having an inner diameter of 20 mm and a length of 200 mm using hexane as a developing solvent, and 6 mL each was fractionated with the same solvent. Each fraction was developed with silica gel TLC (Merck) developing solvent hexane to develop phosphomolybdenum color. The elution fractions 9 and 10 of the silica gel column chromatography with color were combined and concentrated to dryness under reduced pressure of an evaporator (150 mmHg setting) to obtain 4.3 mg of a pure product. This compound was identified as α-copaene represented by the following formula (5) by NMR analysis and GC-MS analysis.

Spectroscopic analysis data of α-copaen:
GC-MS retention time 23.2 min, m / z 204 (M ⁺ ). ¹ H-NMR (CDCl ₃ ): 0.78 (s, 3H, H-12), 0.83 (d, J = 6 .4 Hz, 3H, H-14), 0.84 (d, J = 6.4 Hz, 3H, H-15), 1.52 (m, 1H, H-4), 1.52 (m, 1H, H-13), 1.55 (m, 1H, H-8), 1.56 (m, 1H, H-6), 1.57 (m, 1H, H-7), 1.64 ( m, 1H, H-7), 1.66 (s, 3H, H-11), 1.67 (m, 1H, H-9), 1.72 (m, 1H, H-6), 2. 09 (m, 1H, H-10), 2.15-2.20 (2H, H-1), 5.20 (m, 1H, H-2). ¹³ C-NMR (CDCl ₃ ): 19. 3 (C-12), 19.7 (C-15), 19.9 (C-14), 21.8 (C-7), 23.1 (C-11), 30.0 (C-1 ), 32.2 (C-13), 36.2 (C-6), 36.9 (C-10), 39.4 (C-5), 44.3 (C-9), 44.7 (C-8), 54.2 (C-4), 116.1 (C-2), 144.0 (C-3).

{Determination of camellia scented monoterpene synthase (monoterpene synthase) gene cDNA sequence}
Total RNA was extracted from camellia ( Camellia saluenensis varieties: scented scent) flowers (FIG. 14) using RNeasy Plant Mini Kit (Qiagen). CDNA was prepared from 1.0 μg of total RNA using SMARTer RACE cDNA amplification kit (Takara Bio) according to the manufacturer's instructions. Degenerate oligonucleotide primer pairs (forward: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(SEQ ID NO: 38) and reverse: 5'-TAIGHRTCAWAIRTRTCRTC- based on the sequence of the conserved domain of higher plant sesquiterpene synthase described in Non-Patent Document 3 Using 3 ′ (SEQ ID NO: 39), PCR was performed using cDNA as a template under the conditions as described in Non-Patent Document 3. Thus, an amplified fragment of 409 bp was obtained, and the obtained amplified fragment was designated as pGEM-T Easy Vector. In order to isolate full-length cDNA, the above-mentioned SMARTer RACE cDNA Amplification Kit (Takara Bio), oligonucleotide primer and Advantage2 Polymerase Mix (Clontech) were used. The fragments were extended toward the 5 ′ and 3 ′ ends in accordance with the manufacturer's instructions, and the full-length cDNA sequence was determined. e (5′-TCGAGCTTGATGATGAGAAAGATG-3 ′) (SEQ ID NO: 70) and Pur.3 race (5′-TCACAAAACCCATCTCTTTCATCT-3 ′) (SEQ ID NO: 71) were used.

{Acquisition of linalool synthase (linalool synthase) gene ( CsTps1 ) cDNA}
Oligonucleotide primer pair Pu.Nde-F2 (5′-AGACAGA CATATG CAAATCTTCCACTGTGCAT-3 ′ (SEQ ID NO: 72), underlined represents the Nde I recognition site) specific to the full-length cDNA sequence obtained in Example 12 and Pu Using Kpn-R (5'- GGTACC CTAAGGTAATTTATCATAGAACATAGACTTCATG-3 '(SEQ ID NO: 73), underlined indicates Kpn I recognition site) and Advantage2 Polymerase Mix (Clontech), the camellia scented cDNA was used as a template. PCR (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, 60 ° C. for 30 seconds and 72 ° C. for 4 minutes for 25 cycles). 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: 74, and the amino acid sequence of the polypeptide encoded by the cDNA is shown in SEQ ID NO: 75. The cDNA identified by SEQ ID NO: 74 was named CsTps1, and the protein (polypeptide) identified by SEQ ID NO: 75 encoded by CsTps1 was named CsTPS1.
CsTps1 contained a 1728 bp open reading frame (ORF) and encoded a protein (CsTPS1) with a putative 575 amino acid residue, a molecular weight of 66.1 kDa, and a pI of 6.03. Homology searches against GenBank / EMBL / DDBJ database, CsTPS-1 is known sesquiterpene / monoterpene synthases tea (Camellia sinensis) derived bifunctional ruthenate Lupe isoprenoid synthase (Bifunctional terpenoid synthase) and 97%, in a known monoterpene synthases An amino acid sequence identity of 71% with linalool synthase derived from a certain monkey ( Actinidia arguta ) and 71% with linalool synthase derived from a matabi ( Actinidia polygama ) was shown. Homology with known sesquiterpene synthases confirmed that CsTPS1 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, CsTPS1 is also highly conserved in monoterpenes / sesquiterpene synthases, DDxxD, RDR, and (N / D) Dxx (S / T) xxxE {called NSE / DTE motif} RDR becomes RDQ Although there was a change, etc., it was almost preserved (FIG. 15). On the other hand, like the usual known monoterpene synthase, CsTPS1 was predicted to contain a plastid transit peptide.

(Linalool production by E. coli with plasmid pRSF-CsTps1 and plasmid pAC-Mev / Scidi / Aacl)
Plasmid DNA into which the full-length CsTps1 sequence has been inserted is digested with the restriction enzyme Nde I- Kpn I, and the resulting CsTps1 sequence is ligated to the Nde I- Kpn I site of the pRSFDuet-1 vector (Km resistant; Novagen). PRSF-CsTps1 plasmid was prepared (FIG. 16).
Escherichia coli BL21 (DE3) was transformed with plasmid pRSF-CsTps1 and plasmid pAC-Mev / Scidi / Aacl (Cm resistance; Patent Document 3, Non-Patent Document 7). In addition, E. coli transformation method, recombinant E. coli culture method, extraction of sesquiterpene from cultured E. coli cells, and analysis method using gas chromatograph mass spectrometer (GC-MS: GCMS-QP5050 manufactured by Shimadzu) It is as Patent Documents 2 and 3 or Non-Patent Document 7. However, LAA was used instead of MVL as a substrate for culturing recombinant E. coli, 50 mg / L Km and 30 mg / L Cm were used as drugs, and dodecane was not overlaid during the culture. As a result of analysis using GC-MS, in the control group to which IPTG was added during the cultivation of E. coli containing plasmid pRSFDuet-1 and plasmid pAC-Mev / Scidi / Aacl, a novel substance was extracted into the ethyl acetate extract from the cells. While no sesquiterpene was confirmed, in the experimental group in which IPTG induction was performed during the culture of E. coli containing plasmid pRSF-CsTps1 and plasmid pAC-Mev / Scidi / Aacl, A new peak was observed in (Fig. 17). Peak 1 was analyzed by NMR and identified as linalool (Example 15). From this result, it was found that CsTps1 is a gene encoding linalool synthase. The chemical structure of linalool is shown in FIG. In this example, linalool synthase (CsTPS1) did not produce nerolidol or other by-products in addition to linalool. In addition to the gene of the present invention, the linalool synthase (linalool synthase) gene can be obtained from various plants such as sarunasi ( Actinidia arguta ), matatabi ( Actinidia polygama ), ginger ( Zingiber officinale ), turmeric (autumn turmeric; Curcuma longa ). However, all of the functionally analyzed linalool synthases are known to share the synthesis activity of the sesquiterpene nerolidol (HJ Koo, DR Gang, Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues. PLoS One 7: e51481, 2012). Therefore, linalool synthase is often also referred to as linalool / nerolidol synthase. In addition, linalool has an unpleasant odor while linalool has a pleasant scent. On the other hand, since the linalool synthase (CsTPS1) derived from camellia ( Camellia saluenensis variety name: Inochi fragrance) obtained this time has no nerolidol synthesis activity, CsTPS1 is a functionally novel enzyme. It can be said.

(Identification of linalool)
In addition to plasmid pAC-Mev / Scidi / Aacl, Escherichia coli BL21 (DE3) introduced with plasmid pRSF-CsTps1 containing a gene derived from camellia ( Camellia saluenensis , variety name: Inochi fragrance) was introduced at 50 mg / L. Of 1 ml of TB (Terific Broth) containing 30 mg / L of Cm. At this time, 200 mL of n -octane was added and cultured. After completion of the culture, first culture (broth 1 L + n - octane 200 mL) was stirred well and put in 2 L capacity separating funnel, clear the lower (aqueous layer) was discarded {upper layer at this point (n -Octane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of 50% methanol (0.1 N NaOH 250 mL + methanol 250 mL) were added to the separatory funnel and stirred well. In this operation, the emulsion almost disappeared and separated into two layers, so the lower layer (alkali 50% methanol layer) was discarded. Again, 500 mL of alkali 50% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting).
Next, 20 L of this concentrate was analyzed by PDA-HPLC (high performance liquid chromatography with photodiode array detector) shown below.
Column: Developsil C30-UG-520 mm x 250 mm (Nomura Chemical)
Solvent: 90% CH ₃ CN
Flow rate: 8.0 mL / min
Detection: 200-500 nm (PDA)
As a result, it was estimated that the peak with a retention time of 9.0 min was a monoterpene compound (by preliminary GC-MS analysis), so this peak was divided into about 10 times under the same conditions (the concentrate described above). 2 mL was dispensed 10 times 200 L each time). After completing 10 peak fractions, add the same amount of water as the collected total eluate (80-150 mL) and place in a 500 mL separatory funnel, where the same amount (elution solvent x 2 (160 -300 mL)) n -pentane was added and stirred well to recover the n -pentane layer. The n -pentane layer collected by repeating the same operation once again was transferred to a 1 L Erlenmeyer flask, dried over anhydrous sodium sulfate, and concentrated under reduced pressure (150 mmHg setting) with an evaporator. When the concentrate reaches about 5 mL, transfer the remaining solvent to the 25 mL eggplant flask. Concentration with the evaporator is stopped as soon as n -pentane disappears. Finally, dry nitrogen gas is blown for about 5 minutes to remove the solvent. Distilled completely.
With the above operation, 15.1 mg of a pure compound was obtained. This compound was identified as linalool represented by the following formula (6) by NMR analysis and GC-MS analysis.

Spectroscopic analysis data of linalool:
GC-MS retention time 14.2 min, m / z 154 (M ⁺ ). ¹ H-NMR (CDCl ₃ ): 1.27 (s, 3H, H-10), 1.50-1.63 (2H , H-5), 1.60 (s, 3H, H-9), 1.68 (s, 3H, H-1), 1.95-2.05 (2H, H-4), 5.06 (D, J = 10.7 Hz, 1H, H-8), 5.12 (dd, J = 7.0, 7.1 Hz, 1H, H-3), 5.21 (d, J = 16 .3 Hz, 1H, H-8), 5.91 (dd, J = 10.7, 16.3 Hz, H-7). ¹³ C-NMR (CDCl ₃ ): 17.7 (C-9) , 22.8 (C-4), 25.7 (C-1), 27.9 (C-10), 42.0 (C-5), 73.5 (C-6), 111.7 ( C-8), 124.3 (C-3), 131.9 (C-2), 145.0 (C-9).

(Rakkyo-derived transcript analysis and determination of β-oscymene / α-farnesene synthase gene sequence)
The bulbous tissue of Allium chinense G. Don was chopped and frozen in liquid nitrogen. The frozen tissue was ground in liquid nitrogen using a mortar and pestle, and then total RNA was extracted using FastRNA Pro Green Kit (MP Biomedicals) and RNeasy Plant Mini Kit (Qiagen). The total RNA obtained was quantitatively evaluated for RNA quality by RNA Integrity Number (RIN) using Agilent 2100 Bioanalyzer (Agilent Technology) and Agilent RNA6000 Nano Kit (Agilent Technology). Eight or more samples were subjected to subsequent experiments. 5 μg of the obtained total RNA was subjected to RNA sequence analysis and sequence data assembly. As a result of the sequence and assembly, 217,890 contig sequences were obtained. Results These were subjected to homology search by BlastX, magnolia grandiflora (Magnolia grandiflora) of alpha-terpineol synthase (α-terpineol synthase, Genbank accession B3TPQ7) 46%, Vitis vinifera (Vitis vinifera) of alpha-terpineol synthase (alpha- terpineol synthase, Genbank accession NP_001268216), a gene fragment showing 44% homology was found and named AlcTPS1 . AlcTPS1 encoded a gene predicted to have an 1827 bp open reading frame (ORF), 608 amino acid residues, a molecular weight of 70.1 kDa, and a pI = 6.4. The deduced base sequence is shown in SEQ ID NO: 76, and the deduced amino acid sequence is shown in SEQ ID NO: 77. The deduced amino acid sequence of AlcTPS1, RxR, such DDXXD, were included motifs that are conserved in many terpene synthases. In addition, the Nc terminal amino acid sequence of AlcTPS1 was predicted to have a plastid transfer signal sequence of 55 amino acid residues.

{Isolation of β- osymene / α-farnesene synthase gene ( AlcTPS1 ) and preparation of expression plasmid (pAlcTPS1)}
A reverse transcription reaction was performed on 2 μg of the total RNA sample obtained from the Rakkyo bulb tissue using PrimeScript ^™ II 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.) to prepare a single-stranded cDNA library. An oligonucleotide primer pair (AlcTPS1 Fw, 5′-ATGAGCATTTGTGTGTTAGGTTTTACCCA-3 ′; SEQ ID NO: 78 and AlcTPS1 Rv, 5′-CTAATTCAAACAAGAAAAATCCTTCTGTGCTATATTAATAGG-3 ′; SEQ ID NO: 79) was designed based on the estimated base sequence information of AlcTPS1. Using these oligonucleotide pairs and PrimeSTAR (Japan registered trademark) Max DNA Polymerase, PCR was performed using a single-stranded cDNA library as a template to obtain the target AlcTPS1 fragment. The obtained AlcTPS1 fragment is an oligonucleotide primer pair (pRSF-AlcTPS1 Fw, 5′-TCTTGTTTGAATTAGGCTGCTGCCACCGCTGA-3 ′; SEQ ID NO: 80, and pRSF-AlcTPS1 Rv, 5′-) using the pRSFDuet-1 plasmid (Novagen) as a template. CACACAAATGCTCATATGTATATCTCCTTCTTATACTTAACT-3 '; SEQ ID NO: 81) and DNA fragment obtained by PCR, cloned by in vitro homologous recombination using In-Fusion HD Cloning Kit (Takara Bio) The pAlcTPS1 plasmid was obtained.

(Β-Ocimene / α-farnesene synthesis by E. coli expression system using plasmid pAlcTPS1)
The pAlcTPS1 plasmid is introduced into Escherichia coli BL21 (DE3) together with the pAC-Mev / Scidi / Aacl plasmid according to the method of Patent Document 3 or Non-Patent Document 7, and dodecane is introduced according to the method of Patent Document 3 or Non-Patent Document 7. Culture, production, and product extraction were performed in the added liquid medium. As a result of analyzing 1 μL of the extracted dodecane layer with a gas chromatograph mass spectrometer (GC-MS), in the sample derived from Escherichia coli introduced with the pAlcTPS1 plasmid, the retention times were around 10.75 minutes and 30.2 minutes. A peak not found in the E. coli-derived sample into which pRSFDuet-1 was introduced was detected (FIG. 20; peak 1, FIG. 21; peak 2). As a result of mass spectrum analysis of these two peaks, peak 1 coincided with the spectrum of β-ocimene, and peak 2 coincided with the spectrum of α-farnesene (FIGS. 22 and 23). From this result, it was found that AlcTPS1 is a gene encoding β- ocimene / alpha-farnesene synthase (β-ocimene / α-farnesene synthase).

(Demonstration test 1 of efficient purification method of sesquiterpene using n -alkane having 8 to 10 carbon atoms)
Using the gamma-amorphene synthase gene (γ-amorphene synthase; ZoTps5 ) derived from the radicle stem of ginger (ginger; Zingiber officinale ; variety name: Kintoki ginger) disclosed in Patent Document 1, n having 8 to 10 carbon atoms -The efficiency of the purification method of sesquiterpenes using alkanes was demonstrated. That is, in addition to plasmid pAC-Mev / Scidi / Aacl {chloramphenicol (Cm) resistance; Patent Document 3, Non-patent Document 7}, plasmid pET-Zo501FL2 {ampicillin (Ap) resistance including ginger ZoTps5 gene; Patent Document 1} is introduced into 1 L of TB medium (Terific Broth; J. Sambrook, DW Russel, Molecular Cloning, 3rd edition, Cold, containing 100 mg / L of Ap and 30 mg / L of Cm. Cultured in Spring Harbor, NY, 2001). At this time, n - and cultured with octane (n -octane) 200 mL. After completion of the culture, first, the culture (culture medium 1 L + n -octane 200 mL) was placed in a 2 L separatory funnel and stirred well, and the apparent lower layer (aqueous layer) was discarded {at this time the upper layer (n -Octane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of alkali 90% methanol (50 mL of 0.5 N NaOH + 450 mL of methanol) were added to the separatory funnel and stirred well. In this operation, the emulsion disappeared and it was clearly divided into two layers, so the lower layer (alkali 90% methanol layer) was discarded. Again, 500 mL of alkaline 90% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting).
The same result was obtained even when decane was used instead of the above n -octane. On the other hand, n having 7 carbon - heptane (n -heptane) and less than this n - in the case of using an alkane, inhibited the growth of recombinant E. coli.
Next, 20 μL of this concentrate was analyzed by PDA-HPLC (high performance liquid chromatography with photodiode array detector) under the conditions shown below.
Column: Developsil C30-UG-5 10 mm x 250 mm (Nomura Chemical)
Solvent: 95% CH ₃ CN
Flow rate: 3.0 mL / min
Detection: 200-500 nm (PDA)
The results are shown in FIG. Since it was presumed that the peak group with circled numbers 1 to 4 shown in this figure was a terpene compound (by preliminary GC-MS analysis), each peak was divided into about 10 times under the same conditions ( 2 mL of the concentrate was injected 10 times at a time of 200 μL. (Of these, the numbers 1 to 3 with circles represent a certain amount of each compound, so that each peak was fractionated. After 10 peak fractions were collected, the total eluate (10 - 30 mL) and placed in the same amount of water was added to 200 mL volume separatory funnel, the same amount therein (eluent x 2 (20 - 60 mL) ) of the n - well stirred added pentane n - The n -pentane layer collected by repeating the same operation once again was transferred to a 200 mL Erlenmeyer flask, dried over anhydrous sodium sulfate, and concentrated under reduced pressure (150 mmHg setting) with an evaporator. When the product reaches about 5 mL, transfer the solvent to a 25 mL eggplant flask, stop the evaporator concentration as soon as n -pentane disappears, and finally blow dry nitrogen gas for about 5 minutes to keep the solvent completely. Left.
In the above operation, 2.9 mg of the compound with a circled number 1 {identified as germacrene D (germacrene D) by various analyzes}, 2.8 mg of the compound with a circled number 2 [ cis- muurola-4 (15 by various analyzes) ), 5-diene {identified as cis -Murora-4 (15), 5-diene}], 1.0 mg of compound with circled number 3 {identified as aromadendrene by various analyzes}, circled number Compound 9.9 mg {identified as γ-amorphene (γ-amorphene) by various analyzes} was obtained as a pure product. The structure of each compound is shown in FIG. Although only γ-amorphene could be identified in Patent Document 1, the production of germacrene D, cis- muurola-4 (15), 5-diene, and aromadendrene could be confirmed by this efficient purification method.
As described above, according to the contents disclosed in the present examples and the like, n -alkane having 8 to 10 carbon atoms of n -octane to decane is overlaid on the medium, E. coli for biosynthesis of terpene is cultured, and the produced terpene is obtained. A manufacturing method has been invented for efficient recovery. It should be noted that there has never been a production method in which n -octane or decane is overlaid and the host is cultured to recover the produced terpene.

(Verification test 2 of efficient purification method of sesquiterpene using n -alkane having 8 to 10 carbon atoms)
A method for purifying a sesquiterpene using an n -alkane having 8 to 10 carbon atoms, using a β-cubebene synthase ( AeTps1 ) gene derived from a sprout of Aralia elata disclosed in Patent Document 2 The efficiency was demonstrated. That is, plasmid pAC-Mev / Scidi / Aacl {chloramphenicol (Cm) resistance; Patent Document 3, Non-Patent Document 7} In addition, the plasmid pET-AeTps1a including AeTps1 gene from Aralia elata {ampicillin (Ap) resistance E. coli introduced with Patent Document 2} is prepared by adding 1 L of TB medium containing 100 mg / L of Ap and 30 mg / L of Cm (Terific Broth; J. Sambrook, DW Russel, Molecular Cloning, 3rd edition, Cold Spring Harbor, NY, 2001). At this time, 200 mL of n -octane (or decane) was added and cultured. After completion of the culture, first put the culture {culture medium 1 L + n -octane (or decane) 200 mL} into a 2 L separatory funnel and stir well, discarding the apparent lower layer (aqueous layer) {at this point Then, the upper layer ( n -octane layer or decane layer) is in an emulsion state}. Next, 300 mL of n -hexane and 500 mL of alkali 90% methanol (50 mL of 0.5 N NaOH + 450 mL of methanol) were added to the separatory funnel and stirred well. In this operation, the emulsion disappeared and it was clearly divided into two layers, so the lower layer (alkali 90% methanol layer) was discarded. Again, 500 mL of alkaline 90% methanol was added to the separatory funnel and stirred well, and the lower layer was discarded. The remaining upper layer was collected in a 500 mL Erlenmeyer flask, dehydrated with anhydrous sodium sulfate for about 30 minutes, and then concentrated to about 2 mL under reduced pressure (20 mmHg setting). The concentrate was subjected to column chromatography on silica gel (Silica Gel 60, Kanto Chemical) having an inner diameter of 20 mm and a length of 200 mm using hexane as a developing solvent, and 6 mL each was fractionated with the same solvent. Each fraction was developed with silica gel TLC (Merck) developing solvent hexane, and the phosphomolybdenum coloration was shown in FIG. The silica gel column chromatography elution fractions 9 and 10 were combined and concentrated to dryness under reduced pressure (150 mmHg setting) with an evaporator to obtain pure α-copaene (4.3 mg). When fractions 12 and 13 were combined and concentrated to dryness, a mixture of β-cubebene and δ-cadinene (7.7 mg) was combined into fractions 15-17. After concentration and drying, pure germacrene D (4.9 mg) was obtained. FIG. 27 shows these structures. Although only β-cubebene could be identified in Patent Document 2, the production of α-copaene, δ-cadinene, and germacrene D could be confirmed by this efficient purification method.

(Cloning of acetoacetate hydrolase gene)
Among acetoacetate esters (acetoacetate fatty acid esters), an enzyme gene that can specifically hydrolyze acetoacetate methyl ester (methyl acetoacetate: MAA) or acetoacetate ethyl ester (ethyl acetoacetate: EAA) As candidates, nucleotide sequences of the ester hydrolase genes (Table 5) containing 18 putative genes composed of 12 families classified from the primary amino acid sequences were obtained from the genome sequence database. 36 kinds of synthetic primer DNAs (Table 6) to which NdeI or NheI and BamHI restriction enzyme cleavage sequences were added based on the obtained base sequence were prepared, and the National Institute of Technology and Evaluation Biotechnology Center (NBRC), The target gene fragment was obtained by PCR amplification using genomic DNA purchased from RIKEN BRC or ATCC as a template.

18 ester hydrolase genes including putative genes.

36 kinds of synthetic primer DNAs to which restriction enzyme cleavage sequences of Nde I or Nhe I and Bam HI are added based on the obtained base sequence.

(Production of acetoacetate hydrolase gene-expressing E. coli strain and examination of expression conditions)
Eighteen ester hydrolase (esterase) genes obtained by the above-described method are the ligation method using restriction enzymes NdeI or NheI and BamHI cleavage and Ligation-Convenience Kit (manufactured by Nippon Gene), or In-Fusion HD Cloning Kit. Using an in vitro homologous recombination method using Takara Bio Inc., the plasmid was ligated to the pET21a plasmid to obtain 18 types of ester hydrolase expression plasmids. These plasmids were introduced into E. coli competent cell ECOS competent E. coli BL21 (DE3) (manufactured by Nippon Gene) and selected on LB agar medium containing ampicillin at a final concentration of 100 μg / mL to obtain positive clones. .
Next, each of the above-mentioned clones was inoculated into a 96-well deep well plate to which 500 μL of LB medium containing final concentration of 100 μg / mL ampicillin was added, and was incubated at 37 ° C. and 1700 rpm in a constant temperature shaking incubator for well plates. Pre-cultured for 17 hours. 1% (v / v) of this preculture was added to 500 μL of LB medium containing 100 μg / mL ampicillin at a final concentration and cultured at 37 ° C. and 1700 rpm for 4 hours. It added so that it might become 0.25, 0.5, or 1.0 mM, and it culture | cultivated for 22 to 30 hours by induction temperature 37, 30 or 25 degreeC, 1700 rpm. 500 μL of the cultured cells were transferred to a 1.5 mL tube, and collected by centrifugation at 18,000 × g for 1 minute. 50 μL of BugBuster Master Mix (Merck) was added to the cells after removing the supernatant, gently vortexing at room temperature for 10-20 minutes, and centrifuged at 16,000 × g for 20 minutes at 4 ° C. The piece was removed. The supernatant was used as a crude enzyme extract sample, and protein concentration was quantified using Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific).
As a result of SDS-PAGE analysis of the above-mentioned enzyme crude extract sample, in the samples derived from 13 strains except for 5 strains BC4102, BC4904, Cgl0292, Cgl1099 and Cgl2220 under the conditions of IPTG final concentration of 0.25 mM and induction temperature of 30 ° C. A significant amount of enzyme protein expression was confirmed in the soluble fraction.

(Identification of enzyme function by measuring acetoacetate hydrolase activity)
Acetoacetate hydrolase (esterase) activity was measured for the above-mentioned enzyme crude extract sample using Acetoacetate Colorimetric Assay Kit (Funakoshi). Prepare a reaction solution for activity measurement containing 10% (v / v) of crude enzyme extract (prepared at a protein concentration of 0.5 μg / μL) and MAA or EAA at a final concentration of 100 mM as a substrate. After reacting on a microplate at 4 ° C. for 100 minutes, absorbance at 550 nm was measured with a microplate reader. As a result of activity quantification using an acetoacetate calibration curve, the production of acetoacetate was confirmed in a plurality of expression strains, and in particular, it was confirmed that PnbA, PA3859 and SGO0795 expression strains have high acetoacetate hydrolysis activity. (FIG. 28). The amino acid sequences of PnbA, PA3859 and SGO0795 are shown in SEQ ID NOs: 85 to 87, and the base sequences are shown in SEQ ID NOs: 82 to 84.

(Construction of plasmids pAC-Mev / Scidi / Aacl / pnbA and pAC-Mev / Scidi / Aacl / pnbAopt)
.. Bacillus subtilis subsp were purchased from NBRC subtilis str 168 genomic DNA as a template, pnbA Fw (5'- AGGGCTAGCACTCATCAAATAGTAACGACTCAATACG-3 '; SEQ ID NO: 88) and pnbA Rv (5'-GGCGGATCCTTATTCTCCTTTTGAAGGGAATAG-3 '; SEQ ID NO: 89) The pnbA gene having a HindIII cleavage site at both ends and an SD sequence at the 5 ′ end was amplified by PCR using these two primers. About this pnbA gene, mevalonate pathway enzyme gene group derived from Streptomyces sp. Strain CL190 (mevalonate kinase, diphosphomevalonate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMG-CoA synthetase and HMG-CoA reductase), budding yeast (Saccharomyces cerevisiae) derived from type 1 IPP isomerase, and Hin rat (Rattus norvegicus) expressing the gene from acetoacetate -CoA ligase pAC-Mev / Scidi / Aacl plasmid (Patent Document 3, non-Patent Document 7) The pAC-Mev / Scidi / Aacl / pnbA plasmid was constructed by ligating to the dIII cleavage site (FIG. 29).
Based on the amino acid sequence of PnbA (SEQ ID NO: 85), an artificially synthesized gene was prepared by optimizing the frequency of E. coli codon usage and named pnbAopt . The base sequence is shown in SEQ ID NO: 90. Using this synthetic gene fragment as a template, both PCR were performed using two primers of pnbAopt Fw (5′-GTTAAGCTTAGGAGGCAGCTATGACCCATCAGATTGTTACCAC-3 ′; SEQ ID NO: 91) and pnbAopt Rv (5′-CTCAAGCTTATTCGCCTTTGCTCGGAAAC-3 ′; SEQ ID NO: 92). The pnbAopt gene having a HindIII cleavage site at the end and an SD sequence at the 5 ′ end was amplified. This pnbAopt gene was ligated to the Hin dIII cleavage site of the pAC-Mev / Scidi / Aacl plasmid in the same manner as described above to construct the pAC-Mev / Scidi / Aacl / pnbAopt plasmid (FIG. 29).

(Lycopene production by E. coli strains carrying plasmids pAC-Mev / Scidi / Aacl / pnbA and pAC-Mev / Scidi / Aacl / pnbAopt)
The plasmid constructed by the above-described method is a lycopene production plasmid pCRT-EIB (N. Misawa, M. Nakagawa, K. Kobayashi, S. Yamano, Y. Izawa, K. Nakamura, K. Harashima, J. Bacteriol, 172: 6704-6712, 1990) and introduced into E. coli competent cell ECOS competent E. coli JM109 (Nippon Gene), LB containing ampicillin at a final concentration of 100 μg / mL and chloramphenicol at a final concentration of 30 μg / mL. A positive clone was obtained by selection on an agar medium.
The obtained Escherichia coli clone was inoculated into a test tube containing 2 mL of LB medium containing 100 μg / mL ampicillin at a final concentration of 30 μg / mL and chloramphenicol at a final concentration of 30 μg / mL for 17 hours at 30 ° C. and 180 rpm. Shake culture (preculture). Next, add 1% (v / v) of the preculture to a 100 mL baffled Erlenmeyer flask containing 20 mL of 2 × YT medium containing 100 μg / mL ampicillin and a final concentration of 30 μg / mL chloramphenicol. v) Added and cultured with shaking at 30 ° C. and 140 rpm. When the absorbance at 600 nm reached 0.5, the mixture was cooled to room temperature. After addition of IPTG having a final concentration of 1 mM and MAA or EAA having a final concentration of 5 mM, shaking culture was continued at 20 ° C. and 160 rpm. After 48 hours and 72 hours, 500 μL of cultured cells were transferred to a 1.5 mL tube and collected by centrifugation at 18,000 × g for 10 minutes at room temperature. To the precipitated cells, 0.5 mL of a mixed solution of chloroform: methanol = 1: 1 was added to extract the pigment component. The extract was dried for 1 to 3 hours using a centrifugal evaporator (CC-105, manufactured by Tommy Seiko Co., Ltd.) and then dissolved in 200 μL of ethyl acetate. High-performance liquid chromatograph HPLC LC-2000Plus system (manufactured by JASCO Corporation) Quantitative analysis was performed.
Using a TSK ODS-80Ts column (4.6 × 150 mm, manufactured by Tosoh Corporation) as a separation column, a flow rate of 1.0 mL / min, a temperature of 35 ° C., mobile phase A (methanol: water = 95: 5) And mobile phase B (methanol: tetrahydrofuran = 7: 3) to form a gradient, and detection was performed at a wavelength of 470 nm. Gradient conditions include holding mobile phase A as 100% for 5 minutes, then forming a linear gradient from mobile phase A to mobile phase B over 5 minutes, and holding 100% in mobile phase B for 8 minutes. did. Thereafter, 100% mobile phase A was allowed to flow for 12 minutes to equilibrate the inside of the column.
The pigment component was converted based on the retention time of the high performance liquid chromatograph and the area value created using the lycopene standard sample, and the lycopene concentration in the sample was calculated as the amount of lycopene per E. coli dry weight and per culture broth.
The quantitative results are shown in FIG. As shown in the figure, in the E. coli production strain into which plasmids pAC-Mev / Scidi / Aacl / pnbA and pAC-Mev / Scidi / Aacl / pnbAopt were introduced, even when MAA or EAA as an ester was used as a substrate, The amount of lycopene produced was about 15 mg per gram of dry cell weight (DCW) (12 to 15 mg per liter of the culture solution) as in the case of using the substrate lithium acetoacetate (LAA).

(Sesquiterpene production by E. coli strain carrying plasmid pAC-Mev / Scidi / Aacl / pnbA)
Along with the pAC-Mev / Scidi / Aacl / pnbA plasmid, a sesquiterpene β-bisabolen production plasmid (pET-Zo506FL3, non-patent document 2) E. coli competent cell ECOS competent E. coli BL21 (DE3) (manufactured by Nippon Gene) The positive clone was obtained by selection on an LB agar medium containing ampicillin having a final concentration of 100 μg / mL and chloramphenicol having a final concentration of 30 μg / mL. The obtained Escherichia coli clone was inoculated into a test tube containing 2 mL of LB medium containing 100 μg / mL ampicillin at a final concentration of 30 μg / mL and chloramphenicol at a final concentration of 30 μg / mL for 17 hours at 30 ° C. and 180 rpm. Shake culture (preculture). Next, 1% (v / v) of the pre-cultured solution was added to a 100 mL baffled Erlenmeyer flask containing 20 mL of TB medium containing final concentration of 100 μg / mL ampicillin and final concentration of 30 μg / mL chloramphenicol. The mixture was added and cultured with shaking at 37 ° C. and 180 rpm. When the absorbance at 600 nm reaches 0.8, after cooling to room temperature, add final concentration of 0.1 mM IPTG, final concentration of 5 mM MAA or EAA, 20% (v / v) n -dodecane The shaking culture was continued at 20 ° C. and 180 rpm. After 72 hours, 1 mL of the culture solution was transferred to a 1.5 mL tube and centrifuged at 18,000 × g for 5 minutes at room temperature. The n -dodecane layer was quantitatively analyzed with a gas chromatograph mass spectrometer GCMS-QP2010 (manufactured by Shimadzu Corporation). For the analysis, a sample (1 μL) was injected by split injection (ratio: 10: 1, injector temperature: 250 ° C.). The analysis program was performed under the condition of heating from 60 ° C. to 3 ° C. per minute to 280 ° C. and then holding this temperature for 6.7 minutes. Mass spectrometry measured a range of 40 to 400 m / z at an output of 70 eV and an interface temperature of 250 ° C. From the retention time (30.2 minutes) and the peak area value, quantitative analysis was carried out as an equivalent amount of α-humulene sample (Funakoshi).
The quantitative results are shown in FIG. As shown in the figure, in the E. coli production strain into which the plasmid pAC-Mev / Scidi / Aacl / pnbA was introduced, when MAA or EAA as an ester was used, the production amount was about more than that using LAA of the same concentration as the substrate. Increased 1.8 times.
From these results, this E. coli production system using acetoacetate hydrolase (esterase) whose function was newly identified utilized LAA, which is less expensive than LAA, which is a conventional substrate, as a substrate. It has been shown that terpenes can be produced more efficiently (with higher yields) than when using as a substrate.

{Investigation of sugars involved in enhancement of production per culture by carotenoid (tetraterpene) -producing Escherichia coli}
Plasmids pACCRT-EIB, pACCAR16ΔcrtX, pACCAR25ΔcrtX (N. Misawa et al, J. Bacteriol., 177: 6575-6684, 1995) and plasmid pRK-idi (M. Albrecht et al, Biotechnol. Lett., 21: 791- 795, 1999) Escherichia coli JM101 strain (Sambrook and Russel, Molecular Cloning A Laboratory Manual (Third edition) Cold Spring Harbor Laboratory Press, 2001) Went. The plasmid pACCRT-EIB is obtained by inserting crtE , crtB , crtI genes derived from the soil bacterium Pantoea ananatis into the Eco RV site of the Escherichia coli vector pACYC184 {chloramphenicol (Cm) resistance}, and lycopene Synthesize into E. coli. The plasmid pACCAR16ΔcrtX is obtained by inserting the crtE , crtB , crtI , and crtY genes derived from Pantoea ananatis into the Eco RV site of the E. coli vector pACYC184 to synthesize β-carotene in E. coli. pACCAR25ΔcrtX is obtained by inserting the crtE , crtB , crtI , crtY , crtZ genes derived from Pantoea ananatis into the Eco RV site of the Escherichia coli vector pACYC184 to synthesize zeaxanthin in Escherichia coli. Plasmid pRK-idi inserts the idi (IPP isomerase; type 1; accession no. AB019035) gene (cDNA) of yeast Xanthophyllomyces dendrorhous into the Pst I- Bam HI site of the E. coli vector pRK404 {tetracycline (Tc) resistance} However, when it is made to coexist with said plasmid, the production amount of carotenoid will increase 2 to 3 times.
As a preculture, in a 100 mL LB medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl) / 500 mL Erlenmeyer flask (flask) containing 33 mg / L Cm and 15 mg / L Tc Plasmid pACCRT-EIB, pACCAR16ΔcrtX or pACCAR25ΔcrtX, and 1 mL of a -80 ° C. frozen stock of recombinant Escherichia coli carrying plasmid pRK-idi were added and incubated at 30 ° C. and 150 rpm for 24 hours. As the main culture, inoculate 2% (2 mL) of the preculture solution into a 100 mL LB medium / 500 mL Erlenmeyer flask containing 33 mg / L Cm and 15 mg / L Tc, and rotate at 20 ° C. and 150 rpm. Common cultures include baffled or normal (non-baffled) Erlenmeyer flasks as culture vessels (500 mL Erlenmeyer flasks), and 1 g each of various sugars or nitrogen sources to basic LB media as production media Thus, the effect of these differences on the carotenoid production titer was examined.
To examine the titer, after 24 hours, 48 hours, 72 hours, and 96 hours of each medium, collect 5 mL of the culture solution in a 15 mL falcon tube, and centrifuge the tube at 3000 rpm for 5 minutes to obtain bacterial cells (carotenoids). Collected). After discarding the supernatant culture solution, the produced carotenoids are completely dissolved in an organic solvent by ultrasonically crushing the cells with 8 mL of dichloromethane: methanol (1: 1) solution added to each Falcon tube. I let you. Next, the falcon tube was centrifuged at 3000 rpm for 5 minutes to precipitate the crushed cells again, and then the carotenoid concentration in the supernatant solution was 476 nm (in the case of lycopene) or 450 nm (β-carotene, zeaxanthin) In the case of (1)) by measuring with a spectrophotometer.
An example of the result is shown in FIG. This figure shows the case of using lycopene-producing E. coli (with plasmids pACCRT-EIB and pRK-idi), but β-carotene-producing E. coli (with plasmids pACCAR16ΔcrtX and pRK-idi) and zeaxanthin-producing E. coli (plasmid pACCAR25ΔcrtX). And pRK-idi) were also used.

The results are summarized below.
The sugar added to the LB medium has a significant effect as a carbon source (basic 1 g / flask added).
The carbon source can be classified into the following A to C depending on the magnitude of the influence.
A: The carbon sources whose titer increases remarkably are fructose, galactose, trehalose, and sorbitol.
B: Although not as high as A, the carbon sources whose titer increases are mannitol and mannose.
C: Carbon sources that hardly increase in titer are glucose, sucrose, lactose, maltose, and starch.
* However, even if the carbon source of A is 2 g / flask, in many cases, no further increase in titer is observed. The addition of 4 g / flask will lower the potency rather than 1 g / flask. Even when two carbon sources of A are combined, no further increase in titer is observed.
Aeration is considered important. When cultured in a baffled Erlenmeyer flask under the conditions of fructose addition, the titer increases to about twice that of a normal Erlenmeyer flask.
Three nitrogen sources, namely bacto soytone, peptone, or polypeptone, were added to the LB medium at a concentration of 1 g / flask, but none contributed to the increase in titer (in some cases, it decreased).
Under high titer conditions, an increase in the amount of cells is observed (about 150 mg / flask for LB medium but about 500 mg / flask for fructose added (conical flask with baffle)).

  Fructose, galactose, trehalose, sorbitol as carbon sources that significantly increase carotenoid production per culture broth, and mannitol as a carbon source that increases carotenoid production per culture broth, although not as much as the previous four. The discovery of mannose was unexpected. This is because the effects of glucose, sucrose, and lactose, which are generally considered to contribute to the increase in the amount of cells per culture, are not recognized, and it is difficult to contribute to the increase in the amount of cells per culture compared to the previous three. This is because fructose, galactose, trehalose, sorbitol, mannitol and mannose were effective. Heretofore, there has been no report that these sugars are effective in enhancing the amount of carotenoid production per culture solution. As described above, according to the contents disclosed in the present example, an efficient culture method has been invented in which fructose, galactose, trehalose, sorbitol, mannitol, and / or mannose is added to a medium to culture carotenoid-producing Escherichia coli.

{Rat brain lipid peroxidation inhibition test ( in vitro antioxidant activity test)}
Rat brain lipid peroxidation inhibition test was conducted using α-copaene produced using E. coli BL21 (DE3) introduced with plasmids pRSF-Fh6Tps-1 and pAC-Mev / Scidi / Aacl. This experiment was carried out by Shindo et al. (2008) (K. Shindo, E. Asagi, A. Sano, E. Hotta, N. Minemura, K. Mikami, E. Tamesada, N. Misawa, T. Maoka, Diapolycopenedioic acid xlosyl). esters A, B, and C, novel antioxidative glycol-C30-carotenoic acids produced by a new marine bacterium Rubritalea squalenifaciens . J. Antibiot. 61: 185-191, 2008). The results are shown in FIG. As a result, it was revealed that α-copaene has a clear antioxidant activity (IC50 = 35.1 μM) although it is weaker than the positive control catechin.
Therefore, it was confirmed that the α-copaene produced by the present invention has a physiological function useful for human health.

  According to the present invention, a terpene synthase and a terpene synthase gene are provided, the terpene is efficiently produced in E. coli and the like, and an industrially useful terpene is obtained in E. coli and the like using the acetoacetate hydrolase gene. It can be manufactured efficiently.

Claims (4)

The following (1) to any one of the polypeptides acetoacetic ester having free of (7) (acetoacetic fatty acid ester) hydrolyzing agent:
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 85 ,
(2) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 85 , wherein 1 to 20 amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence set forth in SEQ ID NO: 85 A polypeptide having an activity to hydrolyze acetoacetate substantially the same as
(3) a polypeptide consisting of an amino acid sequence homology of 90% or more to have the amino acid sequence set forth in SEQ ID NO: 85, and comprising the amino acid sequence set forth in SEQ ID NO: 85 polypeptide substantially homogeneous acetoacetate A polypeptide having an activity of hydrolyzing an ester,
(4) a polypeptide comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 82 or 90,
(5) In the base sequence set forth in SEQ ID NO: 82 or 90, a polypeptide comprising an amino acid sequence encoded by a base sequence in which 1 to 50 base sequences are substituted, deleted, inserted and / or added, And a polypeptide having an activity of hydrolyzing an acetoacetate substantially the same quality as a polypeptide consisting of the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 82 or 90,
(6) A polypeptide comprising an amino acid sequence encoded by a base sequence having 85% or more homology with the base sequence described in SEQ ID NO: 82 or 90, and encoded by the base sequence described in SEQ ID NO: 82 or 90 A polypeptide having an activity of hydrolyzing an acetoacetate substantially the same quality as a polypeptide comprising the amino acid sequence
(7) A polypeptide consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 82 or 90, and SEQ ID NO: 82 Or a polypeptide having an activity of hydrolyzing an acetoacetate ester substantially the same quality as a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to 90 .
Recombinant E. coli introduced with the following genes (1) to (5):
(1) Gene or gene group of an enzyme that synthesizes a terpene (isoprenoid, terpenoid) from geranyl diphosphate or farnesyl diphosphate,
(2) type 1 and / or type 2 isopentenyl diphosphate isomerase gene or gene group,
(3) Mevalonate pathway genes that synthesize acetoacetyl-coenzyme A to isopentenyl diphosphate,
(4) Acetoacetate-coenzyme A ligase gene,
(5) acetoacetate ester (acetoacetate fatty acid ester) hydrolase (esterase) gene , wherein the gene is any one of the following (5-1) to (5-7):
(5-1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 85;
(5-2) In the gene encoding the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 85, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and set forth in SEQ ID NO: 85 A gene encoding a polypeptide having an activity to hydrolyze an acetoacetate substantially the same as a polypeptide comprising an amino acid sequence,
(5-3) a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 85, and substantially equivalent to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 85 A gene encoding a polypeptide having an activity to hydrolyze a homogenous acetoacetate,
(5-4) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 82 or 90,
(5-5) In the gene consisting of the base sequence set forth in SEQ ID NO: 82 or 90, 1 to 50 base sequences are substituted, deleted, inserted and / or added, and set forth in SEQ ID NO: 82 or 90 A gene encoding a polypeptide having an activity of hydrolyzing an acetoacetate substantially the same quality as a polypeptide consisting of an amino acid sequence encoded by a base sequence;
(5-6) a gene comprising a nucleotide sequence having 85% or more homology with the nucleotide sequence set forth in SEQ ID NO: 82 or 90, and an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 82 or 90 A gene encoding a polypeptide having an activity of hydrolyzing acetoacetate (acetoacetate fatty acid ester) substantially the same quality as
(5-7) consisting of a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence set forth in SEQ ID NO: 82 or 90, and according to the base sequence set forth in SEQ ID NO: 82 or 90 A gene encoding a polypeptide having an activity of hydrolyzing an acetoacetate ester (acetoacetate fatty acid ester) substantially the same as a polypeptide having an encoded amino acid sequence.
The recombinant Escherichia coli according to claim 2 is cultured in a medium containing methyl acetoacetate and / or ethyl acetoacetate to obtain a culture or cells, and a terpene is obtained from the obtained culture or cells. A method for producing a terpene, Acetoacetic acid ester (acetoacetic acid fatty acid ester) hydrolysis method using any one of the following polypeptides (1) to (7):
(1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 85,
(2) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 85, wherein 1 to 20 amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence set forth in SEQ ID NO: 85 A polypeptide having an activity to hydrolyze acetoacetate substantially the same as
(3) An acetoacetate which is a polypeptide consisting of an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 85 and substantially the same as the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 85 A polypeptide having an activity of hydrolyzing
(4) a polypeptide comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 82 or 90,
(5) In the base sequence set forth in SEQ ID NO: 82 or 90, a polypeptide comprising an amino acid sequence encoded by a base sequence in which 1 to 50 base sequences are substituted, deleted, inserted and / or added, And a polypeptide having an activity of hydrolyzing an acetoacetate substantially the same quality as a polypeptide consisting of the amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 82 or 90,
(6) A polypeptide comprising an amino acid sequence encoded by a base sequence having 85% or more homology with the base sequence described in SEQ ID NO: 82 or 90, and encoded by the base sequence described in SEQ ID NO: 82 or 90 A polypeptide having an activity of hydrolyzing an acetoacetate substantially the same quality as a polypeptide comprising the amino acid sequence
(7) A polypeptide comprising an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 82 or 90, and SEQ ID NO: 82 Or a polypeptide having an activity of hydrolyzing an acetoacetate ester substantially the same quality as a polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence according to 90.