JP2015208273A - Novel elemol synthase gene and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel elemol synthase gene which synthesizes elemol, a kind of a sesquiterpene, to provide a novel elemol synthase which is encoded by the gene, and to provide a technique related to use thereof.SOLUTION: The present invention relates to elemol synthase genes encoding proteins consisting of specific amino acid sequences, recombinant vectors containing the genes, transformants containing the vectors, and elemol production methods comprising a process of culturing the transformant, preferably Escherichia coli, to obtain elemol.

Description

  The present invention relates to a novel elemol synthase gene, a novel elemol synthase encoded by the gene, and use thereof.

  Isoprenoids (also called terpenoids) are the most diverse group of compounds in nature, including over 20,000 species, including over 3,000 sesquiterpenes. Many isoprenoids are industrially useful such as pharmaceuticals, agricultural chemicals, functional foods, fragrances, or their raw materials. However, the accumulated amount in the natural world is small except for some examples, and many items that require enormous costs and labor to prepare a large amount of a single product.

  For this reason, development research for mass production by biotechnology using genetically modified microorganisms or plants has been actively conducted. In particular, among microorganisms, Escherichia coli is the microorganism with the most extensive technologies, materials, and information on genetic recombination, and therefore, techniques for mass-producing isoprenoids using recombinant Escherichia coli have been reported (for example, patent documents). 1, 2).

JP 2009-207376 A JP 2011-125272 A

  Although the technology described above is excellent, it cannot be said that it is sufficient, and further development of production technology for isoprenoids is desired. In particular, there is a strong demand for new genes and the like for producing sesquiterpenes.

  The present invention has been made in view of the above problems, and its purpose is to provide a novel elemol synthase gene that synthesizes elemol, a kind of sesquiterpene, a novel elemol synthase encoded by the gene, and use thereof. It is to provide such technology.

  As a result of intensive studies to achieve the above object, the present inventors synthesize elemol, a sesquiterpene possessed by a camellia plant, using farnesyl diphosphate (hereinafter referred to as “FPP”) as a substrate. A novel gene was identified for the first time in the world, and the present invention was completed. That is, the present invention includes the following inventions.

(1) Any gene selected from the group consisting of the following (a) to (e):
(A) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and converts farnesyl diphosphate to elemol. A gene encoding a protein having activity;
(C) a gene encoding a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having an activity of converting farnesyl diphosphate to elemol;
(D) a gene consisting of the base sequence set forth in SEQ ID NO: 2;
(E) a protein which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any of the genes (a) to (d) above and which has an activity of converting farnesyl diphosphate into elemol A gene encoding

(2) Any protein selected from the group consisting of the following (f) to (i):
(F) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(G) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and farnesyl diphosphate is converted to elemol. An active protein;
(H) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having an activity of converting farnesyl diphosphate to elemol;
(I) A protein encoded by the gene according to claim 1.

  (3) A recombinant vector comprising the gene according to (1) above.

  (4) A transformant comprising the gene according to (1) above or the recombinant vector according to (3).

(5) The transformant according to (4), further introduced and expressed by the following genes (a) to (d):
(A) Mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(A) type 1 isopentenyl diphosphate isomerase gene,
(C) type 2 isopentenyl diphosphate isomerase gene,
(D) Acetoacetate-coenzyme A ligase gene.

  (6) The transformant according to (4) or (5), which is a recombinant Escherichia coli.

  (7) A method for producing Elemol comprising culturing the transformant according to any one of (4) to (6) and obtaining Elemol.

  (8) An antibody that specifically binds to the protein of (2) above.

  According to the present invention, Elemol can be produced efficiently.

It is a figure which shows the structure of plasmid pAC-Mev / Scidi / Aacl. It is a figure which shows the biosynthesis from the chemical structure and farnesyl diphosphate of elemol which is a sesquiterpene which can be produced by this invention. It is a figure showing the (a) flower and the (b) above-ground part of "short pillar tea" (Camellia brevistyla). It is a figure showing the amino acid sequence alignment of CbTPS1 and the known ginger plant sesquiterpene synthase (synthase). FIG. 3 is a diagram showing the structure of plasmid pRSF-CbTps1. It is a figure showing the analysis result of the sesquiterpene product of colon_bacillus | E._coli using GC-MS.

  An embodiment of the present invention will be described in detail below. In addition, all the academic literatures and patent literatures described in this specification are incorporated by reference in this specification. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more (including A and greater than A) and B or less (including B and less than B)”.

  As used herein, the term “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. Here, the gene may be present in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). Further, the gene may be chemically synthesized, and the codon usage may be changed so that expression of the encoded protein is improved. Substitutions can be made between codons encoding the same amino acid. The term “protein” is used interchangeably with “peptide” or “polypeptide”. As used herein, the base and amino acid notation uses the one-letter code or three-letter code defined by IUPAC and IUB as appropriate.

<1. Gene / Protein of the Present Invention>
The gene according to the present invention is any polynucleotide selected from the group consisting of the following (a) to (e):
(A) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has an activity of converting FPP to elemol A gene encoding a protein;
(C) a gene encoding a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 and having an activity of converting FPP to elemol;
(D) a gene consisting of the base sequence set forth in SEQ ID NO: 2;
(E) a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the polynucleotide of any one of (a) to (d) above and has an activity of converting FPP to elemol Gene to do.

The protein according to the present invention is any protein selected from the group consisting of the following (f) to (i):
(F) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(G) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and has an activity of converting FPP to elemol protein;
(H) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having an activity of converting FPP to elemol;
(I) A protein encoded by the gene according to any one of (a) to (e) above.

  The genes (a) to (e) above encode a protein having a function of converting FPP into elemol. For this reason, elemol can be efficiently produced by expressing the gene in plants and microorganisms.

  The gene (a) will be specifically described. SEQ ID NO: 1 is a kind of sexerpene synthase derived from “Camellia brevistyla”, and as shown in FIG. 2, the activity of converting FPP to elemol (hereinafter referred to as “elemol synthesis activity”). A protein composed of a total length of 554 amino acid residues (sometimes referred to herein as “CbTPS1”).

  The gene of (b) above is a fusion with a functionally equivalent variant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein / peptide of the protein having the amino acid sequence shown in SEQ ID NO: 1. As long as it encodes a protein or the like and having a protein having elemol synthesis activity, its specific sequence is not limited. Here, the number of amino acids that may be deleted, substituted or added is not limited as long as the above functions are not lost. However, the number of amino acids may be deleted, substituted or substituted by a known introduction method such as site-directed mutagenesis. This number refers to the number that can be added, usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, more preferably within 7 amino acids, still more preferably within 5 amino acids (for example, 5, 4, 3, 2 or 1 amino acid). Further, in the specification, “mutation” mainly means a displacement artificially introduced by site-directed mutagenesis or the like, but may be a naturally occurring similar mutation.

  The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom-containing side Amino acids having chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) are included (the parentheses indicate single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.

  As used herein, “functionally equivalent” means that the target protein has a biological function or biochemical function equivalent (same and / or similar) to CbTPS1. In the present specification, examples of the biological function and biochemical function of CbTPS1 include a function of converting FPP to elemol and / or a function of catalyzing this reversible reaction. Biological properties may include the specificity of the site to be expressed, the expression level, and the like.

  Whether a protein into which a mutation has been introduced imparts a desired trait to a plant is obtained by obtaining a transformant in which a gene encoding the protein is introduced and expressed, and this transformant can produce elemol using FPP as a substrate. It can be judged by examining whether or not.

  The gene of (c) above also has a functionally equivalent variant, derivative, variant, allele, homolog, ortholog, partial peptide, or other protein / peptide fusion of the protein having the amino acid sequence shown in SEQ ID NO: 1. A specific sequence is not limited as long as the protein is intended and encodes a protein having elemol synthesis activity.

  Amino acid sequence homology refers to at least 80% or more, more preferably 90% or more, and even more preferably 95% or more (for example, 95%, 96%, 97) of the entire amino acid sequence (or a region necessary for functional expression). %, 98%, 99% or more). Amino acid sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. In addition, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. In order to align the base sequence or amino acid sequence to be compared with each other in an optimal state, addition or deletion (for example, a gap) may be allowed.

  In the present specification, the term “homology” intends a ratio of the number of amino acid residues having similar properties (homology, positive, etc.), but more preferably, the ratio of the number of amino acid residues matched, ie, identity. (Identity). The properties of amino acids are as described above.

  Regarding the gene (d) above, SEQ ID NO: 2 shows the base sequence (Open Reading Frame: ORF) of the gene encoding the protein consisting of the amino acid sequence shown by SEQ ID NO: 1.

The gene (e) is intended to be a gene that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any of the genes (a) to (d). Here, stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions for hybridizing at a temperature ranging from the melting temperature (Tm value) of highly homologous nucleic acids, for example, perfectly matched hybrids, to 15 ° C, preferably 10 ° C, more preferably 5 ° C lower. It can be said. For example, the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably in a buffer solution consisting of 0.25 M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, 1 × Denhardt's solution. Hybridize under conditions of 68 ° C. for 16 to 24 hours, and further in a buffer solution comprising 20 mM Na ₂ HPO ₄ , pH 7.2, 1% SDS, 1 mM EDTA, the temperature is 60 to 68 ° C., preferably 65 ° C. A preferable example is a condition in which washing is performed twice for 15 minutes under the condition of 68 ° C. Other examples include 25% formamide, 50% formamide under more severe conditions, 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / mL denatured salmon sperm DNA After prehybridization is performed overnight in a hybridization solution at 42 ° C., a labeled probe is added, and hybridization is performed by incubating at 42 ° C. overnight. The cleaning solution and temperature conditions for the subsequent cleaning are about “1 × SSC, 0.1% SDS, 37 ° C.”, and the more severe conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”. It can be performed at about “0.2 × SSC, 0.1% SDS, 65 ° C.”. As the hybridization washing conditions become more severe, hybridization with higher specificity is achieved. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

  Further, the gene of (e) above includes a gene consisting of DNA having 1-50 base sequences substituted, deleted, inserted and / or added in the DNA consisting of the base sequence shown in SEQ ID NO: 2, and Also included is a gene comprising a DNA having a nucleotide sequence of SEQ ID NO: 2 and a DNA having 90% or more homology.

  As a method for obtaining the above-mentioned gene / protein, a commonly used polynucleotide modification method may be used. That is, a polynucleotide having genetic information of a desired recombinant protein can be prepared by substituting, deleting, inserting and / or adding a specific base of a polynucleotide having protein genetic information. Specific methods for converting polynucleotide bases include, for example, commercially available kits (KOD-Plus Site-Directed Mutagenesis Kit; Toyobo, Transformer Site-Directed Mutagenesis Kit; Clontech, QuickChange Site Directed Mutagenesis Kit; Stratagene, etc. ) Or the use of the polymerase chain reaction (PCR). These methods are known to those skilled in the art.

  The gene may be composed only of a polynucleotide encoding the protein, but may be added with other base sequences. The base sequence to be added is not particularly limited, but includes a label (for example, histidine tag, Myc tag or FLAG tag), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP), a promoter sequence, and Examples thereof include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum transition signal sequence, a secretory sequence, etc.). The site to which these base sequences are added is not particularly limited and may be, for example, the N-terminus or C-terminus of the translated protein.

<2. Recombinant vector, transformant>
The present invention provides a vector containing the gene. This vector includes not only an expression vector for expressing the above gene in a host cell for producing a transformant, but also a vector used for production of a recombinant protein. The target of transformation is not particularly limited, and examples include bacteria, yeasts, insects, animals and plants.

  As the base material vector serving as the base of the vector, various commonly used vectors can be used. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected depending on the introduced cell or the introduction method. That is, the specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. Depending on the type of host cell, an appropriate promoter sequence may be selected in order to ensure the expression of the gene, and those in which this gene and the gene are incorporated into various plasmids may be used as an expression vector. Such expression vectors include, for example, phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors and viral vectors (eg, bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements and viruses (eg, baculo Vectors derived from viruses, papovaviruses, vaccinia viruses, adenoviruses, tripox viruses, pseudorabies viruses, herpesviruses, lentiviruses and retroviruses)) and combinations thereof are available (eg cosmids and phagemids).

  In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, the vector can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. A retroviral vector can also be replicable or replication defective. In the latter case, viral propagation generally occurs only in helper cells.

  The vector is preferably a vector containing a cis-acting regulatory region for the gene of interest. Appropriate trans-acting factors can be supplied by the host, supplied by a complementary vector, or supplied by the vector itself upon introduction into the host. In a preferred embodiment in this regard, it is preferred that the vector provides specific expression that may be inducible and / or cell type specific. Particularly preferred among such vectors are vectors that are inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

  Among the preferred vectors for use in bacteria are, for example, pQE-70, pQE-60 and pQE-9 (available from Qiagen); pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A and pNH46A (Stratagene And ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene). Preferred eukaryotic vectors also include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (available from Addgene).

  Various markers may be used to confirm whether or not the gene has been introduced into the host cell, and whether or not the gene has been reliably expressed in the host cell. That is, the vector preferably contains at least one selectable marker. Such selectable markers include, for example, dihydrofolate reductase or neomycin resistance, e. Examples of the culture in Escherichia coli and other bacteria include drug resistance genes such as a tetracycline resistance gene or an ampicillin resistance gene. In addition, a gene deleted in the host cell may be used as a marker. By introducing a plasmid containing this marker and the gene according to the present invention into the host cell as an expression vector, the introduction of the gene can be confirmed from the expression of the marker gene. The gene may also be linked to a vector containing a selectable marker for growth in the host cell.

  The gene insert is preferably operably linked to a suitable promoter. As other suitable promoters, those known to those skilled in the art can be used, and are not particularly limited. For example, phage λPL promoter, E. coli lacI promoter, lacZ promoter, T3 promoter and T7 promoter, trp promoter and These include the tac promoter, SV40 early promoter and late promoter, and retroviral LTR promoter.

  When E. coli is used as a host for transformation, “ori” for replication in E. coli and a gene for selecting transformed E. coli (for example, drugs (ampicillin, tetracycline, kanamycin, chloramphenicol, etc.) It is preferable to have a resistance gene) on the vector. Examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, pGEM-T, pDIRECT, pGEX (GE Healthcare) pET (Promega), pTrc (Invitrogen) and pT7. It is done.

  The vector preferably further contains a site for transcription initiation and transcription termination, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the vector construct will contain a transcription start AUG at the beginning of the polypeptide to be translated and a stop codon appropriately located at the end.

  Alternatively, transcription of DNA by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10-300 bp, that serves to increase the transcriptional activity of a promoter in a given host cell type. Enhancers include, for example, the SV40 enhancer (which is located 100-270 bp on the late side of the origin of replication), the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer on the late side of the origin of replication. Is mentioned.

  The host into which the vector is introduced is not particularly limited, but various cells can be suitably used. Representative examples of suitable hosts include cells (eg, E. coli cells, Streptomyces cells and Salmonella typhimurium cells), fungal cells (eg, yeast cells), insect cells (eg, Drosophila S2 cells and Spodoptera Sf9 cells). ), Animal cells (eg, CHO cells, COS cells and Bowes melanoma cells) and plant cells. More specifically, not only mammalian cells such as humans and mice but also, for example, silkworm-derived cells, insects such as Drosophila melanogaster, bacteria such as Escherichia coli, yeast (Saccharomyces cerevisiae ) And fission yeast (Schizosaccharomyces pombe)), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and the like, but are not particularly limited. Appropriate culture media and conditions for the above host cells can be used as known in the art.

  A method for introducing the vector into a host cell, ie, a transformation method is not particularly limited, and electroporation method, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, electroporation, Conventionally known methods such as transfection, transduction or infection can be suitably used. Such methods are described in many standard laboratory manuals such as Davis et al., Basic Methods In Molecular Biology (1986).

  In the present invention, a recombinant expression vector containing a polynucleotide encoding the partial fragment of the protein and a recombinant expression vector for recombinant production of the partial fragment (fragment) of the protein are genetically manipulated. Transformants (host cells).

  Further, the present invention may include an invention relating to the production of a protein mutant or fragment thereof obtained by the above-described recombinant technique. That is, the present invention can also include a method of producing a protein mutant or a fragment thereof using recombinant technology.

  Protein variants produced by such techniques can be isolated from host cells or extracellular (such as media) and purified as substantially pure and homogeneous proteins. For the separation and purification of the protein, the separation and purification methods used in normal protein purification may be used. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, ammonium sulfate precipitation or ethanol precipitation, acid extraction, Proteins can be separated and purified by appropriately selecting or combining dialysis and recrystallization. Further, a plurality of these columns can be combined.

  Chromatography includes affinity chromatography, anion or cation ion exchange chromatography, hydrophobic chromatography, phosphocellulose chromatography, hydroxyapatite chromatography, lectin chromatography, gel filtration, reverse phase chromatography and adsorption chromatography. Etc.

  When a protein mutant is expressed in a host cell (such as E. coli) as a fusion protein with GST or a recombinant protein to which 6 × His is added, the expressed recombinant protein is a glutathione column or nickel. It can be purified using a column. After purification of the fusion protein, the region other than the target protein in the fusion protein can be cleaved and removed with thrombin or factor Xa as necessary.

  The present invention also includes a transformant containing the above gene or the above vector. Here, “including a gene or vector” means that the gene is introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). The “transformant” means not only cells / tissues / organs but also individual organisms.

  A method for producing the transformant (production method) includes a method for transforming the above-described vector. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms exemplified in the host cell. In addition, if a promoter or vector is selected, plants or animals can be targeted for transformation.

  Moreover, since the gene of the present invention is derived from a plant, a transformant may be obtained using a plant cell as a host. Plant cells include various forms of plant cells such as suspension culture cells, protoplasts, and cells in plants. In addition, the transformant according to the present invention includes not only plant cells but also whole plants, plant organs (eg, roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (eg, epidermis, sieve parts). , Soft tissue, xylem, vascular bundle, etc.), these slices, callus, shoot primordia, polyblasts, hairy roots, cultured roots and the like.

  As a method for expressing the gene in plant host cells, the gene is incorporated into an appropriate vector, for example, polyethylene glycol method, Agrobacterium method, liposome method, cationic liposome method, calcium phosphate precipitation method, electric pulse perforation. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection method (GIBCO-BRL), microinjection method, particle gun Examples thereof include a method of introduction into a living body by a method known to those skilled in the art such as a method.

  Moreover, the administration into a plant body may be an ex vivo method or an in vivo method. In addition, when the gene of the present invention is introduced into a plant body, the gene can be directly introduced into a plant cell using a microinjection method, an electroporation method, a polyethylene glycol method, etc. It can also be introduced into a plant cell indirectly via a virus or bacterium capable of infecting a plant as a vector. Examples of such viruses include cauliflower mosaic virus, tobacco mosaic virus, gemini virus and the like as typical viruses, and Agrobacterium and the like as bacteria. When introducing a gene into a plant by the Agrobacterium method, a commercially available plasmid can be used. As a method for introducing the gene of the present invention into a plant body using such a vector, the leaf disk method (Jorgensen, RA et al., (1996), preferably introducing a gene via Agrobacterium. ). Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31, 957-973.).

  The “plant” to be transformed in the present invention is not particularly limited, but is preferably an angiosperm, and may be a monocotyledonous plant or a dicotyledonous plant. Moreover, not only herbs but also trees can be included.

  In addition, it is preferable that the above-described transformation method is appropriately selected according to the type of plant or the like serving as a host (for example, monocotyledonous plant or dicotyledonous plant).

  In addition, in the present invention, not only a host cell into which the gene or vector is directly introduced, but also the host cell is a higher plant, etc., the plant body in which the plant cell is grown, the progeny, progeny or Plants that are clones, as well as propagation materials (eg seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) are included. Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells. For example, with regard to the technique for producing a transformed plant body, a method for regenerating a plant body by introducing a gene into protoplasts using polyethylene glycol, a method for regenerating a plant body by introducing a gene into protoplasts using an electric pulse, or a gene for cells by a particle gun method. Can be mentioned, and a method for regenerating a plant body and a method for regenerating a plant body by introducing a gene via Agrobacterium are not particularly limited. The above technique has already been established and widely used in the technical field of the present invention, and the above method can be suitably used in the present invention.

  The method for regenerating a plant body by redifferentiating transformed plant cells differs depending on the type of plant cell. For example, in the case of rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) is used. Examples of corn include the method of Shillito et al. (Bio / Technology 7: 581 (1989)) and the method of Gorden-Kamm et al. (Plant Cell 2: 603 (1990)). The presence of the introduced foreign gene in the transformed plant that has been regenerated and cultivated by the above method is confirmed by a known PCR method or Southern hybridization method, or by analyzing the DNA base sequence in the plant body. can do. In this case, the extraction of DNA from the transformed plant can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

  For example, when the gene of the present invention present in the regenerated plant body is analyzed using the PCR method, the amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction can be carried out in a reaction mixture in which a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the gene of the present invention or a modified gene is used as a primer, and these are mixed. . In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the base sequence of the gene of the present invention. When the reaction solution containing the amplified product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and that the DNA fragments correspond to the gene of the present invention.

  Once a transformed plant into which the gene of the present invention has been introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material from the plant body, its descendants or clones, and mass-produce the plant body based on them. The present invention includes a plant cell into which the gene or recombinant expression vector of the present invention has been introduced, a plant containing the cell, a progeny and clone of the plant, and a propagation material for the plant, its progeny and clone. included. That is, in the present invention, a progeny plant such as “T0 generation” which is a regenerated generation that has undergone transformation treatment, or “T1 generation” which is a self-propagating seed of a plant of T0 generation, or crossed with them as one parent. Includes hybrid plants and progeny plants.

  Although various explanations have been given above, Escherichia coli is particularly preferable as a host cell. In the examples described later, E. coli B strain BL21 (DE3) was used. However, since various strains such as JM109 (DE3) of Escherichia coli K12 exist in Escherichia coli, the strain of Escherichia coli is not limited to BL21 (DE3). In the examples, LB medium and TB medium were used as the culture medium for recombinant Escherichia coli. However, there are many mediums such as 2 × YT medium and M9 medium as the culture medium for E. coli. It is not limited to TB medium. In addition to the implementation manuals provided by the manufacturers shown in the examples, many manuals exist as genetic recombination experiment methods. 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.

Moreover, the transformant according to the present invention is preferably one obtained by introducing and expressing the following genes (a) to (d):
(A) Mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(A) type 1 isopentenyl diphosphate isomerase gene,
(C) type 2 isopentenyl diphosphate isomerase gene,
(D) Acetoacetate-coenzyme A ligase gene.
The genes (a) to (d) will be described later.

<3. Manufacturing method of ELEMOL>
The method for producing Elemol according to the present invention is not particularly limited as long as it includes a step of culturing the above-described transformant and obtaining Elemol, and other specific conditions, materials, equipment used, and the like.

  Hereinafter, Escherichia coli, which is a microorganism with the most extensive materials and information among genetic recombination techniques, will be described as an example. In addition, this invention is not limited to this, About the host, the various materials described in the above-mentioned <2> column can be used.

  The outline in the case of producing a large amount of isoprenoid using recombinant Escherichia coli will be described. Escherichia coli does not have a mevalonate pathway, but is the first isoprenoid due to the non-mevalonate pathway [2-C-methyl-D-erythritol 4-phosphate (hereinafter also referred to as MEP pathway)). The substrate isopentenyl diphosphate (also called isopentenyl pyrophosphate; hereinafter referred to as IPP) is made. IPP is converted to dimethylallyl diphosphate (hereinafter referred to as DMAPP) by IPP isomerase (hereinafter also referred to as Idi), and DMAPP is farnesyl diphosphate (hereinafter referred to as FPP) synthase ( By sequentially condensing with IPP by synthase, it is converted to geranyl diphosphate having 10 carbon atoms (hereinafter referred to as GPP) and FPP having 15 carbon atoms. A monoterpene, which is a volatile component, is branched from GPP. Furthermore, sesquiterpenes and triterpenes are made by branching from the FPP. FPP is further condensed with IPP by geranylgeranyl diphosphate (hereinafter referred to as GGPP) synthase to synthesize GGPP having 20 carbon atoms. From this GGPP, diterpenes and carotenoids (tetraterpenes) are synthesized. Since Escherichia coli does not synthesize the above terpenes, in order to synthesize these isoprenoids into Escherichia coli, it is necessary to introduce and express the biosynthetic enzyme gene (s) responsible for the synthesis from FPP to the isoprenoid into E. coli. . Hereinafter, these genes will be described in detail.

(3-1. Mevalonate pathway gene group)
Examples of mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate include, for example, HMG-CoA synthase gene, HMG-CoA reductase gene, Examples thereof include 5 genes including mevalonate kinase (MVA kinase) gene, phosphomevalonate kinase (PMVA kinase) gene, and diphosphomevalonate decarboxylase (DPMVA decarboxylase) gene.

  As the mevalonate pathway gene group, a mevalonate pathway gene group derived from Streptomyces genus CL190 (Non-patent Document 4, Accession no AB037666) can be used, but besides this, it is derived from Saccharomyces cerevisiae. Mevalonate pathway genes (VJJ Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), and the mevalonate pathway genes from the bacterium Streptococcus pneumoniae ( SH Yoon, YM Lee, JE Kim, SH Lee, JH Lee, JY Kim, KH Jung, YC Shin, JD Keasling, SW Kim, Biotechnology & Bioengineering, 94: 1025-1032, 2006) can also be used.

(3-2.1 type / 2 type isopentenyl diphosphate isomerase gene)
Furthermore, in order to increase the supply amount of FPP, it is preferable to use an IPP isomerase (Idi) gene (idi). As the idi gene, an isopentenyl diphosphate isomerase gene derived from Streptomyces genus CL190 (Non-patent Document 4 Accession no AB037666) can be used, but in addition to this, an isopentenyl diphosphate isomerase gene derived from E. coli (VJJ) Martin, DJ Pitera, ST Withers, JD Newman, JD Keasling, Nature Biotechonosy, 21: 796-802, 2003), isopentenyl diphosphate isomerase gene (S. Kajiwara, PD Fraser, K) from Saccharomyces cerevisiae Kondo, N. Misawa, Biochemical Journal, 324: 421-426, 1997), isopentenyl diphosphate isomerase gene derived from the green alga Haematococcus pluvialis (cited by JJ Martin et al., And S Kajiwara et al.) Can also be used.

  In addition, there are a type 1 (type 1) and a type 2 (type 2) having different structures. In the present invention, any Idi may be used, but the present inventors used both Idi in the following examples.

(3-3. Acetoacetate-coenzyme A ligase gene)
According to the recombinant Escherichia coli introduced with the above-described mevalonate pathway gene group and Idi gene, a large amount of FPP can be obtained by adding mevalonate or mevalonolactone (D-mevalonate lactone) as a substrate in the medium. Can be produced.

  On the other hand, acetoacetate (for example, lithium acetoacetate; LAA), which is cheaper than mevalonolactone, can also be used as a substrate. In order to utilize LAA added to the medium, it is preferable to further introduce an acetoacetate-CoA ligase gene using it as a substrate.

  Acetoacetate-CoA ligase is an enzyme that catalyzes the conversion to acetoacetyl-CoA using ATP using acetoacetate and CoA as substrates (JR Stern, Biochem. Biophys. Res. Commun. 44, 1001-1007). 1971; Bergstrom, JD; Wong, GA; Edwards, PA; Edmond, J., J. Biol. Chem. 259, 14548-14553, 1984). As acetoacetate-CoA ligase genes, genes derived from mammals such as rats (Rattus norvegicus) and humans, certain bacteria, fungi, and the like are known, and these genes can also be used in the present invention. it can. A plasmid containing the full length gene (Accession No. BC061803) encoding rat-derived acetoacetate-CoA ligase can be obtained from Invitrogen as a Mammalian Gene Collection cDNA clone (clone ID: 5598532).

  That is, the present invention includes (a) a mevalonate pathway gene group that synthesizes mevalonic acid or mevalonolactone to isopentenyl diphosphate in addition to the genes described in the section <1> above; A transformant introduced with an isopentenyl diphosphate isomerase gene, (c) type 2 isopentenyl diphosphate isomerase gene, and (d) an acetoacetate-coenzyme A ligase gene is cultured in a medium containing acetoacetate And the manufacturing method of ELEMOL including the process of acquiring ELEMOL is also included.

  In addition to the above, for the genes (a) to (d) described above, for example, in addition to Patent Documents 1 and 2, Non-Patent Documents 1 to 8 which are references described at the end of the present specification The described items can also be used. For example, regarding the source of the mevalonate pathway gene group, the present inventors used those derived from Streptomyces genus CL190 strain in the following examples, but it is also possible to use a corresponding gene group derived from yeast or other bacteria ( Reference: Non-Patent Document 8).

  As described above, the present invention relates to a protein that synthesizes an essential oil component (sesquiterpene) of an aromatic and healthy food and ornamental plant, a gene encoding the protein, and a method for producing the essential oil component using the gene . More specifically, the present invention relates to a protein for synthesizing elequitol, a sesquiterpene that can be expected to have a physiological activity useful for humans, from farnesyl diphosphate, a gene encoding the protein, and production of a sesquiterpene using the gene Regarding the method. According to the present invention, it is possible to produce elemol which is difficult to chemically synthesize using recombinant Escherichia coli or recombinant plants. Elemol, a sesquiterpene that can be produced according to the present invention, is not only a comfortable scent component, but can be expected to have various beneficial physiological activities in humans.

<4. Antibody>
The present invention includes an antibody that specifically binds to the protein. The present antibody may be an antibody obtained by a known method using the protein described in the section <1> or a partial peptide thereof as an antigen. The form of this antibody is not particularly limited.

  Known methods include, for example, literature (Harlow et al. “Antibodies: A laboratory manual (Cold Spring Harbor Laboratory, New York (1988))”, Iwasaki et al. “Monoclonal antibody hybridoma and ELISA, Kodansha (1991)”). The method of description is mentioned. The antibody thus obtained can be used for protein detection and measurement. For example, the protein can be obtained using the sensitizing antigen. The protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. In addition, cells expressing the protein, lysates thereof, or chemically synthesized proteins may be used as the sensitizing antigen. A short peptide can be appropriately combined with a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin and egg white albumin to be used as an antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion in the production of a monoclonal antibody. Rodents such as Rabbits, Rabbits such as Rabbits, or primates such as Rhesus monkeys are used.

  The present invention also includes a polypeptide having the amino acid sequence of the immunogenic epitope portion in the protein. The polypeptide having the amino acid sequence of the epitope-bearing portion of the protein contains at least the mutation site described in the section <1> and has 6, 7, 8, 9, or 10 amino acids. What is necessary is just to include the part of a peptide.

  In the above protein, the immunogenic epitope part that elicits an antibody response can be identified by methods known in the art. For example, Geysen, HM et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984) includes hundreds of peptides that are sufficiently pure to be available for reaction in an enzyme-linked immunosorbent assay. A rapid co-synthesis procedure on a solid support is disclosed. The interaction of the synthetic peptides with the antibody can then be easily detected without removing them from the support. In this manner, peptides carrying immunogenic epitopes of the desired protein can be routinely identified by those skilled in the art. For example, immunologically important immunogenic epitopes in the coat protein of foot-and-mouth disease virus were obtained by elucidating 7 amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Was positioned by. A complete substitution set of peptides, in which all 20 amino acids were sequentially substituted at each position within the epitope, was then synthesized and the specific amino acids that gave specificity for reaction with the antibody were determined. Accordingly, peptide analogs of the epitope-bearing peptides of the present invention can be routinely made by this method. Geysen (1987) U.S. Pat. No. 4,708,871 describes this method in more detail for identifying peptides carrying an immunogenic epitope of the desired protein.

  An “immunogenic epitope” is defined as the part of a protein that elicits an antibody response when the entire protein is an immunogen. These immunogenic epitopes are believed to be limited to a few focal points on the molecule. On the other hand, the region of a protein molecule to which an antibody can bind can be defined as an “antigenic epitope”. The number of immunogenic epitopes in a protein is generally less than the number of antigenic epitopes. This is described, for example, in Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984).

  Peptides carrying the antigenic epitope of the protein are useful for raising antibodies according to the present invention, particularly monoclonal antibodies. Thus, the majority of hybridomas obtained by fusion of spleen cells from donors immunized with antigenic epitope-bearing peptides generally secrete antibodies that are reactive with natural proteins. Antibodies raised by antigenic epitope-bearing peptides are useful for detecting mimetic proteins, and antibodies against different peptides are used to track the end of various regions of the protein precursor that undergo post-translational processing Can be done. Because immunoprecipitation assays have shown that even short peptides (eg, about 9 amino acids) can bind and displace longer peptides, peptides and anti-peptide antibodies have various qualitative properties for mimetic proteins. Or it can be used in quantitative assays, such as competitive assays. This is described, for example, on page 777 of Wilson, I. A. et al., Cell 37: 767-778 (1984). Protein-specific antibodies included in the present invention are also useful for purification of mimetic proteins (eg, by adsorption chromatography using methods well known in the art).

  Antigenic epitope-bearing peptides derived from the protein designed according to the above guidelines are preferably at least 7, more preferably at least 9, and most preferably 15 contained within the amino acid sequence comprising the mutation site of the protein. Includes sequences between ˜30 amino acids. Furthermore, the amino acid sequence including the mutation site of the protein may contain any length and the whole of 30 to 50 amino acids or the whole. Also, the amino acid sequence of the antigenic epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (ie, the sequence contains relatively hydrophilic residues and is highly hydrophobic Are preferably avoided), and sequences containing proline residues are particularly preferred. In addition, the antigenic epitope-bearing peptide of the protein can be easily obtained by a known recombinant protein production technique or the like.

  The antigenic epitope-bearing peptide can be used to induce antibodies by methods well known in the art. This is described, for example, in Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985). ing. In general, animals can be immunized with free peptides. However, anti-protein antibody titers can be boosted by coupling the peptide to a macromolecular carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine can be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides are more like glutaraldehyde. Common linking agents can be used to couple to the carrier. Animals such as rabbits, rats, and mice can be injected either intraperitoneally and / or intradermally with either free or carrier-coupled peptides, eg, emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant. Immunized. In order to provide useful titers of anti-protein antibodies that can be detected by an ELISA assay using, for example, free peptides adsorbed on a solid surface, for example at intervals of about 2 weeks May be needed. The titer of anti-protein antibodies in serum from immunized animals is increased by selection of anti-protein antibodies, for example, adsorption to peptides on solid supports and elution of selected antibodies by methods well known in the art. obtain.

Also, as used herein, the term “antibody” includes immunoglobulins (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) ₂ fragments and Fc fragments), Examples include, but are not limited to, all classes of polyclonal antibodies, monoclonal antibodies, single chain antibodies, anti-idiotype antibodies, humanized antibodies, and humanized antibodies by gene recombination.

Moreover, as long as this antibody couple | bonds with the said protein, an antibody fragment or an antibody modification thing may be sufficient. That is, the present antibodies include complete molecules and antibody fragments (eg, Fab and F (ab ′) ₂ fragments) that can specifically bind to the proteins described above. Fab and F (ab ′) ₂ fragments lack the Fc portion of the complete antibody, are removed more rapidly by circulation, and have little nonspecific tissue binding of the complete antibody (Wahl et al., J Nucl. Med. 24: 316-325 (1983)). Accordingly, these fragments are preferred.

  The antibody can also be produced in a two-step procedure through the use of anti-idiotype antibodies. Such a method uses the fact that the antibody itself is an antigen, and therefore it is possible to obtain an antibody that binds to a secondary antibody. According to this method, the antibody is used to immunize an animal (preferably a mouse). The splenocytes of such animals are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce antibodies whose ability to bind to the antibody can be blocked by the protein antigen. Is done. Such antibodies include anti-idiotype antibodies against the present antibodies and can be used to immunize animals to induce further formation of the present antibodies.

It will be appreciated that Fab and F (ab ′) ₂ and other fragments of the antibodies may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (resulting in Fab fragments) or pepsin (resulting in F (ab ′) ₂ fragments). Alternatively, protein binding fragments according to the present invention can be produced by the application of recombinant DNA technology or by synthetic chemistry.

  In addition, it is added that the contents described in the above items <1> to <4> can be used as appropriate in other items. The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Embodiments are also included in the technical scope of the present invention. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited only to this Example.

[Example 1] Determination of cDNA sequence of sesquiterpene synthase (sesquiterpene synthase) gene derived from camellia short-pillar tea From the flower of "short-column tea" (Camellia brevistyla) (Fig. 3), RNeasy Plant Mini Kit (Qiagen) Total RNA was extracted using Using SMARTer RACE cDNA amplification kit (Takara Bio), cDNA was prepared from 1.0 μg of total RNA according to the manufacturer's instructions. Described in Non-Patent Document 3 using a degenerate oligonucleotide primer pair (forward: 5′-ttycgaytny tnmgrmarca ngg -3 ′ (SEQ ID NO: 3) and reverse: 5′-tanghrtcaw anrtrtcrtc -3 ′ (SEQ ID NO: 4)) PCR was performed using cDNA as a template under the conditions described above to obtain an amplified fragment of 1005 bp.

  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 a reverse primer sequence at both ends and was amplified only with the reverse primer. Since the 5 ′ side contained the starting methionine and the 5 ′ UTR sequence, the above-mentioned SMARTer RACE cDNA Amplification Kit (manufactured by Takara Bio), oligonucleotide primer (White.3race-2) : 5'-TTTAGACAAACAGCTATGGGACAA-3 '(SEQ ID NO: 5)) and Advantage2 Polymerase Mix (Clontech) were used to extend the fragment toward the 3' end according to the manufacturer's instructions to determine the full-length cDNA sequence.

[Example 2] Acquisition of elemol synthase (elemol synthase) gene (CbTps1) cDNA Oligonucleotide primer pair Wh.Bgl-F (5'-AGACAGAAGATCTCATGGCTTCATCTCAAGTTGGTGA -3 specific for the full-length cDNA sequence obtained in Example 1 '(SEQ ID NO: 6), primer “AGATCT” indicates BglII recognition site) and Wh.Xho-R (5′-GAGGTACCTCGAGTCACATGGGAATTGGATCTTCGA -3 ′ (SEQ ID NO: 7), primer “CTCGAG” indicates XhoI recognition site ) And Advantage2 Polymerase Mix (manufactured by Clontech), using the camellia short-brown tea cDNA as a template (reaction composition: according to the manufacturer's instructions; reaction conditions: denaturation at 94 ° C. for 2 minutes, then 94 ° C. For 30 seconds, 55 ° C for 30 seconds, 72 ° C for 4 minutes, then 94 ° C for 30 seconds, 60 ° C for 30 seconds, 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: 2, and the amino acid sequence of the polypeptide encoded by the cDNA is shown in SEQ ID NO: 1. In addition, the cDNA specified by SEQ ID NO: 2 was named CbTps1 gene, and the protein specified by SEQ ID NO: 1 encoded by CbTps1 gene was named CbTPS1.

  The CbTps1 gene contained a 1665 bp open reading frame (ORF) and encoded a protein (CbTPS1) with a putative 554 amino acid residue, a molecular weight of 64.0 kDa, and a pI of 5.19. Based on a homology search against the GenBank / EMBL / DDBJ database, CbTPS1 is a known sesquiterpene synthase, Valeriana officinalis L., and 50%, lavender (Lavandula pedunculata) germinal A synthase ( The amino acid sequence identity was 49% with germacrene A synthase and 51%, and terpene synthase from tomato (Solanum lycopersicum) with 49%.

  From the homology with known sesquiterpene synthases, CbTPS1 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. See Proc. Natl. Acad. Sci. USA 95: 4126-4133). Furthermore, CbTPS1 contained the motifs DDxxD, RDR and (N / D) Dxx (S / T) xxxE (called NSE / DTE motif) highly conserved in sesquiterpene synthase (FIG. 4). On the other hand, like the known sesquiterpene synthase, CbTPS1 was predicted not to contain a plastid transit peptide. Note that ZoGDS represents Z. officinale Roscoe-derived gelmacrene D synthase (AAX40665). ZzZSS1 and ZzZSS2 represent α-humulene synthase (BAG12020) and β-eudesmol synthase (BAG12021) derived from Z. zerumbet Smith, respectively. Amino acids common to all proteins are shown in black. An arrow is added below the sequence corresponding to the degenerate oligonucleotide primer. RDR, DDxxD, and (N / D) Dxx (S / T) xxxE motifs highly conserved in sesquiterpene synthase are indicated by asterisks.

[Example 3] Production of elemol by Escherichia coli having plasmid pRSF-CbTps1 and plasmid pAC-Mev / Scidi / Aacl Plasmid DNA into which the full-length sequence of CbTps1 gene was inserted was digested with restriction enzyme BglII-XhoI, and the resulting CbTps1 gene Was ligated to the BglII-XhoI site of the vector pRSFDuet-1 (Novagen; kanamycin resistance) to prepare a pRSF-CbTps1 plasmid (FIG. 5). A cDNA (1,665 bp) corresponding to the ORF of the CbTps1 gene is inserted between BglI and XhoI of the vector pRSFDuet-1.

  In addition, in order to use lithium acetoacetate (LAA) as a substrate, the rat-derived acetoacetate-CoA ligase gene (Aacl) is inserted into the plasmid pAC-Mev / Scidi, and the final plasmid pAC-Mev / Scidi / Aacl (FIG. 1) was produced (Patent Document 2, Non-Patent Documents 7 and 8).

  Non-patent literature: transformation method of E. coli using plasmid pRSF-CbTps1 and plasmid pAC-Mev / Scidi / Aacl, culture method of recombinant E. coli, extraction of elemol from cultured E. coli cells, and identification method 2 and 7. However, in the culture of recombinant E. coli, LAA was added at a concentration of 1 mg / mL using kanamycin 50 mg / L and chloramphenicol 30 mg / L as drugs to be added to the medium. The specific culture method is as follows.

  First, recombinant Escherichia coli introduced with plasmid pRSF-CbTps1 and plasmid pAC-Mev / Scidi / Aacl was treated with an LB medium containing Km and Cm (bactotryptone 1%, yeast extract 0.5%, NaCl 0.5-1). %) And overnight culture at 20 ° C. and 180 rpm to prepare a preculture. Subsequently, IPTG 0.05 mM (final concentration) was added to TB medium containing Sam, Km, Cm, and LAA (Sambrook and Russel, Molecular Cloning A Laboratory Manual (Third edition) Cold Spring Harbor Laboratory Press, 2001). The main culture was started by adding a preculture solution corresponding to 1% of the medium. The main culture was performed at 18 ° C. and 180 rpm for 70 hours. After completion of the main culture, E. coli was collected and extracted from the cells with ethyl acetate.

  As a result of analysis using a gas chromatograph mass spectrometer (GC-MS), in the control group to which IPTG was added in the E. coli culture containing the empty vector pRSFDuet-1 and the plasmid pAC-Mev / Scidi / Aacl, While no new sesquiterpene was produced in the ethyl acetate extract, in the experimental group in which IPTG induction was carried out in an E. coli culture containing plasmid pRSF-CbTps1 and plasmid pAC-Mev / Scidi / Aacl, A new peak was found in the ethyl acetate extract from the body. The results are shown in FIG. In FIG. 6, (a) is a chromatogram of an ethyl acetate extract of an Escherichia coli strain carrying the plasmid pRSF-CbTps1 containing the CbTps1 gene and the plasmid pAC-Mev / Scidi / Aacl. A new peak 1 is indicated by an arrow. (B) is a chromatogram (control) of an ethyl acetate extract of an Escherichia coli strain carrying the vector pRSFDuet-1 not containing the CbTps1 gene and the plasmid pAC-Mev / Scidi / Aacl. (C) is a mass spectrum of peak 1 (upper figure) indicated by the arrow in (a) and Elemol (lower figure) in the database.

  As shown in FIG. 6, this peak 1 was identified as Elemol by comparison with a database of mass spectra. From this result, it was found that the CbTps1 gene is a gene encoding elemol synthase. The chemical structure of Eremol is shown in FIG.

  From the above results, it was shown that selective production of elemol was possible using Escherichia coli introduced with plasmid pRSF-CbTps1 and plasmid pAC-Mev / Scidi / Aacl.

<References>
[Non-Patent Document 1] S. Picard, ME Olsson, M. Brodelius, PE Brodelius, Arch. Biochem. Biophys. 452: 17-28, 2006
[Non-Patent Document 2] M. Fujisawa, H. Harada, H. Kenmoku, S. Mizutani, N. Misawa, Planta, 232: 121-130; Erratum, 232: 131, 2010
[Non-Patent Document 3] F. Yu, S. Okamoto, K. Nakasone, K. Adachi, S. Matsuda, H. Harada, N. Misawa, R. Utsumi, Planta 227: 1291-1299, 2008
[Non-Patent Document 4] F. Yu, H. Harada, K. Yamasaki, S. Okamoto, S. Hirase, Y. Tanaka, N. Misawa, R. Utsumi, FEBS Lett 582: 565-572, 2008
[Non-Patent Document 5] M. Takagi, T. Kuzuyama, S. Takahashi, H. Seto, J. Bacteriol., 182: 4153-4157, 2000
[Non-Patent Document 6] K. Kakinuma, Y. Dekishima, Y. Matsushima, T. Eguchi, N. Misawa, M. Takagi, T. Kuzuyama, H. Seto, J. Am. Chem. Soc., 123: 1238 -1239, 2001
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  Elemol is not only a comfortable scent component, but can be expected to have various beneficial physiological activities in humans. For this reason, it can be used in various industries such as pharmaceuticals, functional foods, fragrances, agriculture and horticulture, and consumer goods.

Claims (8)

Any gene selected from the group consisting of the following (a) to (e):
(A) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and converts farnesyl diphosphate to elemol. A gene encoding a protein having activity;
(C) a gene encoding a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having an activity of converting farnesyl diphosphate to elemol;
(D) a gene consisting of the base sequence set forth in SEQ ID NO: 2;
(E) a protein which hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to any of the genes (a) to (d) above and which has an activity of converting farnesyl diphosphate into elemol A gene encoding Any protein selected from the group consisting of the following (f) to (i):
(F) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(G) The amino acid sequence shown in SEQ ID NO: 1 consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added, and farnesyl diphosphate is converted to elemol. An active protein;
(H) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 1 and having an activity of converting farnesyl diphosphate to elemol;
(I) A protein encoded by the gene according to claim 1.   A recombinant vector comprising the gene according to claim 1.   A transformant comprising the gene according to claim 1 or the recombinant vector according to claim 3. The transformant according to claim 4, further comprising the following genes (a) to (d) introduced and expressed:
(A) Mevalonate pathway genes that synthesize mevalonic acid or mevalonolactone to isopentenyl diphosphate,
(A) type 1 isopentenyl diphosphate isomerase gene,
(C) type 2 isopentenyl diphosphate isomerase gene,
(D) Acetoacetate-coenzyme A ligase gene.   The transformant according to claim 4 or 5, which is a recombinant Escherichia coli.   A method for producing ELEMOL, comprising the step of culturing the transformant according to any one of claims 4 to 6 to obtain ELEMOL.   An antibody that specifically binds to the protein of claim 2.