CN106987578B - A kind of terpenoid synthase for producing koraiol and its application - Google Patents

Abstract

The invention discloses a terpenoid synthase for producing koraiol and its application, and belongs to the field of synthetic biology. By providing a terpenoid synthase FGJ09920 for synthesizing koraiol, the nucleotide sequence of which is shown in SEQ ID NO: 2, together with the mevalonate pathway-related genes, a strain for producing koraiol is constructed. Overexpression of the atoB gene or idi gene derived from Escherichia coli XL1‑blue in the p-mevalonate pathway can synthesize a large amount of the catalytic substrate farnesyl pyrophosphate FPP, which can further promote the production of koraiol. The terpenoid synthase of the invention has specificity and high efficiency, can increase the yield of koraiol, greatly overcomes the drawbacks of large input of raw materials and low yield of koraiol, reduces research cost, and ensures green environmental protection.

Description

A kind of terpenoid synthase for producing koraiol and its application

technical field

The invention belongs to the field of synthetic biology, and relates to a terpenoid synthase for producing a sesquiterpene compound-koraiol and its application.

Background technique

Terpenoids are a general term for compounds containing isoprene units. It exists widely in nature. So far, about 76,000 terpenoids have been found in animals, plants and microorganisms. The compound has many physiological activities, so it is widely used in the perfume production industry, the health care product industry, the agricultural production field and the medical industry.

The production methods of terpenoids include natural extraction, chemical synthesis and fermentation. However, due to the high production cost of natural extraction method, the possible toxic effects of chemical synthesis method and the increasing respect of natural products in the world, people have begun to study a lot of terpenoids by microbial fermentation.

Organisms use the isopentenyl pyrophosphate isomerase Idi of the 2-methyl-erythritol phosphate (MEP pathway) or mevalonate pathway (MVA pathway) to synthesize IPP and DMAPP, and then IPP and DMAPP are synthesized in isopentenyl. Transferase catalyzes the synthesis of isoprene units with different chain lengths such as GPP, FPP, GGPP and GFPP. Then terpene synthase (TS) can use these isoprene units of different chain lengths as substrates to synthesize monoterpenes (C10), sesquiterpenes (C15), diterpenes (C120), and disesquiterpenes (C25), triterpenes (C30), tetraterpenes (C40) and polyterpenes.

Among them, monoterpenes and sesquiterpenes are the main sources of perfume and fragrance compounds. The mining of biosynthetic genes related to these compounds and the metabolic engineering of terpenoid microbial synthesis high-yield platforms will enable us to efficiently and economically realize Synthesis of these high value-added products.

SUMMARY OF THE INVENTION

In order to overcome the shortcomings and deficiencies of the prior art, the present invention provides a novel terpenoid synthase FgJ09920 and a koraiol-producing strain containing the terpenoid synthase gene, so as to realize the sesquiterpene compound koraiol microbial synthesis, improve yield, reduce The cost is the main source for the preparation of lavender, citrus and other fragrance flavors, and the toxic effect is reduced.

The first aspect of the present invention provides a terpenoid synthase FgJ09920, which has the amino acid sequence shown in SEQ ID NO: 1.

Further, the present invention provides a nucleic acid molecule encoding the above-mentioned terpenoid synthase FgJ09920, and the nucleotide sequence is shown in SEQ ID NO: 2.

The second aspect of the present invention provides the use of the terpenoid synthase FgJ09920 for the production of koraiol.

The third aspect of the present invention provides a strain for producing koraiol, the strain contains genes related to mevalonate pathway (MVA pathway) and koraiol synthesis; the genes related to the mevalonate pathway include (1) The gene atoB that condenses acetyl-CoA to acetoacetyl-CoA, (2) the gene erg13 that condenses acetyl-CoA and acetoacetyl-CoA to HMG-CoA, (3) the gene thmg1 that reduces HMG-CoA to mevalonate , (4) the gene erg12 that phosphorylates mevalonate to mevalonate-5-phosphate, (5) the gene that phosphorylates mevalonate-5-phosphate to mevalonate-5-pyrophosphate erg8, (6) the gene mvd1 that decarboxylates mevalonate-5-pyrophosphate to generate isopentenyl pyrophosphate and (7) the gene idi that isomerizes isopentenyl pyrophosphate to dimethylallyl pyrophosphate; The genes related to the synthesis of koraiol include ispA and terpenoid synthase FgJ09920 gene, and the sequence of the FgJ09920 gene is shown in SEQ ID NO: 2. The synthesized target terpenoid koraiol has the structure of the following formula (I):

The genes related to the mevalonate pathway are preferably atoB gene (AM946981.2), idi (CP010152.1) derived from Escherichia coli XL1-blue, and erg13 gene (CP005477.2) derived from Saccharomyces cerevisiae INVSC1, tHMG1 (CP005464.2), erg12 (CP008027.1), erg8 (CP005426.1), mvd1 (CP005554.2).

The terpenoid synthase FgJ09920 gene is derived from FgJ09920 of the fungus Fusarium graminearum J1-012 (Fusarium graminearum J1-012), its nucleotide sequence is shown in SEQ ID NO: 2, and its amino acid sequence is SEQ ID NO: 1 .

Preferably, the strain for producing koraiol is Escherichia coli containing plasmid pMH1, plasmid pFZ81 and plasmid pGB235; the plasmid pMH1 uses pBBR1MCS as the backbone vector, the promoter is the lac promoter, and the replicon is replaced by the p15A replicon, Comprising atoB, erg13 and thmg1 genes, the sequence of pMH1 (without skeleton vector sequence) is as shown in SEQ ID NO.3; described plasmid pFZ81 uses pBBR1MCS-2 as skeleton vector, promoter as lac promoter, and replicon as plasmid The self-contained pBBR1MCS replicon includes erg12, erg8, mvd1 and idi genes, and the sequence of pFZ81 (without the backbone vector sequence) is shown in SEQ ID NO.4; the plasmid pGB235 takes pET21 as the backbone vector, and the promoter is The T7 promoter and replicon are the high-copy pBR322 replicons that come with the plasmid, including FgJ09920, ispA, and idi genes. The sequence of pGB235 (without the backbone vector sequence) is shown in SEQ ID NO.5.

Preferably, the prokaryotic expression of the koraiol-producing strain overexpresses the atoB gene or the idi gene derived from Escherichia coli XL1-blue to synthesize a large amount of the catalytic substrate farnesyl pyrophosphate FPP.

The fourth aspect of the present invention is to provide the use of the above-mentioned koraiol-producing strain in the production of koraiol.

Compared with the prior art, the present invention has the following advantages and effects: for the first time, the present invention utilizes the terpenoid synthase FgJ09920 gene to combine with the exogenous mevalonate pathway to obtain Escherichia coli that stably produces koraiol. The terpenoid synthase of the invention has specificity and high efficiency, can increase the yield of koraiol, greatly overcomes the disadvantages of large input of raw materials and low yield of koraiol, reduces research cost, and ensures green environmental protection.

Description of drawings

Figure 1 is the GC-MS detection of FgJ09920-FPP-in vitro reaction and FgJ09920 fermentation (pGB235), E.coli K1 in vitro reaction chromatogram;

Fig. 2 is a schematic diagram of the structure of plasmid pMH1;

Fig. 3 is a schematic diagram of the structure of plasmid pFZ81;

Fig. 4 is a schematic diagram of the structure of plasmid pET28a-FgJ09920;

Fig. 5 is a schematic diagram of the structure of plasmid pGB234;

Fig. 6 is a schematic diagram of the structure of plasmid pGB235;

Fig. 7 is the MS figure of bacterial strain fermentation product;

Fig. 8 is the spectrogram of compound koraiol;

a is the structural schematic diagram of koraiol; b is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101 MHz); c is the hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400 MHz).

Detailed ways

The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The examples provided are merely illustrative of the methods of the present invention, and are not intended to limit the remainder of the present disclosure in any way.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

[Example 1] Verification of the function of terpenoid synthase in vitro

1. Construction of plasmid pGB28a-FgJ09920

Using the reverse transcribed cDNA of Fusarium graminearum J1-012 as a template, the coding region FgJ09920 obtained by amplification with primers P27/P29 (see Table 1) was ligated to the plasmid pET28a to obtain the plasmid pGB28a-FgJ09920 (Figure 4). for purified protein.

2. Protein purification

The expression vector pET28a-FgJ9920 containing the gene of interest (nucleotide sequence shown in SEQ ID NO: 2) was transformed into the expression host E. coli BL21 (DE3), and after the transformation, a single clone was picked into the LB medium containing the corresponding antibiotics , 37 ℃, 220rpm overnight culture. Transfer to 1L of fresh LB medium containing the corresponding antibiotics at 1% inoculum, cultivate at 37°C, 220rpm to an OD ₆₀₀ of about 0.6-0.8, cool down to 16°C, add IPTG with a final concentration of 0.1mM, 16°C , 220rpm cultured for 16-18h. Cells were collected by centrifugation at 8 000 rpm for 5 min, and then thoroughly resuspended with 30-40 mL of protein purification buffer A (Buffer A: 50 mM Tris-HCl, 300 mM NaCl, 4 mM β-mercaptoethanol, pH 7.6), and sonicated (pulse 5 s, pause 8 s). , sonicated for 5 min). 4°C, centrifuge at 12,000g for more than 30min, collect the supernatant, centrifuge at 4°C, 20,000rpm for 1 h, collect the supernatant, filter with a 0.45μm filter, add 6% buffer B (Buffer: 500mM imidazole is added to Buffer A) to make Imidazole is about 30 mM, mix well for use.

Histidine-tagged proteins were purified using Bio-Rad's Biologic DuoFlow Chromatography System. The protein separation column was loaded onto the FPLC for control, and the flow rate of the FPLC was always 1.5 mL/min, while the flow rate of the automatic sample loading was 2 mL/min. The obtained supernatant sample was purified by Biorad with 5mL Hitrap HP Ni-NTA column. The nickel ion chelating column was first passed through 30mL (6 column volumes) buffer A (Buffer A: 50mM Tris-HCl, 300mM NaCl, 4 mM β-mercaptoethanol, pH 7.6), then 30 mL of the prepared supernatant was loaded onto the column by an autosampler, and the column was washed with 20 mL of buffer A (4 column volumes) , then start the linear gradient of buffer B (50mM Tris-HCl, 150mM NaCl, 250mM Imidazole pH 7.6), in the flow of 100mL (20 column volumes), buffer B increases from 0% to 100%, and then use The column was washed with 20 mL (4 column volumes) of 100% buffer B. Histidine-tagged target proteins were collected according to UV absorption and detected by SDS-PAGE. The relatively pure fractions were collected and concentrated to 2.5mL by centrifugation through Millipore's centrifugal concentrator Amicon Centricon-10 (the molecular weight below 10,000 will be filtered out), and then desalted and exchanged through Pharmacia's PD-10 column. into buffer C (50 mM phosphate buffer containing 10% glycerol, pH 7.6), quick-frozen in liquid nitrogen after aliquoting and stored in a -80°C refrigerator.

3. In vitro catalytic reaction

We set up the following in vitro enzymatic reaction system: 10 μM purified protein, 100 μM substrate GPP, FPP or GGPP, and ² mM Mg were added to 200 μL final concentration of 50 mM PBbuffer (pH 7.6) containing 10% glycerol ⁺ , 30℃ overnight reaction. It was then extracted twice with an equal volume of n-hexane, the organic phases were combined and the resulting product was detected by GC-MS.

The GC-MS used for the detection of terpenoids was a Thermo TRACE GC ULTRA gas chromatography equipped with a TSQQUANTUM XLS MS, and the gas chromatography column was a TRACE TR-5MS (30m×0.25mm×0.25um). 1 μL was injected for each analysis, high-purity helium was used as the carrier gas, and the flow rate was set to 1 mL/min. The GC conditions were maintained at 80°C for 1 min, then ramped to 220°C at a rate of 10°C/min, and maintained at 220°C for 15 min. The injector and transfer line temperatures were set at 230°C and 240°C, respectively.

The results showed that the terpenoid synthase expressed by FgJ09920 could use FPP as a substrate to synthesize sesquiterpene products (Figure 1).

[Example 2] Construction of expression vector

Escherichia coli XL1-blue genomic DNA and Saccharomyces cerevisiae INVSC1 genomic DNA were purified with the Blood and Cell Culture DNA Mini Kit of Qiagen Company.

Plasmid pMH1 contains the first three genes of the mevalonate pathway: atoB gene (acetoacetyl-CoA thioesterase, AM946981.2) from Escherichia coli XL1-blue, erg13 from Saccharomyces cerevisiae INVSC1 (HMG-CoAsynthase, CP005477 .2) and tHMG1 (HMG-CoA reductase, deleted transmembrane region of HMG1, CP005464.2).

Plasmid pFZ81 contains the last four genes of the mevalonate pathway: erg12 (mevalonate kinase, CP008027.1), erg8 (mevalonate-5-phosphate kinase, CP005426.1) and mvd1 from Saccharomyces cerevisiae INVSC1 (Mevalonate-5-pyrophosphate kinase, CP005554.2), derived from the idi (isoamyl pyrophosphate isomerase, CP010152.1) gene of Escherichia coli XL1-blue.

Plasmid pGB235 contains three genes for synthesizing sesquiterpene compounds, which are FgJ09920 (SEQ ID NO: 2) derived from the fungus Fusarium graminearum J1-012 (Fusarium graminearum J1-012), and its amino acid sequence is SEQ ID NO: 1 ; Idi derived from Escherichia coli XL1-blue; ispA derived from Escherichia coli XL1-blue, able to base on the mevalonate products isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) Synthesis of farnesyl pyrophosphate for the synthesis of sesquiterpenes.

All genes were amplified by PCR, and the primers used are shown in Table 1.

Table 1 Primer sequence list

The specific construction method is as follows:

①Construction of plasmid pMH1

The replicon of the pBBR1MCS plasmid was first replaced with the p15A replicon derived from the pMSD15 plasmid. The plasmid pBBR1MCS was used as a template for amplification with primers P1/P2, and the p15A replicon was amplified with primers P3/P4 (see Table 1 for primer sequences). After the PCR product was purified, the DNA concentration was measured with Nanodrop, and then 20ng of pCR was amplified The p15A fragment and equimolar pBBR1MCS fragment were mixed, and after one round of PCR amplification, the amplification conditions were: 98°C, 2min pre-denaturation, and then 30 PCR cycles of 98°C, 20s; 60°C, 20s; 72°C, 6min, The final extension was fully extended at 72°C for 10 min. Subsequently, E. coli XL1-blue was transformed to obtain plasmid pBBR1MCS/p15A.

The pMH1 plasmid backbone was amplified with primers P5/P6 and pBBR1MCS/p15A as the template, while the corresponding genes were amplified with primers P7/P8, P9/P10 and P11/P12. After the PCR product was purified, 50ng of pBBR1MCS/p15A amplification product was mixed with equimolar amplification products of each gene, and the volume was adjusted to 5 μL with deionized water, then added to 15 μL of Gibson buffer and mixed, and reacted at 50°C for 1 h. E. coli XL1-blue was transformed, clones were picked, and positive clones were sequenced to obtain plasmid pMH1 (Figure 2).

②Construction of plasmid pFZ81

The pFZ81 plasmid backbone was amplified with primers P13/P14 and pBBR1MCS-2 as the template, while the corresponding genes were amplified with primers P15/P16, P17/P18, P19/P20 and P21/P22. After the PCR product was purified, 50ng of pBBR1MCS-2 amplification product was mixed with equimolar amplification products of each gene, and the volume was adjusted to 5 μL with deionized water, then added to 15 μL of Gibson buffer and mixed, and reacted at 50°C for 1 h. After transformation of E. coli XL1-blue, clones were picked, and positive clones were sequenced to obtain plasmid pFZ81 (Figure 3).

③ Construction of plasmid pGB234

Using the reverse transcribed cDNA of Fusarium graminearum J1-012 as a template, the coding region of FgJ09920 was obtained by amplification with primers P27/P28 and ligated to plasmid pET21a to obtain plasmid pGB234 (Figure 5).

④Construction of plasmid pGB235

The ispA and idi genes were amplified from E. coli BL21 (DE3) genome with primers P23/P24 and P25/P26, respectively, and then ispA was cloned into pET21a to obtain plasmid pGB305; idi was cloned into pET21a(+) to obtain plasmid pGB306. Plasmids pGB305 and pGB306 were digested with XbaI/XhoI and SpeI/XhoI, respectively, and then the idi fragment from pGB306 was ligated to plasmid pGB305 by isocaudal enzyme to obtain plasmid pGB308. The ispA-idi fragments were subsequently cleaved from pGB308 with XbaI/XhoI and ligated into plasmid pGB234 by isocaudal enzymes to obtain plasmid pGB235 (Fig. 6).

[Example 3] In vivo synthesis of sesquiterpenoids derived from FgJ09920 in Escherichia coli

In order to produce sesquiterpenoids, two plasmids of the mevalonate pathway, pMH1 and pFZ81, were simultaneously transformed into E. coli BL21(DE3) to obtain BL21(DE3)/pMH1/pFZ81, named PS, and then pGB235 was transformed into the strain In PS, strain K1 was obtained, and then single clones were picked into 10 mL of LB medium (containing 100 μg/mL ampicillin, 50 μg/mL kanamycin and 34 μg/mL chloramphenicol at the same time), 37 ° C, 220 rpm overnight Cultivated, then inoculated into the same fresh medium at 1% inoculum at 37°C, continued to culture at 220rpm until the OD ₆₀₀ was about 0.6 to 0.8, cooled to 16°C and added IPTG with a final concentration of 0.1mM to induce expression. After expression for 18h, the temperature was raised to 28°C for fermentation for 72h, followed by fermentation and product extraction. The bacteria and fermentation broth were collected and extracted twice with an equal volume of n-hexane. After distillation under reduced pressure, methanol was reconstituted (add a small amount of DMSO before adding methanol). co-solubilization) for product purification.

The results showed that the mutant strain E. coli K1 containing FgJ09920 could synthesize the compound koraiol with a retention time of 12.06 min using FPP as a substrate (Figures 1 and 7).

[Example 4] Compound identification

The results of ¹ H NMR and ¹³ C NMR showed (Fig. 8) that the compound with a retention time of 12.06 min by GC-MS was a sesquiterpene alcohol, koraiol. ¹ H NMR (400MHz, CDCl ₃ ) δ 2.58 (q, J=9.5Hz, 1H), 2.07-1.99 (m, 1H), 1.98 (dd, J=11.5, 7.8Hz, 1H), 1.93 (ddd, J=12.9, 6.2, 1.8Hz, 1H), 1.83–1.65 (m, 3H), 1.56–1.46 (m, 3H), 1.46–1.37 (m, 2H), 1.35–1.27 (m, 1H), 1.24 ( d, J=0.9Hz, 3H), 1.20 (d, J=0.8Hz, 3H), 1.12 (s, 3H), 0.85 (s, 3H). ¹³ C NMR (101 MHz, CDCl3) δ 73.99, 53.22, 50.37, 46.27, 42.77, 42.33, 37.62, 34.01, 32.30, 31.04, 24.18, 23.40, 22.28, 22.16, 18.66. The theoretical molecular weight of the sesquiterpene alcohol koraiol is 223.2056, and the high-resolution mass spectrometry results show that its actual molecular weight is 223.2048.

SEQUENCE LISTING

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cagcgggatc gctaaccaac cctttagaaa tagccttctt ggaataactc ttgtaaactt 2400

gatcaagagc cttgacgtaa caagttaatg aaaaatgacc atcgacgtaa ggatattcgc 2460

tggtgaaatc tggcttgtaa aaatcgtagg cgtgttccat gtaagaagct cttacagagt 2520

caaatacaat tggagcatca ggaccgatcc acatagcaac agtaccggca ccaccggttg 2580

gtcttgcggc acccttatcg tagatggcaa tatcaccgca aactacaatg gcgtctctac 2640

catcccatgc gttagattca atccagttca aagagttgaa caacgcgttg gtaccaccgt 2700

aacaggcatt aagcgtgtca ataccttcga cgtcagtgtt ttcaccaaac aattgcatca 2760

agacagactt gacagacttg gacttgtcaa tcagagtttc agtaccgact tctaatctac 2820

caattttgtt ggtgtcgatg ttgtaactct tgatcaactt agacaaaaca gttagggaca 2880

tcgagtagat atcttctctg tcattgacaa aagacatgtt ggtttggccc agaccaattg 2940

tgtatttacc ttgagaaacg ccatcaaatt tctctagctc agattggttg acacattgag 3000

ttgggatgta aatttggata cctttaatac cgacattttg aggtctggtt ttttgttcag 3060

cggtcttttg tttttttagt tcagtcattt gcaagtttgt attgtgtaat tgttgttgct 3120

tttgcggcct aagtcttcct ttaataccac accaacaaag tttagttgag agtttcattt 3180

agctgtcctc cttaattcaa ccgttcaatc accatcgcaa ttccctgacc gccgccaatg 3240

cacagtgttg ccagccccag cgttttatcg cgtgcctgca tggcatgtaa tagtgtgacc 3300

agaatacgag caccactggc accgatagga tgcccgagcg cgatggcccc gccgttgaca 3360

ttcactttct cagaatcaaa gcccaggttt ttcccaacgg caaggaactg tgcagcaaat 3420

gcttcattag cctcaatgag atcaatatcc gccagttgca gccccgccag ttgtaacgct 3480

ttttgcgtgg caggtactgg ccccataccc atcaatgcgg ggggcacgcc accgctggca 3540

taacttttaa tgcgagccag gggggtaagg cctgctgcca gcgccgcaga ttcttccata 3600

atcaccagag cggcagcacc gtcgttaata ccagacgcgt tcccagcggt gactgttcct 3660

gctttatcga aggccgggcg caatgcacct aacgcttcag ccgttgaatt cgctttcggg 3720

aattcgtctt gactgaagac gaaggttttc tttcgagtga caacatttac cgggacgatt 3780

tcggctgtaa aagcaccgga ctcaattgcg gctgccgctt tacgctgtga atgtagcgcc 3840

agttcatcct gcatttcacg ggtaattccg tactctttag ccacgttttc ggcggtaatc 3900

cccatatgat aaccatgggt ggcgcacatc aggccatcgc gcaggattac gtcataaacc 3960

tgtccgtctc caagacgata accagagcgt gcttttgcat cgagtaagta gggggctaaa 4020

ctcatatttt ccataccccc cgccacaatg ctctgcgcct gacctgcctg aatggcctgg 4080

gcggcaagcg ccacactttt aagacccgaa ccacatactt tattgaccgt gaatccgcac 4140

accgtttctg ccagcccgct ttttaacagt gcctgacgcg ccggattttg ccccagcccg 4200

gcttgtaaca cgttacccat aatcacttca tcaacgtgtt gtgaatcgat ttttgcacgt 4260

tcaatggcgg ctttaattac tgtcgccccc aggtcgatgg cgctggtgga agcgagtgaa 4320

ccgttaaaac taccgatagc agtacgtacc gcactgacga tgacacaatt tttcatttta 4380

tattcctcct agtcgactct agaggatccc cgggctgcag gaattcgata tcaagcttat 4440

cgataccgtc gacctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt 4500

taattgcgcg ctggg 4515

<210> 4

<211> 4667

<212> DNA

<213> Artificial sequences

<400> 4

cgcgcgtaat acgactcact atagggcgaa ttggagctct tatttaagct gggtaaatgc 60

agataatcgt tttctggctt cgcgatttgt cgcctgcatc accatccacg gactgaacgc 120

ccacggcgtg gcatcaatac cgtgtaatac atctgctaaa tcacaccatt gataatccat 180

cacttcatca tcattgatct gtaacgcact agtggtgcgt gcggcaaata ccggacacac 240

ttcattttcc acaatgccac tcggatcggt ggcgcggtag cgaaagtcag gatagataga 300

ttcaggaggc gtaatttcca cgccaagctc ataacggcaa cggcggatca ctgcgtcttc 360

gttgctttct cccagttgtg ggtgcccaca aaccgagtta gtccacacgc caggccatgc 420

ttttttgctc agtgcgcggc gggtaactaa taattgtcct ttggcattaa acagccaact 480

ggagaacgcg agatgtaagc gggtgtctgc cgtgtgtgcg gcatactttt ccagcgtacc 540

cgtgggaact ccctgtgcat tcaataaaat gacgtgttcc gtttgcatat ggctgccgcg 600

cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatat agtaatcctc 660

ctcccgggct gcagttattc ctttggtaga ccagtctttg cgtcaatcaa agattcgttt 720

gtttcttgtg ggcctgaacc gacttgagtt aaaatcactc tggcaacatc cttttgcaac 780

tcaagatcca attcacgtgc agtaaagtta gatgattcaa attgatggtt gaaagcctca 840

agctgctcag tagtaaattt cttgtcccat ccaggaacag agccaaacaa tttatagata 900

aatgcaaaga gtttcgactc attttcagct aagtagtaca acacagcatt tggacctgca 960

tcaaacgtgt atgcaacgat tgtttctccg taaaactgat taatggtgtg gcaccaactg 1020

atgatacgct tggaagtgtc attcatgtag aatattggag ggaaagagtc caaacatgtg 1080

gcatggaaag agttggaatc catcattgtt tcctttgcaa aggtggcgaa atctttttca 1140

acaatggctt tacgcatgac ttcaaatctc tttggtacga catgttcaat tctttcttta 1200

aatagttcgg aggttgccac ggtcaattgc ataccctgag tggaactcac atccttttta 1260

atatcgctga caactaggac acaagctttc atctgaggcc agtcagagct gtctgcgatt 1320

tgtactgcca tggaatcatg accatcttca gcttttccca tttcccaggc cacgtatccg 1380

ccaaacaacg atctacaagc tgaaccagac ccctttcttg ctattctaga tatttctgaa 1440

gttgactgtg gtaattggta taacttagca attgcagaga ccaatgcagc aaagccagca 1500

gcggaggaag ctaaaccagc tgctgtagga aagttatttt cggagacaat gtggagtttc 1560

cattgagata atgtgggcaa tgaggcgtcc ttcgattcca tttcctttct taattggcgt 1620

aggtcgcgca gacaattttg agttctttca ttgtcgatgc tgtgtggttc tccatttaac 1680

cacaaagtgt cgcgttcaaa ctcaggtgca gtagccgcag aggtcaacgt tctgaggtca 1740

tcttgcgata aagtcactga tatggacgaa ttggtgggca gattcaactt cgtgtccctt 1800

ttcccccaat acttaagggt tgcgatgttg acgggtgcgg taacggatgc tgtgtaaacg 1860

gtcatgagta ttacctccta tttatcaaga taagtttccg gatctttttc tttcctaaca 1920

ccccagtcag cctgagttac atccagccat tgaaccttag aaaatctttt gtcattagcg 1980

gtttgagccc taagatcaac atcttgctta gtaatcactg caatggcgtc ataaccacca 2040

gcaccaggta ttaagcaagt aagaactcct tttaaggtct ggcaatcatc caataagcta 2100

gtttgtacgg gaggttcgat atcggcacca gattctttag ttatttttct aaaggaacgt 2160

ctaattgtgg caactgcatc tctaacttct gtgatttcag gatacttttg acaggtacag 2220

tcattcctct caagagactc aaatatctga tcgctgtaat cgtcatgagt ctcgtgtaag 2280

cgatctagtt tagatagtcc atccataaat ctagaatttg catgatcgag ttctgtatat 2340

attttcaagc tttctggcat atgcgaatca taccaatttt ttaccttctg gaccagtttt 2400

actgtttctg aaccattctt aatatcgccc atccataaag ttaatcccga aggtaaatgg 2460

ttacttttaa tcgtaatatt ccagtcttct tcatcaacca aatgcgccag tttactgccg 2520

taagtagcac ttccaatatc tggcaaatta gagattaatg cgggtgggaa tcttctatat 2580

ctgatagatc catatgctgc cgccgctaca tcaaacccgc ttccaatttt accctgagct 2640

tgacaatgag caacttgtgc taaattatga ataacttctc tatatttgtc tacattattt 2700

tccaggtccg atacaaaaaa ggaggccaaa gctgtagtta aaactgtgac taaacctgcc 2760

gaggagccca gccctgtttt gggaacttct tcaattctgt gcgaatgaaa actcaatctt 2820

ctgttgccac gatgttcggt aacgctatcc tcctgagaat ggtaggcatc atcagagaaa 2880

atatcaataa cgaacaagtt tctattgcag tagtcgtcca tgttaggttt aaagtagcta 2940

aatacgttag cgataacttt ttcaatgaaa gggttcttag atccgcctat cgaaacagga 3000

atgaagccac ttttaggact tatatggtac agccactccc catctttaaa ttgtttactt 3060

ttcacacgca cttcaaactt atcagaccct tgcaatgaac cgtaaggatg ggctacagca 3120

tgcattcttg ccgataatcc gactacaaat gcttcatatt ttgtatctaa aactaaatat 3180

ccaccagcta gtaacgcttt ccctggggca ctgaaggctc tcaactctga catttgatct 3240

gcctcctatg aagtccatgg taaattcgtg tttcctggca ataatagatc gtcaatttgt 3300

tgctttgtgg tagttttatt ttcaaataat tggaatacta gggatttgat tttaggatct 3360

ttattcaaat tttttgcgct taacaaacag cagccagtcc cacccaagtc tgtttcaaat 3420

gtctcgtaac taaaatcatc ttgcaatttc tttttgaaac tgtcaatttg ctcttgagta 3480

atgtctcttc gtaacaaagt caaagagcaa ccgccgccac cagcaccggt aagttttgtg 3540

gagccaattc tcaaatcatc gctcagattt ttaataagtt ctaatccagg atgagaaaca 3600

ccgattgaga caagcagtcc atgatttatt cttatcaatt ccaatagttg ttcatacagt 3660

tcattattag tttctacggc ctcgtcatcg gtgcctttac atttacttaa ctttgtcatg 3720

atctctaagc cttgtagggc acattcaccc atggcatcta gaattggctt cataacttca 3780

ggaaatttct cggtgaccaa cacacgaacg cgagcaacaa gatcttttgt agaccttgga 3840

attctagtat aggttaggat cattggaatg gctgggaaat catctaagaa cttaaaattg 3900

tttgtattta ttgttccatt atgtgagtct ttttcaaata gcagggcatt accataagtg 3960

gccacagcgt tatctattcc tgaaggggta ccgtgaatac acttttcacc tatgaaggcc 4020

cattgattca ctatatgctt atcgttttct gacagctttt ccaagtcatt agatcctatt 4080

aaccccccca agtaggccat agctaaggcc agtgatacag aaatagaggc gcttgagccc 4140

aacccagcac cgatgggtaa agtagacttt aaagaaaact taatattctt ggcatggggg 4200

cataggcaaa caaacatata caggaaacaa aacgctgcat ggtagtggaa ggattcggat 4260

agttgagcta acaacggatc caaaagacta acgagttcct gagacaagcc atcggtggct 4320

tgttgagcct tggccaattt ttgggagttt acttgatcct cggtgatggc attgaaatca 4380

ttgatggacc acttatgatt aaagctaatg tccgggaagt ccaattcaat agtatctggt 4440

gcagatgact cgcttattag caggtaggtt ctcaacgcag acacactagc agcgacggca 4500

ggcttgttgt acacagcaga gtgttcacca aaaataataa cctttcccgg tgcagaagtt 4560

aagaacggta atgacatggt taattcctcc tactgcagga attcgatatc aagcttatcg 4620

ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccc 4667

<210> 5

<211> 3052

<212> DNA

<213> Artificial sequences

<400> 5

caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc 60

ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg 120

gtggtggtgg tgctcgagtg cggccgcaag cttgtcgacg gagctcgaat tcttatttaa 180

gctgggtaaa tgcagataat cgttttctgg cttcgcgatt tgtcgcctgc atcaccatcc 240

acggactgaa cgcccacggc gtggcatcaa taccgtgtaa tacatctgct aaatcacacc 300

attgataatc catcacttca tcatcattga tctgtaacgc actagtggtg cgtgcggcaa 360

ataccggaca cacttcattt tccacaatgc cactcggatc ggtggcgcgg tagcgaaagt 420

caggatagat agattcagga ggcgtaattt ccacgccaag ctcataacgg caacggcgga 480

tcactgcgtc ttcgttgctt tctcccagtt gtgggtgccc acaaaccgag ttagtccaca 540

cgccaggcca tgctttttttg ctcagtgcgc ggcgggtaac taataattgt cctttggcat 600

taaacagcca actggagaac gcgagatgta agcgggtgtc tgccgtgtgt gcggcatact 660

tttccagcgt acccgtggga actccctgtg cattcaataa aatgacgtgt tccgtttgca 720

tatggctgcc gcgcggcacc aggccgctgc tgtgatgatg atgatgatgg ctgctgccca 780

tggtatatct ccttcttaaa gttaaacaaa attatttcta gttatttatt acgctggatg 840

atgtagtccg ctagcgcttc cagtgccgag gtatcgagtg actgttcagc cagttgtttc 900

agcgactgac gggcatcgtc gatcagatcc cgggctttct tccgggcttg ctcaagaccc 960

agaagtgcag ggtaggtact ttaccaagt tgctggtcgg caccctggcg ttttcccaac 1020

gttgcagtat ctcccaccac atccaggatg tcatcctgaa cctggaaggc aaggccgatg 1080

ctctctgcat acttgtcgag taccggcaga gcacgacgtc ctttatctcc ggcgcttaat 1140

gcaccaaggc gaacggcggc gcgaatcaat gcgccggttt tatgacgatg aatacgctca 1200

agcgcgtcca gaggtacgtg tttgccttcc gcgtctaaat ctaatgcctg accaccgcac 1260

attccggcaa taccactggc gctcgccagt tcagaaatca tcgaaattct gtcgcggtcc 1320

gacacttccg gcatatcggc atcgcttaaa atcgagaacg ccagcgtttg taaagcgtcg 1380

ccagcgagaa tcgcgtttgc ttcgccaaac ttcacatggc aggttggcaa accgcgacgc 1440

agatcgtcat catccattgc cggtaaatca tcatgaatta atgagtaagc gtggatacac 1500

tcaacggcgg cagcgggtgc gtccagcgtg tttgtgctaa cgccgaacat atgaccggtg 1560

gcataaacca ggaaaggtcg caggcgctta ccacctaata atgcgccata ctgcatggtt 1620

tcgaccacgg gagtgttctg aaagggcagt ggggcgataa aacggctcag cgcctggttg 1680

gcctgcttaa cgcaggcttc gagttgctgc ggaaagtcca tggatccgcg acccatttgc 1740

tgtccaccag tcatgctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga 1800

tgatgatgat ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc 1860

tagttacggc agacgcatca tacgggtttt cttcacctgc gggccttcgt cacccagata 1920

acggaaggtg tacaggctcc agtgcaggtt gcccagcgcg atgttacggc aaccgttcac 1980

aaatttaata acatcacggt ccacgccctc gccccagaac ggcaggttcg ccatcgcggt 2040

gtgccaacga cgatagcaat cgtacagcat ttcgccgatc tcgtccgccg cttcttgatc 2100

ggtcataccc tggtcacgca gaataaacat gatgttgctg tcctcgcctt gaatcagatc 2160

cttacgatag ctgcacaggt cgttctgcag cgtcaccaga tcgcaggtaa tacggctgat 2220

cgcttccagg ctcgggtgat ccataacgtc ttgcggcagg tcgatgcctt ccgcgaactc 2280

catcagacca atgcacggat acgcacccac gcaacccgca cgcatatcca tgtattcctc 2340

aacgctcaga cggctcgcgg tgttctgaac gcccacttgc tgcagaacac cgatgcaata 2400

catggtcagg tgtttcttcc aacggtattg cagcgccggg ctcgcacgct gacgaaaacg 2460

gttccagcag ctttgcaggg tgtgacgcag cgggttctgg tccgggctaa ccaccgggtg 2520

aatgttatcc agggtcgcca gggtgtagat cacttcctcc gccgctttaa tcgggttgtt 2580

cgcaaagctg ccttcgtcga acggatcgtc aaacgcgaaa acccaaccgt tccagtcgtt 2640

catcagcttc agggtctcca gatccgcgtt cggcgcgcag atcgcgctca tatacgcaat 2700

gtccagacgg ctccagatgc tcgcggtttt atcgttcata cgcagcgcat ccttcagcca 2760

cgcgtcgctc agcgctttgc tggtcttgta catcgggtta acgtccggtt ccaccgccat 2820

gatgctgctg aacagcgcct ccggcagacg aatccaacga ccatccgcca gctccggaat 2880

atccacgtgg tcgctgcaaa tgctgctcgg gctgatcggg ccacccgcgc agctcggcgg 2940

cggggtaatg atgctcggaa ccatatgtat atctccttct taaagttaaa caaaattatt 3000

tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat ta 3052

<210> 6

<211> 52

<212> DNA

<213> Artificial sequences

<400> 6

catcttatta atcagataaa atatttctcg agctccggca aaaagtggcc cc 52

<210> 7

<211> 50

<212> DNA

<213> Artificial sequences

<400> 7

catcttccag gaaatctccg ccccgctcga gaaacccacg gcggcaatgc 50

<210> 8

<211> 50

<212> DNA

<213> Artificial sequences

<400> 8

gcattgccgc cgtgggtttc tcgagcgggg cggagatttc ctggaagatg 50

<210> 9

<211> 52

<212> DNA

<213> Artificial sequences

<400> 9

ggggccactt tttgccggag ctcgagaaat attttatctg attaataaga tg 52

<210> 10

<211> 55

<212> DNA

<213> Artificial sequences

<400> 10

tttgaaagat gggtccgtca cctgcattaa atcctaagga tccactagtt ctaga 55

<210> 11

<211> 59

<212> DNA

<213> Artificial sequences

<400> 11

ttttatattc ctcctagtcg actctagagg atccccgggc tgcaggaatt cgatatcaa 59

<210> 12

<211> 59

<212> DNA

<213> Artificial sequences

<400> 12

cccggggatc ctctagagtc gactaggagg aatataaaat gaaaaattgt gtcatcgtc 59

<210> 13

<211> 49

<212> DNA

<213> Artificial sequences

<400> 13

gttgagagtt tcatttagct gtcctcctta attcaaccgt tcaatcacc 49

<210> 14

<211> 46

<212> DNA

<213> Artificial sequences

<400> 14

acggttgaat taaggaggac agctaaatga aactctcaac taaact 46

<210> 15

<211> 49

<212> DNA

<213> Artificial sequences

<400> 15

tggctgctgc ccatagtgta atcctcctta ttttttaaca tcgtaagat 49

<210> 16

<211> 47

<212> DNA

<213> Artificial sequences

<400> 16

atgttaaaaa ataaggagga ttacactatg ggcagcagcc atcatca 47

<210> 17

<211> 23

<212> DNA

<213> Artificial sequences

<400> 17

ttaggattta atgcaggtga cgg 23

<210> 18

<211> 59

<212> DNA

<213> Artificial sequences

<400> 18

cagaaaacga ttatctgcat ttacccagct taaataagag ctccaattcg ccctatagt 59

<210> 19

<211> 55

<212> DNA

<213> Artificial sequences

<400> 19

ttaagaacgg taatgacatg gttaattcct cctactgcag gaattcgata tcaag 55

<210> 20

<211> 42

<212> DNA

<213> Artificial sequences

<400> 20

ctgcagtagg aggaattaac catgtcatta ccgttcttaa ct 42

<210> 21

<211> 44

<212> DNA

<213> Artificial sequences

<400> 21

ctcaactctg acatttgatc tgcctcctat gaagtccatg gtaa 44

<210> 22

<211> 51

<212> DNA

<213> Artificial sequences

<400> 22

ttaccatgga cttcatagga ggcagatcaa atgtcagagt tgagagcctt c 51

<210> 23

<211> 53

<212> DNA

<213> Artificial sequences

<400> 23

gatgctgtgt aaacggtcat gagtattacc tcctatttat caagataagt ttc 53

<210> 24

<211> 50

<212> DNA

<213> Artificial sequences

<400> 24

atcttgataa ataggaggta atactcatga ccgtttacac agcatccgtt 50

<210> 25

<211> 55

<212> DNA

<213> Artificial sequences

<400> 25

tgcccatata gtaatcctcc tcccgggctg cagttattcc tttggtagac cagtc 55

<210> 26

<211> 51

<212> DNA

<213> Artificial sequences

<400> 26

ggaataactg cagcccggga ggaggattac tatatgggca gcagccatca t 51

<210> 27

<211> 24

<212> DNA

<213> Artificial sequences

<400> 27

ttatttaagc tgggtaaatg caga 24

<210> 28

<211> 31

<212> DNA

<213> Artificial sequences

<400> 28

atatggatcc atggactttc cgcagcaact c 31

<210> 29

<211> 38

<212> DNA

<213> Artificial sequences

<400> 29

atatgaattc actagtttat ttattacgct ggatgatg 38

<210> 30

<211> 34

<212> DNA

<213> Artificial sequences

<400> 30

atcgcatatg caaacggaac acgtcatttt attg 34

<210> 31

<211> 36

<212> DNA

<213> Artificial sequences

<400> 31

atatctcgag actagttatt taagctgggt aaatgc 36

<210> 32

<211> 27

<212> DNA

<213> Artificial sequences

<400> 32

atatcatatg gttccgagca tcattac 27

<210> 33

<211> 30

<212> DNA

<213> Artificial sequences

<400> 33

atatgaattc ttacggcaga cgcatcatac 30

<210> 34

<211> 35

<212> DNA

<213> Artificial sequences

<400> 34

atatctcgag actagttacg gcagacgcat catac 35

Claims (5)

1. Use of a terpenoid synthase FgJ09920, the terpenoid synthase has the amino acid sequence shown in SEQ ID NO: 1, and is characterized in that, it is used for the production of koraiol. 2. a bacterial strain for producing koraiol, is characterized in that, this bacterial strain contains the related gene of mevalonate approach and koraiol synthesis; The related gene of described mevalonate approach comprises atoB , erg13, thmg1, erg12, erg8, mvd1 and idi genes; the genes related to koraiol synthesis include ispA and terpenoid synthase FgJ09920 gene, the sequence of which is shown in SEQ ID NO:2. 3. the bacterial strain of producing koraiol according to claim 2, is characterized in that, the bacterial strain of producing koraiol is the Escherichia coli containing pMH1, plasmid pFZ81 and plasmid pGB235; The plasmid pMH1 uses pBBR1MCS as the backbone vector, the promoter is the lac promoter, the replicon is p15A, and contains atoB, erg13 and thmg1 genes; The plasmid pFZ81 uses pBBR1MCS-2 as the backbone vector, the promoter is the lac promoter, and the replicon is the pBBR1Rep replicon, including erg12, erg8, mvd1 and idi genes; The plasmid pGB235 uses pET21 as the backbone vector, the promoter is the T7 promoter, and the replicon is the pBR322 high-copy replicon, and contains FgJ09920, ispA and idi genes. 4. The strain for producing koraiol according to claim 2 or 3, wherein the strain overexpresses the atoB gene or the idi gene derived from Escherichia coli XL1-blue to synthesize a large amount of the catalytic substrate farnesyl pyrophosphate FPP. 5. The application of the strain for producing koraiol according to claim 4 in the production of koraiol.

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