JP5219025B2 - Nucleic acid encoding a polypeptide having sesquiterpene synthase activity - Google Patents

Description

  The present invention relates to a nucleic acid encoding a polypeptide having sesquiterpene synthase activity, a polypeptide having sesquiterpene synthase activity, a method for producing a useful sesquiterpene using such a polypeptide, and the like.

  Wild perennial root herb native to Southeast Asia has been widely used as a “shampoo ginger” because the milky substance contained in persimmon has been used as a natural shampoo or as a component of some commercial shampoos. Are known. In addition, fresh rhizomes of shampoo ginger have been widely used as folk medicine for sprains, indigestion, toothache and so on. The scent is said to have a mental refreshing effect. Because of its wide range of properties, shampoo ginger is cultivated in many tropical and subtropical regions around the world.

In recent years, shampoo ginger has received much attention because terpenoids present in abundance in rhizome essential oil are pharmacologically useful. Terpenoids are natural plant products with a wide variety of structures. Most of the terpenoids are in the interaction between plants and pathogens (Non-Patent Document 1), the interaction between plants and herbivores (Non-Patent Document 2), and the interaction between plants and plants (Non-Patent Document 3). It is considered to play an essential role. Terpenoids are also effective as antibacterial agents and anticancer agents. For example, monoterpenoids (C ₁₀ ) are anti-carcinogenic agents limonene (Non-patent Document 4), and sesquiterpenoids (C ₁₅ ) are antimalarial drugs artemisinin (Non-patent Document 5). In addition, diterpenoid (C ₂₀ ) is used as an active ingredient of the anticancer agent taxol (Non-patent Document 6).

  Shampoo ginger rhizome oil contains a complex mixture of monoterpenes, sesquiterpenes, and diterpenes containing zerumbone, α-humulene and camphene as main components (Non-patent Document 7). Zelmbon, a unique sesquiterpene found exclusively in shampoo ginger, has been regarded as a promising anticancer agent for colon cancer and skin cancer (Non-Patent Documents 8 and 9). Since 5-hydroxy zerumbone, which is a humulene derivative, inhibits the production of nitric oxide induced by lipopolysaccharide in mouse macrophages, its potential as an anti-inflammatory agent has been suggested (Non-patent Document 10). . However, despite their usefulness, little is known about the mechanism and physiological function of terpenoid biosynthesis in shampoo ginger.

  Because of the small amount of terpenoids produced in natural plants, purification results in low yields, low purity, and large amounts of plant waste. Furthermore, since terpenoids have a complicated structure, their biosynthesis is difficult and expensive (Non-patent Document 11). In order to solve these problems, development of a method for producing a large amount of terpenoids using plants is desired.

Terpenoid biosynthetic pathways in plants are monoterpenes, sesquiterpenes of C ₁₀ , C ₁₅ , and C ₂₀ acyclic precursors geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), respectively. And conversion to diterpenes (Non-Patent Documents 12 and 13), which are catalyzed by terpene (terpenoid) synthase (TPS). Among such terpene synthases, α-humulene synthase is known as an enzyme that converts farnesyl diphosphate into α-humulene and β-caryophyllene. However, α-humum can be obtained as a main product. Ren synthase has not been identified. Moreover, the enzyme which can obtain (beta) -eudesmol as a main product among this terpene synthase was not identified.
Chen, XY, Chen, Y., Heinstein, P. and Davisson, J. (1995) Cloning, expression and characterization of (+)-δ-cadinene synthase: A catalyst for cotton phytoalexin biosynthesis. Arch. Biochem. Biophys. 20 : 255-266. Pare, PW and Tumlison, JH (1999) Plant volatiles as a defense against insect herbivores.Plant Physiol.121: 325-331. Kalsi, PS, Goyal, R., Talwar, KK and Chhabra, BB (1989) Stereostructures of two biologically active sesquiterpene lactones from Inula Racemosa. Phytochemistry. 28: 2093-2096. Crowell, P. and Gould, MN (1994) Chemoprevention and therapy of cancer by d-limonene. Crit. Rev. Oncog. 5: 1-22. Klayman, DL (1985) Qinghaosu (artemisinin): an antimalarial drug from China.Science 228: 1049-1055. Wani, MC, Taylor, HL, Wall, ME, Coggon, P. and McPhail, AT (1971) Plant antitumor agents.VI.The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia.J. Am. Chem. Soc. 93: 2325-2327. Chane, MJ, Vera, R. and Chalchat, JC (2003) Chemical composition of the essential oil from rhizomes, leaves and flowers of Zingiber zerumbet Smith from Reunion Island.J. Essent. Oil. Res. 15: 202-205. Tanaka, T., Shimizu, M., Kohno, H., et al. (2001) Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet.Life Sci. 69: 19351945. Murakami, A., Tanaka, T., Lee, J.-Y., et al. (2004) Zerumbone suppresses skin tumor initiation and promotion stages in mice: Involvement of induction of anti-oxidative and phase II drug metabolizing enzymes and inhibition of NF-kB-mediated cyclooxygenase-2 expression. Int. J. Cancer. 110: 481-490. Jang, DS, Min, HY, Kim, MS, Han, AR, Windono, T., Jeohn, GH, Kang, SS, Lee, SK and Seo, EK (2005) Humulene Derivatives from Zingiber zerumbet with the Inhibitory Effects on Lipopolysaccharide -Induced Nitric Oxide Production. Chem. Pharm. Bull. 53: 829-831. Martin, VJJ, Pitera, DJ, Withers, ST, Newman, JD and Keasling, JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21: 796-802. Cane, DE (1999) Sesquiterpene biosynthesis: cyclization mechanisms.In DE Cane, Comprehensive Natural Products Chemistry.Isoprenoids Including Carotenoids and Steroids, Vol 2 Pergamon Press, Oxford, pp 155-200. Wise, ML and Croteau, R. (1999) Monoterpene biosynthesis.In DE Cane, ed., Comprehensive Natural Products Chemistry.Isoprenoids Including Carotenoids and Steroids, Vol 2.Pergamon Press, Oxford, pp 97153.

  The problem to be solved by the present invention was to provide an enzyme that produces useful terpenes and a nucleic acid that encodes them, and to produce useful terpenes in large quantities using them.

  As a result of intensive studies in view of the above circumstances, the present inventor has preceded the isolation of a nucleic acid encoding the following enzyme from Hana ginger. That is, the present inventors succeeded in isolating a nucleic acid encoding α-humulene synthase that can generate α-humulene with little formation of β-caryophyllene from farnesyl diphosphate. Furthermore, the present inventors have succeeded in isolating a nucleic acid encoding a sesquiterpene synthase that can produce β-eudesmol with little by-product from farnesyl diphosphate. From these facts, the present inventor has completed the present invention.

That is, the present invention provides:
(1) a nucleic acid encoding a polypeptide having sesquiterpene synthase activity,
(A) a nucleic acid having any one of the nucleotide sequences of SEQ ID NOs: 1 to 6,
(B) a nucleic acid having a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in any one of the nucleotide sequences of SEQ ID NOs: 1 to 6,
(C) a nucleic acid capable of hybridizing with the nucleic acid of (a) or (b) under stringent conditions, and (d) having 50% or more homology with any one of the nucleotide sequences of SEQ ID NOs: 1 to 6 A nucleic acid having a nucleotide sequence,
A nucleic acid selected from the group consisting of:
(2) a nucleic acid encoding a polypeptide having α-humulene synthase activity,
(A) a nucleic acid having the nucleotide sequence of SEQ ID NO: 1,
(B) a nucleic acid having a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1,
(C) a nucleic acid capable of hybridizing with the nucleic acid of (a) or (b) under stringent conditions, and (d) a nucleic acid having a nucleotide sequence having 50% or more homology with the nucleotide sequence of SEQ ID NO: 1. ,
The nucleic acid according to (1), selected from the group consisting of:
(3) The nucleic acid according to (2), comprising the nucleotide sequence of SEQ ID NO: 1;
(4) a nucleic acid encoding a polypeptide having β-eudesmol synthase activity,
(A) a nucleic acid having the nucleotide sequence of SEQ ID NO: 5,
(B) a nucleic acid having a nucleotide sequence in which one or several nucleotides have been deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 5;
(C) a nucleic acid capable of hybridizing with the nucleic acid of (a) or (b) under stringent conditions, and (d) a nucleic acid having a nucleotide sequence having 50% or more homology with the nucleotide sequence of SEQ ID NO: 5 ,
The nucleic acid according to (1), selected from the group consisting of:
(5) The nucleic acid according to (2), comprising the nucleotide sequence of SEQ ID NO: 5;
(6) A polypeptide having sesquiterpene synthase activity,
(A) a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 8 to 13,
(B) a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 8 to 13; and (c) of SEQ ID NOs: 8 to 13 A polypeptide having an amino acid sequence having 50% or more homology with any amino acid sequence,
A polypeptide selected from the group consisting of:
(7) A polypeptide having α-humulene synthase activity,
(A) a polypeptide having the amino acid sequence of SEQ ID NO: 8;
(B) a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 8, and (c) 50% or more of the amino acid sequence of SEQ ID NO: 8 A polypeptide having an amino acid sequence having homology,
The polypeptide according to (6), selected from the group consisting of:
(8) The polypeptide according to (7), comprising the amino acid sequence of SEQ ID NO: 8;
(9) A polypeptide having β-eudesmol synthase activity,
(A) a polypeptide having the amino acid sequence of SEQ ID NO: 12,
(B) a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 12, and (c) 50% or more of the amino acid sequence of SEQ ID NO: 12 A polypeptide having an amino acid sequence having homology,
The polypeptide according to (6), selected from the group consisting of:
(10) The polypeptide according to (9), comprising the amino acid sequence of SEQ ID NO: 12;
(11) A vector comprising the nucleic acid according to any one of (1) to (5);
(12) Production of a sesquiterpene, wherein the nucleic acid according to any one of (1) to (5) or the vector according to (11) is introduced into a cell or plant, and the cell or plant is cultured or grown. Method;
(13) A method for producing α-humulene, wherein the nucleic acid according to (2) or (3) or a vector containing the nucleic acid is introduced into a cell or plant, and the cell or plant is cultured or grown;
(14) A method for producing β-eudesmol, comprising introducing the nucleic acid according to (4) or (5) or a vector containing the nucleic acid into a cell or plant, and culturing or growing the cell or plant;
(15) A method for producing a sesquiterpene, comprising reacting the polypeptide according to (6) with a substrate;
(16) A method for producing α-humulene, comprising reacting the polypeptide according to (7) or (8) with a substrate;
(17) Provided is a method for producing β-eudesmol, which comprises reacting the polypeptide according to (9) or (10) with a substrate.

  The present invention provides enzymes for producing terpenes, nucleic acids encoding them, methods for mass production of terpenes using them, and the like. Specifically, there are provided enzymes that produce sesquiterpenes, nucleic acids that encode them, methods for mass production of sesquiterpenes using them, and the like. Many sesquiterpenes are useful as fragrances and insect repellents, such as farnesol with a scent of lily of the valley, humulenes as the main component of hops, and α-santonin as a roundworm control agent. Furthermore, as mentioned above, zelmbon, a unique sesquiterpene found exclusively in shampoo ginger, is promising as an anticancer agent against colon cancer and skin cancer, and 5-hydroxy zerumbone, a humulene derivative, is an anti-inflammatory agent. The possibility is suggested. In particular, according to the present invention, a polypeptide capable of producing α-humulene having physiological activity such as antibacterial activity as a main product, a nucleic acid encoding the same, a vector or cell containing the nucleic acid, and α-humulene using these. And the like. Furthermore, according to the present invention, a polypeptide capable of producing β-eudesmol having physiological activity such as antidepressant action as a main product, a nucleic acid encoding the same, a vector or a cell containing the nucleic acid, and using these A method for producing β-eudesmol is provided.

In one aspect, the present invention relates to a nucleic acid encoding a polypeptide having sesquiterpene synthase activity,
(A) a nucleic acid having any one of the nucleotide sequences of SEQ ID NOs: 1 to 6,
(B) a nucleic acid having a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in any one of the nucleotide sequences of SEQ ID NOs: 1 to 6,
(C) a nucleic acid capable of hybridizing with the nucleic acid of (a) or (b) under stringent conditions, and (d) having at least 50% homology with any one of the nucleotide sequences of SEQ ID NOs: 1 to 6 A nucleic acid having a nucleotide sequence having,
It relates to a nucleic acid selected from the group consisting of In the present specification, the nucleic acid means single-stranded or double-stranded DNA or RNA. The nucleic acids of the invention can be produced and obtained by various methods known to those skilled in the art.

  The sesquiterpene synthase activity refers to the ability or property of catalyzing a reaction for producing sesquiterpenes from a substrate, and the substrate is not particularly limited. For example, sesquiterpene synthase activity refers to the ability or property of catalyzing a reaction that produces sesquiterpenes using farnesyl diphosphate as a substrate. The sesquiterpene synthase activity includes, for example, α-humulene synthase activity and β-eudesmol synthase activity as described later. Sesquiterpene synthase means any polypeptide (enzyme) having the above activity. Sesquiterpenes are also known to those skilled in the art, and include α-humulene and β-eudesmol.

α-humulene synthase activity refers to the ability or property to catalyze the reaction of converting a substrate, preferably farnesyl diphosphate, to α-humulene. Accordingly, any polypeptide (enzyme) having α-humulene synthase activity, ie, α-humulene synthase, can be used as long as it can catalyze the reaction of converting a substrate, for example, farnesyl diphosphate into α-humulene. It may be. α-humulene synthase activity can be determined, for example, by incubating farnesyl diphosphate with the polypeptide and examining the presence or amount of the resulting α-humulene. Preferably, the nucleic acid of the invention is such that the encoded polypeptide is capable of converting α-humulene as a major product (eg, greater than 50% of the total product) from a substrate, eg, farnesyl diphosphate. is there. For example, the nucleic acid of the present invention is introduced into a plant to increase the amount of α-humulene produced in the plant, or the nucleic acid of the present invention is incorporated into a vector and introduced into a cell such as Escherichia coli. It becomes possible to manufacture humulene in large quantities.
It becomes possible.

  β-eudesmol synthase activity refers to the ability to catalyze the reaction of converting a substrate, preferably farnesyl diphosphate, to β-eudesmol. Thus, a polypeptide (enzyme) having β-eudesmol synthase activity, ie, β-eudesmol synthase, can catalyze the reaction of converting a substrate, for example, farnesyl diphosphate into β-eudesmol. Any of them may be used. β-eudesmol synthase activity can be determined, for example, by incubating farnesyl diphosphate with the polypeptide and examining the presence or amount of the obtained β-eudesmol. Preferably, the nucleic acids of the invention allow the encoded polypeptide to convert β-eudesmol as a major product (eg, greater than 50% of the total product) from a substrate, eg, farnesyl diphosphate. Is. For example, the nucleic acid of the present invention is introduced into a plant to increase the amount of β-eudesmol produced in the plant, or the nucleic acid of the present invention is incorporated into a vector and introduced into a cell such as E. coli. It becomes possible to produce a large amount of β-eudesmol.

  The nucleic acid of the present invention specifically includes a nucleic acid having any one of the nucleotide sequences of SEQ ID NOs: 1 to 6; one or more, preferably 1 or any of the nucleotide sequences of SEQ ID NOs: 1 to 6 A nucleic acid having a nucleotide sequence in which several, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides are deleted, substituted, or added; hybridizes with the nucleic acid under stringent conditions A nucleic acid capable of soybean; and at least 50%, preferably 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99% of any nucleotide sequence of SEQ ID NOs: 1-6, Or a nucleic acid having a nucleotide sequence having a homology of 99.5% or more. Regardless of the nucleotide sequence, the nucleic acid of the present invention encodes a polypeptide having sesquiterpene synthase activity.

  Examples of the stringent conditions include conditions described in J. Sambrook et al., “Molecular Cloning: A Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory Press. For example, after hybridization with a probe at 68 ° C. in a solution containing 6 × SSC, 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml denatured salmon sperm DNA, the washing conditions are 2 × SSC, Examples include conditions for changing from room temperature in 0.1% SDS to 0.1 × SSC, 68 ° C. in 0.5% SDS.

  The homology of the nucleotide sequence can be measured using a program such as Gendoc, for example.

  A preferred nucleic acid encoding an enzyme having α-humulene synthase activity in the present invention is: a nucleic acid having the nucleotide sequence of SEQ ID NO: 1; one or more, preferably one or several in the nucleotide sequence of SEQ ID NO: 1 For example, a nucleic acid having a nucleotide sequence in which about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides are deleted, substituted, or added; hybridizes with the nucleic acid under stringent conditions. A nucleic acid to be obtained; and at least 50%, preferably 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99%, or 99.5% or more with the nucleotide sequence of SEQ ID NO: 1 A nucleic acid having a nucleotide sequence with homology.

  A more preferred nucleic acid encoding an enzyme having α-humulene synthase activity in the present invention is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1.

  Preferred nucleic acids encoding an enzyme having β-eudesmol synthase activity in the present invention are: a nucleic acid having the nucleotide sequence of SEQ ID NO: 5; one or more, preferably 1 or a number in the nucleotide sequence of SEQ ID NO: 5 A nucleic acid having a nucleotide sequence in which about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides are deleted, substituted, or added; hybridizes with the nucleic acid under stringent conditions As well as at least 50%, preferably 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99%, or 99.5% or more with the nucleotide sequence of SEQ ID NO: 5 A nucleic acid having a nucleotide sequence having the homology of

  A more preferred nucleic acid encoding an enzyme having β-eudesmol synthase activity in the present invention is a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 5.

In another aspect, the present invention provides a polypeptide having sesquiterpene synthase activity comprising:
(A) a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 8 to 13,
(B) a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 8 to 13, and (c) any of SEQ ID NOs: 8 to 13 A polypeptide having an amino acid sequence having 50% or more homology with any amino acid sequence,
It relates to a polypeptide selected from the group consisting of The polypeptide of the present invention has the aforementioned α-humulene synthase activity. Accordingly, α-humulene and β-caryophyllene can be obtained from farnesyl diphosphate using the polypeptide of the present invention. For example, when the polypeptide of the present invention is injected into cells or plants containing farnesyl diphosphate, the amount of α-humulene and β-caryophyllene obtained is increased, or the production rate of α-humulene and β-caryophyllene is increased. It can also be promoted. Preferably, the polypeptide of the present invention has α-humulene as the main product from farnesyl diphosphate, for example, more than 50% of the product, preferably 60, 70, 75, 80, 85, 90, 93, 96, or 99% or more can be converted.

  Specifically, the polypeptide of the present invention has one or more, preferably a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 8 to 13 and any one of the amino acid sequences of SEQ ID NOs: 8 to 13, preferably, A polypeptide having an amino acid sequence in which one or several, for example about 2, 3, 4, 5, 6, 7, 8, or 9 amino acids have been deleted, substituted or added, and SEQ ID NOs: 8-13 At least 50% or more, preferably 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99%, or 99.5% or more of homology. Examples thereof include a polypeptide having an amino acid sequence and a polypeptide encoded by a nucleic acid encoding the above-mentioned polypeptide having α-humulene synthase activity. Regardless of the amino acid sequence, the polypeptide of the present invention has α-humulene synthase activity.

  The homology of the amino acid sequences can be measured using, for example, Gendoc.

  Preferred enzymes having α-humulene synthase activity in the present invention are: a polypeptide having the amino acid sequence of SEQ ID NO: 8; one or more, preferably 1 or several, for example, in the amino acid sequence of SEQ ID NO: 8, A polypeptide having an amino acid sequence in which as few as 2, 3, 4, 5, 6, 7, 8, or 9 amino acids have been deleted, substituted or added; and at least 50% or more of the amino acid sequence of SEQ ID NO: 8, Preferably, it is a polypeptide having an amino acid sequence having homology of 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99, 99.5% or more.

  A more preferred enzyme having α-humulene synthase activity in the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8.

  Preferred enzymes having β-eudesmol synthase activity in the present invention are: a polypeptide having the amino acid sequence of SEQ ID NO: 12; one or more, preferably 1 or several in the amino acid sequence of SEQ ID NO: 12, For example, a polypeptide having an amino acid sequence in which as few as 2, 3, 4, 5, 6, 7, 8, or 9 amino acids have been deleted, substituted or added; and at least 50% with the amino acid sequence of SEQ ID NO: 12 The polypeptide having an amino acid sequence having a homology of 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99, 99.5% or more is preferable.

  A more preferred enzyme having β-eudesmol synthase activity in the present invention is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12.

  Therefore, in a further aspect, the present invention relates to a nucleic acid encoding the above-mentioned polypeptide having sesquiterpene synthase activity of the present invention. Such a nucleic acid is also included in the nucleic acid of the present invention referred to in the present specification. In a further aspect, the present invention relates to a nucleic acid encoding a polypeptide having the α-humulene synthase activity or β-eudesmol synthase activity of the present invention.

  In another aspect, the present invention relates to a vector containing the nucleic acid of the present invention. The vector of the present invention may be any as long as it contains the nucleic acid of the present invention. Constituent elements other than the nucleic acid can be appropriately selected according to various conditions such as the object and purpose of the cell using the vector. The sesquiterpene synthase activity of the present invention is recovered by introducing the vector of the present invention into, for example, a bacterial cell such as Escherichia coli or a plant cell such as ginger plant and recovering the produced polypeptide having sesquiterpene synthase activity. It is also possible to obtain a polypeptide having

In particular, a nucleic acid having the nucleotide sequence of SEQ ID NO: 1; in the nucleotide sequence of SEQ ID NO: 1, one or more, preferably one or several, eg 2, 3, 4, 5, 6, 7, 8, Or a nucleic acid having a nucleotide sequence in which about 9 nucleotides are deleted, substituted or added; a nucleic acid capable of hybridizing with the above nucleic acid under stringent conditions; or at least 50%, preferably with the nucleotide sequence of SEQ ID NO: 1. Is a nucleic acid having a nucleotide sequence with homology of 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99%, or 99.5% or more, or alternatively SEQ ID NO: 1. A vector containing a nucleic acid having the nucleotide sequence shown is introduced into a cell and cultured in a medium containing a substrate, such as farnesyl diphosphate The Rukoto, it is possible to obtain the α- humulene as the main product. An agent that promotes the conversion of farnesyl diphosphate to α-humulene or β-eudesmol, for example, MgCl ₂ , MnCl ₂ , NaWO ₄ , NaF, or the like, may be contained in the medium.

  Further, a nucleic acid having the nucleotide sequence of SEQ ID NO: 5; in the nucleotide sequence of SEQ ID NO: 5, one or more, preferably one or several, for example 2, 3, 4, 5, 6, 7, 8, Or a nucleic acid having a nucleotide sequence in which about 9 nucleotides are deleted, substituted or added; a nucleic acid capable of hybridizing with the above nucleic acid under stringent conditions; or at least 50%, preferably with the nucleotide sequence of SEQ ID NO: 5 Is a nucleic acid having a nucleotide sequence with homology of 60, 70, 75, 80, 85% or more, more preferably 90, 93, 96, 99%, or 99.5% or more, or alternatively to SEQ ID NO: 5 A vector containing a nucleic acid having the nucleotide sequence shown is introduced into a cell and cultured in a medium containing a substrate such as farnesyl diphosphate. By, it is possible to obtain a β- user Death mall as the major product.

  In a further aspect, the present invention relates to a cell into which the nucleic acid of the present invention or the vector of the present invention has been introduced. The nucleic acid or vector of the present invention can be introduced into cells using methods known in the art. There are no particular restrictions on the cells into which the vector is introduced, and examples include plant cells such as plants belonging to ginger, bacterial cells such as E. coli, yeast cells, insect cells, and mammalian cells. . By culturing such cells and recovering the produced polypeptide, the polypeptide of the present invention can be obtained.

  In another aspect, the present invention relates to a plant into which the nucleic acid of the present invention or the vector of the present invention has been introduced. Such plants can be produced by methods known to those skilled in the art. Such a plant of the present invention produces sesquiterpene synthase, for example, α-humulene synthase or β-eudesmol synthase, thereby increasing the amount of sesquiterpenes compared to a normal plant, For example, α-humulene and β-eudesmol are produced.

  Since α-humulene is known to have an antibacterial action, a plant containing a nucleic acid or vector encoding α-humulene synthase of the present invention can reduce the amount of pesticide used. Have. Furthermore, β-eudesmol is said to have an antidepressant action, and is depressed by the action of β-eudesmol-containing essential oil released from plants containing the nucleic acid or vector encoding β-eudesmol synthase of the present invention. The effect of alleviating the disease is also expected.

  The cells and plants used in producing α-humulene may further include, for example, a nucleic acid encoding an enzyme that catalyzes a reaction of a pathway that promotes the production of farnesyl diphosphate such as the mevalonate pathway, This makes it possible to increase production of sesquiterpenes such as α-humulene and β-eudesmol.

  In another aspect, the present invention provides a polypeptide having α-humulene synthase activity by culturing a cell containing the nucleic acid or vector of the present invention or growing a plant containing the nucleic acid or vector of the present invention. And a polypeptide having sesquiterpene synthase activity, such as a polypeptide having β-eudesmol synthase activity. Cell culture conditions can be appropriately selected according to various conditions such as cell type. Similarly, plant growth conditions can be selected as appropriate. In order to increase the purity of the polypeptide obtained by the above method, production methods known to those skilled in the art can be applied. In a further aspect, the present invention also relates to a polypeptide having sesquiterpene synthase activity, which can be obtained by the above-described method for obtaining sesquiterpene synthase.

  In another aspect, the present invention relates to a method for producing sesquiterpenes by reacting the polypeptide of the present invention or the polypeptide obtainable by the above method with a substrate. For example, α-humulene can be produced by reacting a polypeptide having α-humulene synthase activity with farnesyl diphosphate, and reacting a polypeptide having β-eudesmol synthase activity with farnesyl diphosphate. Thus, β-eudesmol can be produced. Preferably, in the method of the present invention, α-humulene is the main product, more than 50% of the product, preferably 60, 70, 75, 80, 85, 90, 93, 96, or 99% or more. It occurs in. Preferably, in the process of the present invention, β-eudesmol is the main product, more than 50% of the product, preferably 60, 70, 75, 80, 85, 90, 93, 96, or 99. % Or higher.

  The method for producing the sesquiterpenes of the present invention may include a step of purifying the obtained sesquiterpenes. Means and methods used for purification are well known to those skilled in the art. Furthermore, this invention relates to the sesquiterpenes which can be obtained by the manufacturing method of said sesquiterpenes in the further aspect.

  EXAMPLES The present invention will be described specifically and in detail below with reference to examples, but the examples should not be construed as limiting the present invention.

(1) Acquisition of α-humulene synthase cDNA Total RNA was prepared from the rhizomes of Japanese ginger using Spectrum (trademark) Plant Total RNA Kit (Sigma-Aldrich). Using SuperScript (trademark) III First-strand Synthesis Kit (Invitrogen), 3 μg of RNA was reverse-transcribed into cDNA in 20 μl of reaction mixture solution by poly (dT) priming. Based on the sequences of the two conserved domains of angiosperm sesquiterpene synthase closely related to rape ginger, the following primer pairs were designed: 5′-AYCWYTYGAIRAIIGARAT-3 ′ (forward; SEQ ID NO: 14) and 5′- TAIGHRTCAWAIRTRTCRTC-3 ′ (reverse; SEQ ID NO: 15). A primary PCR reaction was performed using 50 μl of a reaction mixture solution containing 1 μl cDNA, 1 μM each primer, 200 μM each dNTP, and 1 U ExTag polymerase (TaKaRa) under the following conditions: denaturation at 94 ° C. for 2 minutes, then 94 ° C for 30 seconds, 35 ° C for 1 minute and 30 seconds, 72 ° C for 1 minute for 4 cycles, 94 ° C for 30 seconds, 40 ° C for 1 minute and 30 seconds, 72 ° C for 1 minute for 30 cycles, and finally at 72 ° C Extend for 3 minutes. Using 5 μl of the obtained primary reaction solution as a template, a secondary PCR reaction was performed under the same conditions to obtain a 670 bp fragment. The resulting fragment was purified, cloned into pGEM-Teasy vector (Promega) and sequenced. To isolate full-length cDNAs, Smart RACE cDNA Amplification Kit (BD Biosciences Clontech), 5′-GCATCTCCATTCAAGGTCGGCAA-3 ′ (5′RACE; SEQ ID NO: 16) and 5′-GAGCAGCCTTTAGCAATAGAGTGTTC-3 ′ (3′RACE; SEQ ID NO: 17) and Advantage® 2 Polymerase Mix (Clontech) were used to extend the fragment toward the 5 ′ and 3 ′ ends according to the manufacturer's instructions to obtain six different full-length cDNAs . The nucleotide sequences of these cDNAs are shown in SEQ ID NOs: 1 to 6, and the amino acid sequences of the polypeptides encoded by the cDNAs are shown in SEQ ID NOs: 8 to 13. The cDNA specified by SEQ ID NO: 1 was named ZSS1. The homology of the nucleotide sequence of SEQ ID NO: 2-6 to the nucleotide sequence of SEQ ID NO: 1 and the homology of the amino acid sequence of SEQ ID NO: 9-13 to the amino acid sequence of SEQ ID NO: 8 were determined using Gendoc. The results are shown in Tables 1 and 2, respectively.

  ZSS1 contains a putative ORF of 1647 bp (including the stop codon) that encodes a polypeptide of 548 amino acids with a predicted molecular weight of 64.5 kDa and PI 5.25, and is similar to known sesquiterpene synthases. It was. A sequence similarity search of the GenBank database showed that ZSS1 is most closely related to Zingiber officinale and has 63% amino acid sequence identity. Significant identity was also observed for sesquiterpene synthases from other angiosperm species, which ranged from 50% (Elaeis oleifera sesquiterpene synthase) to 34% (Arabidopsis thaliana β-caryophyllene / α-humulene synthase (Chen, F., Tholl, D., Auria, JCD, Farooq, A., Pichersky, E. and Gershenzon, J. (2003) Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. The Plant Cell 15: 481-494.)) It showed the identity of. From the amino acid sequence and phylogenetic analysis (results not shown), ZSS1 is a TPS subfamily (Bohlmann, J., Meyer-Gauen, G. and Croteau, R.), a group of angiosperm sesquiterpenes and diterpene synthases. (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95: 4126-4133. Furthermore, the apparent deletion of the encoded plastid transit peptide is due to the hypothetical cytoplasmic biosynthesis site of many sesquiterpenes (Lichtenthaler, HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 4765.). Table 3 shows the homology of the ZSS1 nucleotide sequence with nucleotide sequences derived from other angiosperms.

(2) Acquisition of ZSS1 genomic DNA sequence
Using the Plant Genomic Isolation Mini Kit (Qiagen), genomic DNA was isolated from the leaves of Japanese ginger. PCR reaction using primer pair 5′-ATGGAGAGGCAGTCGATGGCCCCTTG-3 ′ (forward; SEQ ID NO: 18) and 5′-AATAAGAAAGATTCAACAAATATGAGAG-3 ′ (reverse; SEQ ID NO: 19) designed based on the cDNA sequence of SEQ ID NO: 1. And ZSS1 genomic DNA was isolated. Comparison of the nucleotide sequences of cDNA and genomic DNA revealed 7 exons and 6 introns (see FIG. 1). ZSS1 introns and exons are composed of 5-epi-Aristolochen synthase from N. tabacum, Betispyradiene synthase from H. muticus, and Casvensynthase from Ricinus communis (Back, K. and Chappell, J (1995) Cloning and Bacterial Expression of a Sesquiterpene Cyclase from Hyoscyamus muticus and Its Molecular Comparison to Related Terpene Cyclases. J. Biol. Chem. 13: 7375-7371; Facchini, PJ and Chappell, J. (1992) Gene family for Anclicitor-induces sesquiterpene cyclase in tobacco. Proc. Natl. Acad. Sci. USA. 89: 11088-11092 .; Mau, CJ and West, CA (1994) Cloning of casbene synthase cDNA: evidence for conserved structural features among terpenoid cyclases in plants. Proc. Natl. Acad. Sci. USA. 91: 8497-8501.). In addition, the fact that plant sesquiterpene synthase has a highly conserved exon-intron composition (Back, K. and Chappell, J. (1995) Cloning and Bacterial Expression of a Sesquiterpene Cyclase from Hyoscyamus muticus and Its Molecular Comparison to Related Terpene Cyclases. J. Biol. Chem. 13: 7375-7371). These findings could be extended to the angiosperm species diterpene synthase (Mau and West, 1994).

  Further upstream and downstream regions of the ZSS1 genomic DNA sequence were analyzed by nested PCR using the DNA Walking SpeedUp ™ Premix Kit (Seegene, Seoul, South Korea) annealing control primer set and the following three pairs of target-specific primers: Obtained; upstream; 5'-TCTCTTTTATCATCTCTTGAGTGG-3 '(SEQ ID NO: 20), 5'-ATCCAGCAATATCTCACTTGATCACC-3' (SEQ ID NO: 21), 5'-CTTCATGAGGGAATTATTGAATAAATGTGAG-3 '(SEQ ID NO: 22), and downstream; 5'-AGTCAACCCTTTCAAAATCAAATG-3 '(SEQ ID NO: 23), 5'-CGATACGTACACCAATTCTGACAC-3' (SEQ ID NO: 24), 5'-CACT CGATGAAAGATAATATCTCTTTGG-3 '(SEQ ID NO: 25). The resulting amplification product was sequenced (SEQ ID NO: 7), a putative TATA box (TTATAAT) 43-48 bp upstream of the translation start site, as well as a 29 bp polyA tail, and three potential AATAAA polyadenylations The signal was revealed (see Figure 1).

(3) S1 nuclease assay The transcription start site of ZSS1 was identified by S1 nuclease. Forward primer (5′-ATCTCTTCCTTATCACCACAC-3 ′ (SEQ ID NO: 26)) and reverse primer (5′-TAACAGATATAGTACACCACACCC-3) 5′-end labeled with T4 polynucleotide kinase (ToYoBo) and 10 μCi [γ- ³² P] ATP '(SEQ ID NO: 27)) was used to carry out a PCR reaction to obtain a labeled PCR fragment. The obtained labeled fragment was incubated with 100 μg of banana ginger leaf total RNA in a hybridization buffer (80% formamide, 0.4 M NaCl, 20 mM HEPES [pH 6.4]) at 75 ° C. for 10 minutes, to 37 ° C. Further incubation overnight and then digested with S1 nuclease. Undigested DNA was precipitated with ethanol, dissolved in formamide dye solution, and determined to have a fragment size of 161-165 bp by electrophoresis on a 6% denaturing polyacrylamide gel. This suggested that the transcription start site is located 117 to 122 bp upstream of the translation start site.

(4) ZSS1 enzyme activity The full-length ORF of ZSS1 was converted into Advantage (registered trademark) HF 2 polymerase (Clontech), forward primer 5′-CACCCATGGAGAGGCAGTCGATGGC-3 ′ (SEQ ID NO: 28) and reverse primer 5′-AATAAGAAAGATTCAACAAATATGAGAG-3 ′ ( The amplified product was cloned into pET101 / DTOPO directional expression vector (Invitrogen). E. coli BL21-CodonPlus (DE3) (Stratagene) was transformed with the obtained recombinant plasmid pET-ZSS1.

Recombinant E. coli cells were grown in 50 ml LB medium containing ampicillin (50 μg / mL) at 37 ° C. to OD ₆₀₀ = 0.5-0.6. Next, IPTG (final concentration 1 mM) was added and further grown at 18 ° C. for 20 hours. The cells were collected by centrifugation and chilled extraction buffer (5 mM MgCl ₂ , 5 mM sodium ascorbate, 0.5 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, using an ultrasonic disrupter (Branson W185 D, NY). Cells were disrupted by treatment for 10 × 10 seconds in 50 mM MOPSO, pH 7.0) and 10% (v / v) glycerol. Cell debris was removed by centrifugation at 15,000 g to obtain α-humulene synthase. The resulting enzyme was labeled with a His tag and purified on a nickel-nitrilotriacetic acid agarose column (Qiagen) according to the manufacturer's instructions. The eluate was desalted through an Econopac 10DG column (Bio-Rad) into assay buffer (10 mM MOPSO, pH 7.0, 1 mM dithiothreitol, and 10% (v / v) glycerol) and the resulting enzyme The solution was used for the following experiments.

To capture volatile products in a Teflon-sealed screw cap glass test tube, 0.5 mL of pentane was overlaid. 900 μl enzyme solution, 20 mM MgCl ₂ , 0.2 mM MnCl ₂ , 0.2 mM NaWO ₄ , 0.1 mM NaF, and 20 μM [1- ³ H]-(E, E) -FPP (555 GBq / mol, American Radiolabeled Chemicals) The enzyme assay was carried out by incubating 1 ml of the reaction solution containing 2 hours at 30 ° C. After incubation, the reaction mixture was quickly frozen in liquid nitrogen to stop the reaction. After thawing, the mixed solution was extracted three times with pentane, and the combined extracts were subjected to column chromatography using anhydrous MgSO ₄ and a silica gel column (60Å, Merck) to obtain a sesquiterpene hydrocarbon free of oxygenated products. A fraction was obtained. Subsequently, the mixed solution was extracted three times with diethyl ether, and the combined extracts were similarly passed through a MgSO ₄ -silica gel column to recover the oxygenated product. Aliquots of each fraction were collected and subjected to liquid scintillation counting to determine enzyme activity. As a negative control, an extract of E. coli BL21-CodonPlus (DE3) transformed with the pET101 / DTOPO plasmid not containing the ZSS1 insert was used. As described later, it was found that the enzyme has an activity to synthesize α-humulene from FPP.

(5) Identification of product In order to obtain a product sufficient for analysis by GC-MS, an enzyme was extracted from 250 ml of recombinant E. coli cell culture. The reaction was performed in 4 ml of a solution containing 3.8 ml of enzyme solution and 80 μM unlabeled FPP (Echelon Research Laboratories Inc. Salt Lake City, UT). Buffers, salts, and incubation conditions are as described above. After overnight incubation, extraction with pentane (3 × 1 ml) gave a hydrocarbon fraction which was then passed through a MgSO ₄ -silica gel column as described above. Finally, the combined pentane extracts were concentrated to about 100 μl on ice under a gentle stream of nitrogen.

  The product was analyzed by GC-MS (split injection (2 μL) ratio on a Shimadzu QP5050A GC / MS system equipped with a DB-WAX column (0.25 mm × 0.25 mm × 30 m, J & W Scientific, Folsom, Calif.). 22: 1, injector temperature 250 ° C .; temperature; starting temperature 40 ° C. (maintained for 3 minutes) to 80 ° C. 3 ° C. per minute, 180 ° C. 5 ° C. per minute, 240 ° C. (maintained for 5 minutes) 10 ° C. per minute Temperature increase; helium flow; 1.8 mL / min). Mass spectra were measured at a mass range of m / z 40-400, a voltage of 70 eV, and an interface temperature of 230 ° C. The enzymatic reaction yields two sesquiterpene products, which are α-humulene (93.75%) and β-caryophyllene (6.25%) by comparison of retention times and mass spectra with standards. (See FIG. 2).

(6) Identification of ZSS1 expression
Total RNA was extracted from the leaf of a ginger using RNAqueous® Micro Kit (Ambion) and from the stem and rhizome of the plant of Ginger using Spectrum ™ Plant Total RNA Kit (Sigma-Aldrich), CDNA was synthesized as described above. RT-PCR (annealing temperature) using 5′-GGTGGAACTTGAGCAGCCCTTTAGCAAT-3 ′ (forward; SEQ ID NO: 29) and 5′-GTCTCTTTGTTGCATCATTCTCCCCAA-3 ′ (reverse; SEQ ID NO: 30) designed to eliminate genomic DNA amplification : 30 ° C. at 68 ° C.), and the difference in ZSS1 expression in different tissues was examined. Ubiquitin was amplified using 5′-AAGGAGTGCCCCAACGCCGAGTG-3 ′ (forward; SEQ ID NO: 31) and 5′-GCCTTCTGGTTGAGCGTAGTAGTGAG-3 ′ (reverse; SEQ ID NO: 32) as an internal standard (at the same temperature conditions as ZSS1 at 25). cycle). The results are shown in FIG. ZSS1 was found to be expressed in leaves and rhizomes but not in the stem. Furthermore, the ZSS1 expression level in the rhizome was significantly higher than that in the leaves. These results show that the content of α-humulene in rhizome oil (14.4%) is higher than that in leaf oil (2.0%) (Chane, MJ, Vera, R. and Chalchat, JC (2003) Chemical composition of the essential oil from rhizomes, leaves and flowers of Zingiber zerumbet Smith from Reunion Island. J. Essent. Oil. Res. 15: 202-205.).

(7) ZSS1 expression induction pattern In order to further investigate the expression induction pattern in leaves and rhizomes, real-time PCR was performed using MeJA-treated and mechanically damaged Hana ginger-derived RNA. The MeJA treatment was carried out by spraying 300 gM MeJA dissolved in 0.1% Tween 20 on hana ginger. Controls were sprayed with 0.1% Tween 20 only. Mechanical damage was done by cutting the leaves with a scissors. After 0, 4, 8, 24, 48, and 72 hours of treatment, leaves and rhizomes were collected, frozen in liquid nitrogen, and stored at -80 ° C until RNA extraction. Using SuperScript ™ III First-strand Synthesis Supermix for qRT-PCR (Invitrogen), cDNA was synthesized from 1 μg of total RNA extracted from leaves and rhizomes according to the manufacturer's instructions, and SYBR® Green PCR Master Mix (Applied Biosystems) was used for quantitative PCR with ABI 7300 Sequence Detection system (95 ° C., 10 minutes, then 95 ° C. for 15 seconds, 67 ° C. for 40 seconds for 40 cycles). Ubiquitin was used as an internal standard. For ZSS1 amplification, a primer pair: 5′-GCAAGTGATGGGTGGAGCAAAA-3 ′ (forward; SEQ ID NO: 33) and 5′-CCGAGCACTACTTGTGTGGACGGT-3 ′ (reverse; SEQ ID NO: 34) was used, and ubiquitin amplification was performed as described above. Primers (SEQ ID NOs: 31 and 32) were used. The relative expression level was determined by the ΔΔCt method (Livak, KJ (1997) Comparative Ct method. ABI Prism 7700 Sequence Detection System. User Bulletin no. 2. PE Applied Biosystems.). The results are shown in FIG. In leaves, transcription was induced by MeJa treatment and reached a peak (approximately 8 times) 48 hours after MeJA treatment. Transcription in rhizomes was induced up to 2 to 3 times 24 hours after MeJA treatment. On the other hand, in the control, transcription levels were only slightly affected by mechanical damage in the leaves and rhizomes. From this, it was found that the enzyme was increased in plants by treatment with MeJA or mechanical damage.

(8) Production of significant amount of α-humulene in E. coli Biotransformation of D-mevalonolactone to FPP in E. coli was promoted, and α-humulene production was examined. derived from Streptmyces sp. CL190 in pAC plasmid (distributed from Dr. Misawa, Marine Biotechnology) with pACY184 as the basic skeleton, tac promoter (Ptac), rrnB terminator (TrrnB), and chloramphenicol resistance gene (Cm ^r ) PAC-Mev was obtained by inserting a mevalonate pathway gene cluster (6.5 kb; distributed by Associate Professor Katsuyama of the University of Tokyo) (see FIG. 5). The mevalonate pathway gene cluster used includes nucleic acids encoding MVA kinase, DPMVA decarboxylase, PMVA kinase, IPP isomerase, HMG CoA reductase, and HMG CoA synthase, which are enzymes that catalyze the reaction of the mevalonate pathway. pAC-Mev and the above-described pET-ZSS1 were introduced into E. coli BL21-CodonPlus (DE3) and cultured overnight at 30 ° C. in LB medium containing 100 μg / mL ampicillin and 30 μg / mL chloramphenicol. . 500 μL of the culture solution (corresponding to about 1% of the whole culture solution) is inoculated into 50 mL of Terrific broth medium containing 100 μg / mL ampicillin and 30 μg / mL chloramphenicol, and OD ₆₀₀ = 0.5 at 30 ° C. Cultured. IPTG (final concentration 1 mM), 0.5 mg / mL D-mevalonolactone, 20% v / v dodecane was added, and cultured at 25 ° C. for 2 days. The dodecane layer was recovered, and 1 μl of the recovered dodecane layer was analyzed by GC-MS as described above. GC analysis detected a peak not seen in the control system at a retention time of about 26.25 minutes (indicated by an arrow in FIG. 6A). FIG. 6B is an enlarged view of the vicinity of this peak (portion surrounded by a square in FIG. 6A). FIG. 7A shows the result of fractionation of this peak and MS analysis. The pattern coincided with the MS analysis result of the α-humulene sample (FIG. 7B), and it was confirmed that the product obtained in the experiment was α-humulene. From these results, it was confirmed that α-humulene was produced in a particularly large amount in E. coli introduced with ZSS1 and pAC-Mev.

(1) Acquisition of β-eudesmol synthase cDNA and genomic DNA These DNAs could be obtained by the following method. Total RNA was prepared from the rhizomes of hana ginger using Spectrum ™ Plant Total RNA Kit (Sigma-Aldrich). Using SuperScript (trademark) III First-strand Synthesis Kit (Invitrogen), 3 μg of RNA was reverse-transcribed into cDNA in 20 μl of reaction mixture solution by poly (dT) priming. Based on the sequences of the two conserved domains of angiosperm sesquiterpene synthase closely related to rape ginger, the following primer pairs were designed: 5'-TTYCGAYTIYTIMGRMARCAIGG-3 '(forward; SEQ ID NO: 35) and 5'- TAIGHRTCAWAIRTRTCRTC-3 ′ (reverse; SEQ ID NO: 15). A primary PCR reaction was performed using 50 μl of a reaction mixture solution containing 1 μl cDNA, 1 μM each primer, 200 μM each dNTP, and 1 U ExTag polymerase (TaKaRa) under the following conditions: denaturation at 94 ° C. for 2 minutes, then 94 ° C for 30 seconds, 35 ° C for 1 minute and 30 seconds, 72 ° C for 1 minute for 4 cycles, 94 ° C for 30 seconds, 40 ° C for 1 minute and 30 seconds, 72 ° C for 1 minute for 30 cycles, and finally at 72 ° C Extend for 3 minutes. Using 5 μl of the obtained primary reaction solution as a template, a secondary PCR reaction was carried out under the same conditions to obtain a 581 bp fragment. The resulting fragment was purified, cloned into pGEM-Teasy vector (Promega) and sequenced. To isolate full-length cDNAs, Smart RACE cDNA Amplification Kit (BD Biosciences Clontech), 5′-CCCAATAATAACCTCCCACAACTCGG-3 ′ (5′RACE; SEQ ID NO: 36) and 5′-CCGAGTTGTGGAAGGTTTATTTGGG-3 ′ (3′RACE; SEQ ID NO: 37) and Advantage® 2 Polymerase Mix (Clontech) were used to extend the fragment toward the 5 ′ and 3 ′ ends according to the manufacturer's instructions to obtain a full length cDNA, designated as ZSS5 did. The nucleotide sequence of this cDNA is shown in SEQ ID NO: 5.

  ZSS5 contained a predicted ORF of 1662 bp encoding a polypeptide consisting of 554 amino acids (SEQ ID NO: 12) with an estimated molecular weight of 64.4 kDa and PI 5.09. The amino acid sequence of the polypeptide encoded by ZSS5 (ZSS5 polypeptide) had a region conserved in plant terpene synthases and contained a metal ion binding motif DDXXXD. The ZSS5 polypeptide is most similar to angiosperm sesquiterpene synthase, with 82% similarity to ZSS1 of the same Japanese ginger that was used to isolate ZSS5, Zingiber officinale's Germaclen D synthase (Picaud, S., Olsson, ME, Brodelius, M. and Brodelius, PE (2006) Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale. Arch. Biochem. Biophys. 452, 17-28) 68% similarity to sesquiterpene synthase from Elaeis oleifera (Cha, TS and Shah, FH (2000) A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase. Plant. Sci. 154, 153 -160) showed 62% similarity.

  In addition, genomic DNA was isolated from the leaves of banana ginger using the Plant Genomic Isolation Mini Kit (Qiagen). PCR reaction using a primer pair designed based on the cDNA sequence of SEQ ID NO: 5, 5'-ATGGGAGAAGCAATCACTAAC-3 '(forward; SEQ ID NO: 38) and 5'-CTTATTGAAGTAGCACAAGATTC-3' (reverse; SEQ ID NO: 39) And ZSS5 genomic DNA was isolated.

(2) Expression of ZSS5 in E. coli and generation of β-eudesmol from farnesyl diphosphate in vitro ZSS5 full-length ORF was converted to Advantage (registered trademark) HF 2 polymerase (Clontech), forward primer 5'-CACCATGGGAAGCAATCACTAAC -3 ′ (SEQ ID NO: 40) and reverse primer 5′-CTTATTGAAGTAGCACAAGATTCAAC-3 ′ (SEQ ID NO: 41) and the amplified product was cloned into the pET101 / DTOPO vector (Invitrogen). E. coli BL21-CodonPlus (DE3) (Stratagene) was transformed with the obtained recombinant plasmid pET-ZSS5.

Recombinant E. coli cells were grown in 50 ml LB medium containing ampicillin (50 μg / mL) at 37 ° C. to OD ₆₀₀ = 0.5-0.6. Next, IPTG (final concentration 1 mM) was added and further grown at 18 ° C. for 20 hours. The cells were collected by centrifugation and chilled extraction buffer (5 mM MgCl ₂ , 5 mM sodium ascorbate, 0.5 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, using an ultrasonic disrupter (Branson W185 D, NY). The cells were disrupted by treatment for 10 × 10 seconds in 50 mM MOPSO, pH 7.0) and 10% (v / v) glycerol. Cell debris was removed by centrifugation at 15,000 g to obtain a β-eudesmol synthase-containing supernatant. The obtained supernatant was purified with a nickel-nitrilotriacetic acid agarose column (Qiagen). The eluate was desalted into assay buffer (10 mM MOPSO, pH 7.0, 1 mM dithiothreitol, and 10% (v / v) glycerol) by passing through an Econopac 10DG column (Bio-Rad). The enzyme solution was used in the following experiment.

To capture volatile products in a Teflon-sealed screw cap glass test tube, 0.5 mL of pentane was overlaid. 900 μl enzyme solution, 20 mM MgCl ₂ , 0.2 mM MnCl ₂ , 0.2 mM NaWO ₄ , 0.1 mM NaF, and 20 μM [1- ³ H]-(E, E) -FPP (555 GBq / mol, American Radiolabeled Chemicals) The enzyme assay was carried out by incubating 1 ml of the reaction solution containing 2 hours at 30 ° C. After incubation, the reaction mixture was quickly frozen in liquid nitrogen to stop the reaction. After thawing, the mixed solution was extracted three times with pentane, and the combined extracts were subjected to column chromatography using anhydrous MgSO ₄ and a silica gel column (60Å, Merck) to obtain a sesquiterpene hydrocarbon free of oxygenated products. A fraction was obtained. Subsequently, the mixed solution was extracted three times with diethyl ether, and the combined extracts were similarly passed through a MgSO ₄ -silica gel column to recover the oxygenated product. Aliquots of each fraction were collected and subjected to liquid scintillation counting to determine enzyme activity. As described below, the same reaction was scaled up (final volume 4 mL) overnight using 80 μM unlabeled FPP as a substrate for GC-MS analysis of the product. The enzyme was found to have activity to synthesize β-eudesmol from FPP.

(3) Identification of product The product was analyzed by GC-MS on a Shimadzu QP5050A GC / MS system equipped with a DB-WAX column (0.25 mm x 0.25 mm x 30 m, J & W Scientific, Folsom, CA). (Split injection (1 μL) ratio 22: 1, injector temperature 250 ° C .; temperature; starting temperature 40 ° C. (maintained for 3 minutes) to 80 ° C. 3 ° C./min, 180 ° C. 5 ° C./min, 240 ° C. (5 min. Maintained) at a rate of 10 ° C. per minute; helium flow; 1.8 mL / min). Mass spectra were measured at mass range m / z 40-400, voltage 70 eV, and interface temperature 230 ° C. Compounds were identified by retention time with standards or by comparison of mass spectral databases. The main product was β-eudesmol, accounting for 62.6% of the total product. Seven minors including 10-epi-γ-eudesmol (16.8%), α-eudesmol (10%), aristolene (5.6%) and two unknown products (5%) A large peak was also observed (see FIG. 8). As a negative control, the reaction was carried out using an extract of E. coli BL21-CodonPlus (DE3) transformed with the pET101 / DTOPO plasmid not containing the ZSS5 insert, and the above product was not found when analyzed in the same manner. .

(4) Significant production of β-eudesmol in E. coli A gene cluster of the mevalonate pathway was constructed according to the method of Harada et al. The procedure will be described below. The tac promoter (Ptac) and rrnB terminator (TrrnB) were amplified from pTTQ18 by PCR and transferred to the EagI-SalI and HindIII-ClaI sites of pACYC184, respectively. Hydroxymethylglutaryl CoA synthase (HMGS), hydroxymethylglutaryl CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MPD) and isopentenyl The gene encoding pyrophosphate isomerase (IPPI) was provided by Dr. T. Kuzuyama (Takagi, M., Kuzuyama, T., Takahashi, S. and Seto, H. (2000) A gene cluster for the mevalonate pathway from Streptomyces sp. strain CL190. J. Bacteriol. 182, 4153-4157). Each gene was ligated using the overlapping extension and inserted between Ptac and TrrnB to obtain pAC-Mev. Co-transformation with pET-ZSS5 and pAC-Mev coli BL21 (DE3) was obtained. The metabolic pathway modified Escherichia coli thus obtained was shaken at 37 ° C. in terrific broth containing ampicillin (100 μg / ml) and chloramphenicol (30 μg / ml) until OD ₆₀₀ reached 0.5. Cultured. After supplementing with 1 mM IPTG, 0.5 mg / ml D-mevalonolactone (Tokyo Kasei) and 20% (v / v) dodecane, the cells were further grown by shaking culture at 25 ° C. for 48 hours. Thereafter, the dodecane layer was extracted and subjected to GC-MS analysis.

  The result of GC-MS analysis is shown in FIG. β-eudesmol was the main product, accounting for 72.4% of the total product. It was then in the order of 10-epi-γ-eudesmol (11.2%), α-eudesmol (7.1%) and aristolene (6.1%). The amount of β-eudesmol produced was 100 mg / L. No sesquiterpene product was detected in E. coli co-transformed with pET and pAC-Mev.

  According to the present invention, a nucleic acid encoding a polypeptide having sesquiterpene synthase activity, a vector and a cell containing the nucleic acid, a polypeptide encoded by the nucleic acid, and these nucleic acids, polypeptides, cells, etc. Therefore, the present invention can be used in the field of pharmaceuticals, the field of quasi-drugs, the field of cosmetics, the field of fragrances, and the like.

FIG. 1 shows the genomic sequence of ZSS1. FIG. 2A is a graph showing retention times of the obtained α-humulene and β-caryophyllene. FIG. 2B is a graph showing a mass spectrum of the obtained α-humulene. FIG. 2C is a graph showing a mass spectrum of the obtained β-caryophyllene. FIG. 3 is an electrophoretic image of ZSS1 derived from leaves, stems and rhizomes of banana ginger. FIG. 4A is a graph showing the expression level of ZSS1 in MeJA-treated leaves and rhizomes. In the figure, diamonds indicate treated samples and triangles indicate controls. FIG. 4B is a graph showing the expression level of ZSS1 in mechanically damaged leaves and rhizomes. In the figure, diamonds indicate treated samples and triangles indicate controls. FIG. 5 is a map of pAC-Mev. FIG. 6A shows the results of gas chromatography (GC) of a dodecane extract of an E. coli culture solution into which pAC-Mev and pET-ZSS1 were introduced. In the figure, the gray line indicates the experimental system, and the black line indicates the control. The control is a system using E. coli into which no plasmid has been introduced. FIG. 6B is an enlarged view around the peak surrounded by a square in the chart of FIG. 6A. In the figure, the gray line indicates the experimental system, and the black line indicates the control. The control is a system using E. coli into which no plasmid has been introduced. FIG. 7A is a graph showing a mass spectrum (MS) of a dodecane extract of the obtained culture broth. FIG. 7B is a graph showing the MS of the α-humulene standard. FIG. 8A shows a GC chart of products from E. coli transformed with pET-ZSS5. ? Indicates the peak of the unidentified substance. FIG. 8B shows the MS of each substance corresponding to each peak in FIG. 8A (each upper stage) and the MS of each standard substance or database (each lower stage). Rt is the peak retention time (in minutes) in GC. FIG. 9A shows a GC chart of products from E. coli co-transformed with pET-ZSS5 and pAC-Mev. ? Indicates the peak of the unidentified substance. FIG. 8B shows the MS results of the substance corresponding to the β-eudesmol peak shown in FIG. 8A. Rt is the peak retention time (in minutes) in GC.

SEQ ID NO: 14: PCR primer, n at position 11 is inosine, and n at position 13 is inosine
SEQ ID NO: 15: PCR primer, n at position 3 is inosine, and n at position 12 is inosine
SEQ ID NO: 16: PCR primer
SEQ ID NO: 17: PCR primer
SEQ ID NO: 18: PCR primer
SEQ ID NO: 19: PCR primer
SEQ ID NO: 20: PCR primer
SEQ ID NO: 21: PCR primer
SEQ ID NO: 22: PCR primer
SEQ ID NO: 23: PCR primer
SEQ ID NO: 24: PCR primer
SEQ ID NO: 25: PCR primer
SEQ ID NO: 26: PCR primer
SEQ ID NO: 27: PCR primer
SEQ ID NO: 28: PCR primer
SEQ ID NO: 29: PCR primer
SEQ ID NO: 30: PCR primer
SEQ ID NO: 31: PCR primer
SEQ ID NO: 32: PCR primer
SEQ ID NO: 33: PCR primer
SEQ ID NO: 34: PCR primer
SEQ ID NO: 35: PCR primer, n at position 9 is inosine, n at position 12 is inosine, and n at position 21 is inosine.
SEQ ID NO: 36: PCR primer
SEQ ID NO: 37: PCR primer
SEQ ID NO: 38: PCR primer
SEQ ID NO: 39: PCR primer
SEQ ID NO: 40: PCR primer
SEQ ID NO: 41: PCR primer

Claims (16)

A nucleic acid encoding a polypeptide having sesquiterpene synthase activity, the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 . 請 Motomeko 1 nucleic acid according that have a α- Hm Len synthase activity. A nucleic acid encoding a polypeptide having sesquiterpene synthase activity, the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 5. 請 Motomeko 3 nucleic acid according that having a β- user Death Mall synthase activity. A polypeptide having sesquiterpene synthase activity, comprising the amino acid sequence of SEQ ID NO: 8 . that having a α- Hm Len synthase activity 請 Motomeko 5, wherein the polypeptide. A polypeptide having sesquiterpene synthase activity, comprising the amino acid sequence of SEQ ID NO: 12. 請 Motomeko 7 wherein the polypeptide that have a β- user Death Mall synthase activity. A vector comprising the nucleic acid according to claim 1 . A vector comprising the nucleic acid according to claim 3 . The vector according to claim 9 or 10, and a vector containing a mevalonate pathway gene cluster that promotes bioconversion of D-mevalonolactone to farnesyl diphosphate in Escherichia coli is introduced into Escherichia coli and cultured in Escherichia coli . Manufacturing method of sesquiterpene. The vector according to claim 9, and a vector containing a mevalonate pathway gene cluster that promotes bioconversion of D-mevalonolactone to farnesyl diphosphate in E. coli is introduced into E. coli, and the E. coli is cultured. A method for producing humulene. A vector comprising the vector according to claim 10 and a vector containing a mevalonate pathway gene cluster that promotes bioconversion of D-mevalonolactone to farnesyl diphosphate in Escherichia coli into Escherichia coli, and cultivating Escherichia coli. Uedes Mall manufacturing method. A method for producing a sesquiterpene, comprising reacting the polypeptide according to claim 5 or 7 with farnesyl diphosphate . A method for producing α-humulene, comprising reacting the polypeptide according to claim 5 with farnesyl diphosphate . A process for producing beta- eudesmol , comprising reacting the polypeptide according to claim 7 with farnesyl diphosphate .

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