KR20110068292A - Dammarenediol synthase gene from centella asiatica l. urban and uses thereof - Google Patents

Abstract

PURPOSE: A Centella asiatica L. Urban-derived dammarenediol synthease gene is provided to produce a large amount of dammarenediol. CONSTITUTION: A Centella asiatica L. Urban-derived dammarenediol synthease gene has a base sequence of sequence number 1. A recombinant vector contains a gene encoding the synthease. A transformed cell is prepared by transforming host cells with the recombinant vector. The host cell is yeast or E.coli.

Description

Dammarenediol synthase gene from Centella asiatica L. Urban and uses approximately}

The present invention relates to a yammarendiol synthase gene derived from a camouflage and its use, and more particularly, to a yammarendiol synthase protein derived from a camouflage, a gene encoding the protein, a recombinant vector comprising the gene, and the recombinant A host cell transformed with a vector, and transforming the host cell with the recombinant vector, the present invention relates to a method for producing dammarenediol, comprising overexpressing a dammareenediol synthase gene.

Centella asiatica L. Urban is a ursane-type tree such as asiaticoside, madecassoside, asiatic acid and madecassic acid. Triterpene saponin is contained in the leaves in large amounts. Since the leaves of Centella have such useful pharmacological ingredients, they are excellent for treating skin diseases and wounds and are effective in preventing dementia, asthma, headache, gonorrhea, syphilis, skin diseases, leprosy and gastrointestinal diseases. In particular, it has been used as a raw material for the treatment of skin diseases because of its excellent ability to regenerate damaged skin.

Triterpene saponins, which show important pharmacological activity in plants, have recently been reported in the form of oleanane, ursane, and dammarane compounds. The main saponins of ginseng are the tetracyclic cyclic malemalediol. It is reported that it is saponin derived from (FIG. 1). Briefly describing these biosynthesis processes, triterpene saponin and phytosterol biosynthesis synthesize squalene, a C30 unit, by squalene synthase. In the next step, a 30-carbon oxidose squalene (2,3-oxidosqualene) cyclization reaction is completed. This reaction is a crucial step in saponin and phytosterol biosynthesis, and oxidosqualene cyclase Beta-amyrin synthase, alpha-amyrin synthase, dammarenediol synthase and cycloartenol synthase And used as a precursor of saponin and phytosterol (FIG. 2). This step is also known to regulate overall saponin production while present in small amounts in plants as a limiting step. In ginseng, dammarenediol is a compound that leads to the synthesis of about 29 ginsenosides reported so far. Therefore, the dammarenediol synthesis gene is reported to be the most important gene for ginsenoside biosynthesis of ginseng. Although the dammarane-type triterpene compounds have been reported in other plants, the gene for synthesizing dammarenediol is Only reported in ginseng (Han et al. 2006, Plant Cell Physiol 47: 1653-1662; Tansakul et al. 2006, 580: 5143-5149). The gene isolated from the cultivars was estimated to beta-amirin synthase through gene expression analysis and analysis of this gene-related substance, but did not provide direct evidence (Kim et al. 2005, Plant Cell Rep 24: 304-311). . Therefore, the present invention was to investigate the function of the gene encoding the yammarendiol synthase derived from Centella asiatic and to develop a strain for mass production of this material.

Korean Patent Publication No. 2009-0078050 discloses a method for producing high efficiency Asian Ticoside from a centella, and Korean Patent Publication No. 2009-0122049 discloses a method for extracting and separating Asiantic acid and Asian Ticoside from a centella.

The present invention is derived from the above-mentioned demands. The present inventors have prepared cDNA from the leaves of the herbaceous plant, and isolated the entire nucleotide sequence encoding the dammarerendiol synthase using gene amplification and inserted into the yeast expression vector. After overexpression, the product was analyzed and identified to determine that it was a dhamrenedidiol synthase, thereby completing the present invention.

In order to solve the above problems, the present invention provides a dammarendiol synthase protein derived from Centella.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

In another aspect, the present invention provides a method for producing dammarenediol comprising the step of transforming the host cell with the recombinant vector overexpressing the dammareene diol synthase gene.

When introduced into the genome of other crops, including ginseng and rice, the plant extract derived from the centella as described in the present invention, there is a possibility of producing saponin of the dammareendiol type, and also dammarendiol antiviral It has been reported to show various pharmacological activities such as action. Therefore, it is expected to have a high industrial ripple effect because it can be introduced into microorganisms such as yeast and E. coli to produce a large amount of dammarenediol.

In order to achieve the object of the present invention, the present invention provides a dhammarenediol synthase protein derived from the centella, consisting of the amino acid sequence represented by SEQ ID NO: 2.

The range of dammarenediol synthase proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from a centella and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. By "substantially homogeneous physiological activity" is meant the activity of converting oxidose squalene to dammarerendiol in plants.

In addition, the present invention provides a gene encoding the dalmenediol synthase protein. The gene of the present invention includes both genomic DNA and cDNA encoding the dammarenediol synthase protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

In another aspect, the present invention provides a recombinant vector comprising a dammarenediol synthase gene according to the present invention. The recombinant vector may be preferably a yeast recombinant vector, but is not limited thereto.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as a host cell, a promoter and an operator site of the E. coli tryptophan biosynthetic pathway, and a phage λ left promoter (pLλ promoter) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains. The host cell is preferably E. coli.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast ( Saccharomyce) as a host cell cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. The host cell is preferably yeast.

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also provides a method for producing dammarerendiol, comprising overexpressing a dammareenediol synthase gene by transforming a host cell with a recombinant vector comprising a stem-derived dammareenediol synthase gene. In the method of the present invention, the dammarenediol synthase gene and the recombinant vector are as described above. In addition, the host cell is preferably yeast or E. coli.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: Dammarenediol  Complete coding for the enzyme cDNA Separation of

Centellar plants were incubated for 5 weeks in MS liquid medium adjusted to 3% sucrose, pH 5.8, and only leaf portions were cut and frozen in liquid nitrogen, and total RNA was extracted using Trizol reagent. The cDNAs of the cultivars include 2 μg total RNA, 2 ng oligo-dT (15) primers, 10 mM dNTP, 30 units of AMV reverse-transcriptase and 40 units of RNase inhibitor. It was synthesized by reacting for 1 hour at 42 ℃ in a buffer. CDNA synthesized to amplify the complete dameneradiol synthase gene of the centella as a template, the forward primer (5'-ATGTGGAAGCTGAAGATA-3 '; SEQ ID NO: 3) and the reverse primer (5'-TCAATTGGAGAGCCACAAG-3'; The complete gene was amplified by DNA synthase (Ex-Taq DNA polymerase, TAKARA) using SEQ ID NO: 4).

Example  2: Yeast Overexpression Vector Production

The complete amplified DNA gene was amplified in the above process and inserted into the yeast expression vector pYES2 TOPO TA (Invitrogen) using the T4 ligase enzyme, and transformed into E. coli . Transformed cells were selected 10 clones after 15 hours incubation at 37 ℃. After extracting the plasmid from the 10 selected clones, the DNA sequence was decoded to finally select one clone that completely matches the nucleotide sequence of the Dammarenediol synthase gene, and the plasmid prepared therefrom was used for yeast transformation. .

Example 3: Yeast Transformation and Extract Preparation

Mutant yeast strain that cannot synthesize lanosterol ( MATa erg7 ura3-1 trp1 -1 ) was distributed from F.Kars and Colmar of France and used for transformation. Transformation of yeast was carried out by the method performed by Kushiro et al. (1998, Eur J Biochem 238-244). Clones selected from uracil-free medium contained 20 mg / L of ergosterol, 13 mg / L of hemin, and 13 g / L of Tween80 for overexpression of the introduced dammarenediol synthase gene. Incubated for 2 days at 30 ℃ 220 rpm in 1000 mL of SC (synthetic complete) medium containing. After incubation, 2% of galactose was added to the same medium described above to induce gene expression for 1 day. After induction, incubated for one day in 0.1M potassium phosphate solution, yeasts were collected in one tube, and 50mL of 20% KOH / 50% KOH was reacted at 100 ° C for 30 minutes. An equal amount of hexane was added to the reaction solution, followed by extraction three times. The solutions were concentrated and used for liquid chromatography analysis.

Example  4: Dhammarendiol  Liquid chromatography analysis and LC - APCI - MS  analysis

The yeast extract described in Example 3 above was used to identify the dammarenediol compound. Using a Surveyor liquid chromatography system (Thermo Finnigan) instrument, the ratio of Capcell pak C18 column, acetonitrile and water was 90:10 (v / v), the flow rate was 0.2 ml / min, and the column temperature was adjusted to 40 ° C. Yeast extract was analyzed. As a result, there was no peak of dammareenediol in the extract of yeast containing the vector without the insertion of dammareenediol synthase gene, but the extract of yeast containing the vector into which the dammareenediol synthase gene was inserted was consistent with the standard. Peaks could be observed (FIG. 3). Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) analysis was performed to more clearly confirm that the peak was the peak of dammarenediol. Mass spectrum analysis confirmed that the gene inserted into the yeast coincided with the mass spectrum of the reported densemarendiol of another document (Dammarenediol synthase gene) (Fig. 4).

Figure 1 shows the structural formula of dammarenediol.

Figure 2 shows the biosynthesis process of dammarenediol.

Figure 3 shows the liquid chromatography (LC) analysis of the yeast extract to which the Dammarenediol synthase gene derived from Centella asiatica.

A: authentic dammarenediol-II, B: β-amirin, C: yeast extract transformed with an empty vector, D: yeast extract comprising a vector into which the dammareenediol synthase gene is inserted

Figure 4 confirms the mass spectrum of dammarenediol through LC-APCI-MS analysis.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Dammarenediol synthase gene from Centella asiatica L. Urban and          uses according <130> PN09270 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2562 <212> DNA <213> Centella asiatica <400> 1 aaagaagatt ttattttatt tttttcagca aaaaaaagtt gaaaaaataa gaaattgata 60 gatcgagcta ggggagatgt ggaagctgaa gatagcagag ggtaatggag catacttgta 120 cagcaccaac aactttgtgg ggagacagat atgggagtat gatcctgatg ctggaactcc 180 tgaagagcga ctagaggtcg agaaacttcg agaaacttac aaatataatc tcatcaacaa 240 tgggattcac ccttgtggtg atatgctcat gaggttgcag ctgataaagg agagtgggct 300 ggatcttttg agcataccgc cggtgagact tggagaacaa gaagaagtga attatcaagt 360 agtgacgacg gctgttaaga aagctctgcg gttaaaccgc gcaatccaag ctcacgacgg 420 tcactggcca gctgaaaatg ctggacctat gttttttaca ccacccctca tcatagcgct 480 atacatcagc ggagcaattg acactcatct aacaatacaa cacaagaagg agatgattcg 540 ttttatttac ctccaccaaa acaaagatgg aggatgggga ttctatatag agggacatag 600 cacgatgata gggtcagcac ttagctacgt ggcgttacgt ttgctgggag aagggcctga 660 tgacggcgat ggtgcagtgg agagagcaag aaaatggatc cttgaccatg gtggtgctgc 720 ttctataccc tcctggggta agacttatct tgcggttctt ggggtatacg agtgggaagg 780 gtgtaacccc ctgcccccag aattttggct tttccctgaa gctttacctt atcatccagc 840 aaaaatgtgg tgttactgtc gcacaacata catgccgatg tcgtatttgt atgggaagaa 900 atatcatggt ccaattacgg atcttgttat atctttaaga aaagaaatac accccattcc 960 ttatgagaag ataaattgga acaaacagcg ccataactgt aacaaggagg atctttacta 1020 ccctcatagc tttatacagg atttgctatg ggatggtctt cactatttta ctgaacctat 1080 cattaaaatg tggcccttca ataagttgcg aaagaaaggg atgaaaagag ccattgaact 1140 tatgcgctac ggaggttatg agagcagatt cattaccatt ggatgtgtat ccaagagtct 1200 agatatgatg tgttggtggg cagagaaccc gaatggtcca gaattcaaac atcacttagc 1260 tagagtacct gattacttgt ggcttgcaga ggatggaatg aagatgcaga gttttggtag 1320 tcaattatgg gactgtgttc ttgctactca agctgtcatg tctactggta tggttgatga 1380 atatggggat tgtcttaaga aagcacattt ctatattaaa gaatcacagt gcaagaaaaa 1440 tccgtcagga gattatgcaa gtatgtgccg gtattttaca aaaggatcat ggacattttc 1500 tgatcaagat cagggttggg ttgtctctga ttgcacagct gaagcgctga agtgtctatt 1560 agcactttct caaatgccag aggaaattgc aggggaaaag gcagatgttg agcgattata 1620 tgatgccgta aacgtcctcc tctacttgca aagccctata agtgggggtt ttgccatttg 1680 ggagccacca gttccaagac catacttgca ggtgttgaat ccttcggaga tttttgccga 1740 catcgttgtc gaaaaagagc atacggagtg cacagcatca ataatagcag ctctggtagc 1800 attcaaacgt ttgcatccgg gtcatcggtc gaaagaaata agtgttgcca tcgcaaaagc 1860 tgtacatttt cttgaaggaa aacaattgga agatggttca tggtatggct actggggaat 1920 atgctttttg tatggcacat tttttgcgtt agctgggtta gcttctgtgg gacagactta 1980 tgaaaacagt gaaaccgttc gtaaagctgt taagtttttc ctttctacac aaaatgaaga 2040 aggtggttgg ggagaaagtc ttgaatcatg tccgagcgag atattcacac cattagaagg 2100 aaacagaaca aatttagtac aaacatcatg ggcaatgctt ggtctcatgt ttggtggaca 2160 ggccacgaga gatccaactc cattgcatag agcagcaaag ttattgatta atgcacaatt 2220 gaataacgga gatttccctc agcaggaaac aactggagtg tacatgaaga attgtatgtt 2280 gcattatgcc gagtatagaa atgtatttcc gttatgggca cttggagagt accgcaaacg 2340 cttgtggctc tccaattgaa gtactttggt tccaaaacat atatcgatcc atttataaat 2400 gcatatatat ggggtgtatg tttaagagtc tcaagttatt gtaatcttta tatatattta 2460 ttaattaatt gtaatgtcaa agagaacatt tgaaaacatt tgtagttgct taattgatgc 2520 atatgtcatt gcaatttact tttaaaaaaa aaaaaaaaaa aa 2562 <210> 2 <211> 760 <212> PRT <213> Centella asiatica <400> 2 Met Trp Lys Leu Lys Ile Ala Glu Gly Asn Gly Ala Tyr Leu Tyr Ser   1 5 10 15 Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Tyr Asp Pro Asp Ala              20 25 30 Gly Thr Pro Glu Glu Arg Leu Glu Val Glu Lys Leu Arg Glu Thr Tyr          35 40 45 Lys Tyr Asn Leu Ile Asn Asn Gly Ile His Pro Cys Gly Asp Met Leu      50 55 60 Met Arg Leu Gln Leu Ile Lys Glu Ser Gly Leu Asp Leu Leu Ser Ile  65 70 75 80 Pro Pro Val Arg Leu Gly Glu Gln Glu Glu Val Asn Tyr Gln Val Val                  85 90 95 Thr Thr Ala Val Lys Lys Ala Leu Arg Leu Asn Arg Ala Ile Gln Ala             100 105 110 His Asp Gly His Trp Pro Ala Glu Asn Ala Gly Pro Met Phe Phe Thr         115 120 125 Pro Pro Leu Ile Ile Ala Leu Tyr Ile Ser Gly Ala Ile Asp Thr His     130 135 140 Leu Thr Ile Gln His Lys Lys Glu Met Ile Arg Phe Ile Tyr Leu His 145 150 155 160 Gln Asn Lys Asp Gly Gly Trp Gly Phe Tyr Ile Glu Gly His Ser Thr                 165 170 175 Met Ile Gly Ser Ala Leu Ser Tyr Val Ala Leu Arg Leu Leu Gly Glu             180 185 190 Gly Pro Asp Asp Gly Asp Gly Ala Val Glu Arg Ala Arg Lys Trp Ile         195 200 205 Leu Asp His Gly Gly Ala Ala Ser Ile Pro Ser Trp Gly Lys Thr Tyr     210 215 220 Leu Ala Val Leu Gly Val Tyr Glu Trp Glu Gly Cys Asn Pro Leu Pro 225 230 235 240 Pro Glu Phe Trp Leu Phe Pro Glu Ala Leu Pro Tyr His Pro Ala Lys                 245 250 255 Met Trp Cys Tyr Cys Arg Thr Thr Tyr Met Pro Met Ser Tyr Leu Tyr             260 265 270 Gly Lys Lys Tyr His Gly Pro Ile Thr Asp Leu Val Ile Ser Leu Arg         275 280 285 Lys Glu Ile His Pro Ile Pro Tyr Glu Lys Ile Asn Trp Asn Lys Gln     290 295 300 Arg His Asn Cys Asn Lys Glu Asp Leu Tyr Tyr Pro His Ser Phe Ile 305 310 315 320 Gln Asp Leu Leu Trp Asp Gly Leu His Tyr Phe Thr Glu Pro Ile Ile                 325 330 335 Lys Met Trp Pro Phe Asn Lys Leu Arg Lys Lys Gly Met Lys Arg Ala             340 345 350 Ile Glu Leu Met Arg Tyr Gly Gly Tyr Glu Ser Arg Phe Ile Thr Ile         355 360 365 Gly Cys Val Ser Lys Ser Leu Asp Met Met Cys Trp Trp Ala Glu Asn     370 375 380 Pro Asn Gly Pro Glu Phe Lys His His Leu Ala Arg Val Pro Asp Tyr 385 390 395 400 Leu Trp Leu Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln                 405 410 415 Leu Trp Asp Cys Val Leu Ala Thr Gln Ala Val Met Ser Thr Gly Met             420 425 430 Val Asp Glu Tyr Gly Asp Cys Leu Lys Lys Ala His Phe Tyr Ile Lys         435 440 445 Glu Ser Gln Cys Lys Lys Asn Pro Ser Gly Asp Tyr Ala Ser Met Cys     450 455 460 Arg Tyr Phe Thr Lys Gly Ser Trp Thr Phe Ser Asp Gln Asp Gln Gly 465 470 475 480 Trp Val Val Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys Leu Leu Ala                 485 490 495 Leu Ser Gln Met Pro Glu Glu Ile Ala Gly Glu Lys Ala Asp Val Glu             500 505 510 Arg Leu Tyr Asp Ala Val Asn Val Leu Leu Tyr Leu Gln Ser Pro Ile         515 520 525 Ser Gly Gly Phe Ala Ile Trp Glu Pro Pro Val Pro Arg Pro Tyr Leu     530 535 540 Gln Val Leu Asn Pro Ser Glu Ile Phe Ala Asp Ile Val Val Glu Lys 545 550 555 560 Glu His Thr Glu Cys Thr Ala Ser Ile Ile Ala Ala Leu Val Ala Phe                 565 570 575 Lys Arg Leu His Pro Gly His Arg Ser Lys Glu Ile Ser Val Ala Ile             580 585 590 Ala Lys Ala Val His Phe Leu Glu Gly Lys Gln Leu Glu Asp Gly Ser         595 600 605 Trp Tyr Gly Tyr Trp Gly Ile Cys Phe Leu Tyr Gly Thr Phe Phe Ala     610 615 620 Leu Ala Gly Leu Ala Ser Val Gly Gln Thr Tyr Glu Asn Ser Glu Thr 625 630 635 640 Val Arg Lys Ala Val Lys Phe Phe Leu Ser Thr Gln Asn Glu Glu Gly                 645 650 655 Gly Trp Gly Glu Ser Leu Glu Ser Cys Pro Ser Glu Ile Phe Thr Pro             660 665 670 Leu Glu Gly Asn Arg Thr Asn Leu Val Gln Thr Ser Trp Ala Met Leu         675 680 685 Gly Leu Met Phe Gly Gly Gln Ala Thr Arg Asp Pro Thr Pro Leu His     690 695 700 Arg Ala Ala Lys Leu Leu Ile Asn Ala Gln Leu Asn Asn Gly Asp Phe 705 710 715 720 Pro Gln Gln Glu Thr Thr Gly Val Tyr Met Lys Asn Cys Met Leu His                 725 730 735 Tyr Ala Glu Tyr Arg Asn Val Phe Pro Leu Trp Ala Leu Gly Glu Tyr             740 745 750 Arg Lys Arg Leu Trp Leu Ser Asn         755 760 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 atgtggaagc tgaagata 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 tcaattggag agccacaag 19  

Claims (8)

A dammarenediol synthase protein derived from a herbaceous plant, consisting of the amino acid sequence of SEQ ID NO: 2. Gene encoding the protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. The transformed host cell of claim 5, wherein the host cell is yeast or E. coli. A method of producing dammarenediol, comprising overexpressing a dammareenediol synthase gene by transforming a host cell with a recombinant vector comprising a stem-derived dammareene diol synthase gene. 8. The method of claim 7, wherein said host cell is yeast or E. coli.

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