CN113402357B

CN113402357B - 5-12-5 Tricyclic diterpene skeleton compound and preparation thereof

Info

Publication number: CN113402357B
Application number: CN202110710008.7A
Authority: CN
Inventors: 刘雪婷; 蒋岚; 吕康杰; 张立新; 张雪; 朱国良; 王芷馨; 杨欢婷; 徐�明
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2024-09-17
Anticipated expiration: 2041-06-25
Also published as: CN113402357A

Abstract

The invention provides a 5-12-5 tricyclic diterpene skeleton compound and a preparation method thereof, wherein the structural formula of Schultriene is shown in the specification, the amino acid sequence of synthetase CcCS is shown in SEQ ID NO.3, the synthetic gene is cloned from a CS12565 strain (Cytospora schulzeri 12565) genome, and the polynucleotide sequence is shown in SEQ ID NO. 1. CcCS protein has the functions of catalyzing chain length extension and structural cyclization of a substrate, and can assist the synthesis of Schultriene compound parent nucleus. The CcCS gene found by the invention catalyzes and generates a novel sesterterpene skeleton compound, thereby providing valuable lead compound resources for enriching a natural product compound library and discovering novel antibiotics.

Description

5-12-5 Tricyclic diterpene skeleton compound and preparation thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a 5-12-5 tricyclic diterpene skeleton compound Schultriene synthetic gene CcCS cloned from Cytospora schulzeri12565,12565 and application thereof.

Background

Terpenes are a collective term for all isoprene polymers and derivatives thereof. As the most abundant class of small molecule natural products, terpenoids play an important role ^[1] in the medical, food and cosmetic industries. The sesterterpene is a terpenoid synthesized by catalyzing GFPP as a precursor, and only occupies a very small part (less than 2%) of the terpenoid, and the sesterterpene generally has a complex cyclization structure and has higher patent medicine potential ^[2]. Such as: ophiobolinA isolated from the Bipolaris oryzae has, in addition to cytotoxic and antimalarial activity, calmodulin and antimalarial activity ^[3-5]. Whereas the sesterterpenes synthesized catalytically by the bifunctional terpene synthases (BFTSs) are often favored by their novel catalytic mechanisms.

The traditional chemical synthesis of the sesterterpene is faced with the problems of complex synthesis steps, poor catalytic specificity, limited acquisition capacity of a new skeleton and the like, and the current technical situation of low yield and high cost makes the sesterterpene compound of the novel skeleton difficult to directly acquire from the nature. The rapid development of genomic technology and bioinformatics has led to the awareness that microorganisms also have considerable terpenoid synthesis potential. By combining the whole genome analysis of the microorganism with a genome mining strategy, a plurality of bifunctional terpene synthases with brand-new catalytic mechanisms can be mined from the microorganism, and a large number of novel skeleton terpene products can be obtained by combining a high-efficiency terpene biosynthesis gene cluster heterologous expression system, so that the method becomes an important method for discovering novel drug lead compounds.

Cytospora schulzeri belonging to Ascomycota (Ascomycota), phytosporaceae (Sordariomycetes), ascomycetes (Diaporthales), and Ascomycetes (Cytospora) of the family Hei Humicola (VALSACEAE) can cause apple tree rot ^[6]. No isolation of the sesquiterpene-active natural product from Cytospora schulzeri has been reported to date. The mining Cytospora schulzeri of the secondary metabolic biosynthesis gene cluster helps to enrich the natural product compound library and provides valuable lead compounds for new drug discovery.

Disclosure of Invention

The invention provides 5-12-5 tricyclic diterpene skeleton compounds, synthetases thereof, genes encoding the synthetases, and heterologous expression methods of the compounds.

The idea of the invention is as follows: in the biosynthesis process of the metabolite sesterterpenoid of the excavating strain C.schulzeri12565, the CcCS gene is related to the terpene synthesis through the functional analysis of the gene, which indicates that the CcCS gene participates in the biosynthesis of the 5-12-5 tricyclic sesterterpenoid skeleton compound. Further, the CcCS protein is obtained through heterologous expression CcCS gene to perform in vitro enzymatic reaction, and the CcCS gene is verified to be a synthetic gene of 5-12-5 tricyclic diterpene skeleton compound Schultriene.

The primary object of the present invention is to provide a structure of 5-12-5 tricyclic diterpene skeleton compound; a second object is to provide an enzyme and a gene for synthesizing the compound; a third object is to provide a method for the heterologous expression of 5-12-5 tricyclic diterpene skeleton compounds.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect of the present invention, there is provided a 5-12-5 tricyclic diterpene backbone compound Schultriene having the formula C ₂₅H₄₀, the structural formula is as follows:

In a second aspect of the present invention, there is provided a synthetase CcCS of the above 5-12-5 tricyclic diterpene skeleton compound, wherein the amino acid sequence of the synthetase is shown in SEQ ID NO.3, and the N-terminal and the C-terminal of the synthetase are responsible for terpene cyclization and prenyl transfer functions, respectively.

Further, the synthase contains two conserved domains: the terpene cyclase domain contains two characteristic conserved motifs DDACE and NDYWSWPRE that recognize Mg ²⁺ and substrates, and the E-IPPS domain also contains two characteristic conserved motifs DDIED and DDYMN that have similar functions.

In a second aspect of the present invention, there is provided a gene encoding the above-described synthetase, cloned from the genome of the CS12565 strain (C.schulzeri 12565), the polynucleotide sequence of which is shown in SEQ ID NO. 1. The CS12565 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.21944. The gene contains 3 introns, the cDNA size is 2280bp, and the sequence is shown as SEQ ID NO. 2.

In a third aspect of the invention, there is provided a recombinant expression vector of a 5-12-5 tricyclic diterpene skeleton compound, which is a eukaryotic or prokaryotic expression vector carrying the above-mentioned synthetases or genes, such as E.coli, yeast systems and filamentous fungi.

In a fourth aspect of the invention, a recombinant expression host cell of a 5-12-5 tricyclic diterpene skeleton compound is provided, which contains the recombinant expression vector as described above, and realizes heterologous expression of the compound.

In a fifth aspect of the invention, there is provided the use of a synthetase or gene as described above in the synthesis of terpenoids, in particular in the synthesis of 5-12-5 tricyclic sesterterpene framework compounds.

In a sixth aspect, the present invention provides a method for the heterologous expression of a 5-12-5 tricyclic diterpene skeleton compound, comprising the steps of:

A. construction of CcCS Gene heterologous expression vector

Amplifying by using a C.schulzeri12565 genome as a template through a PCR technology to obtain a gene sequence containing CcCS, wherein the primer sequences used for amplification are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

primer sequences used for amplification:

CcCS-F：cgGAATTCGAGCTCGATGCTTGAAGCAAACGAGCT；

CcCS-R：tactacaGATCCCCGGTCAAACTCGCAAGGTTTCCACCAG。

Connecting the amplified fragment with pUARA carrier by homologous recombination to construct pUARA-CcCS expression plasmid, transferring the connection product into colibacillus DH10B, screening positive transformant, culturing, extracting plasmid PCR to verify and obtain pUARA-CcCS plasmid,

B. protoplast transformation

Culturing Aspergillus oryzae Aspergillus oryzae NSAR1, collecting protoplast, mixing with pUARA-CcCS plasmid, culturing, performing PCR verification on the grown transformant, wherein the positive transformant is CcCS heterologous expression strain AO-CcCS,

C. Culture of heterologous expression strain AO-CcCS and product isolation

Inoculating heterologous expression strain AO-CcCS, screening with pUARA plasmid screening liquid culture medium, fermenting, separating and purifying the obtained crude fermentation extract by forward silica gel column chromatography, TLC rapidly detecting each fraction, mixing the same fractions, concentrating under reduced pressure, rotary evaporating to dryness, transferring into a weighed sample bottle, weighing sample, and recording weight.

The beneficial effects of the invention are as follows:

The invention discovers that the CcCS gene for synthesizing the 5-12-5 tricyclic diterpene skeleton compound Schultriene, and the coded CcCS protein can assist the synthesis of Schultriene compound parent nucleus. The invention provides a new resource for the biosynthesis of 5-12-5 tricyclic diterpene compounds and provides a choice for the synthesis of the compounds.

The CcCS gene found by the invention catalyzes and generates a novel sesterterpene skeleton compound, thereby providing valuable lead compound resources for enriching a natural product compound library and discovering novel antibiotics.

Drawings

FIG. 1 is a chart showing the HR-EI-MS spectrum of compound Schultriene of the present invention.

FIG. 2 is a ¹ H-NMR spectrum of compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 3 is a ¹³ C-NMR spectrum of compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 4 is a ¹³ C-DEPT 135 spectrum of a compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 5 is a ¹H-¹ H COSY spectrum of a compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 6 is a spectrum of HSQC of compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 7 is a chart showing the HMBC pattern of compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 8 is a NOESY spectrum of a compound Schultriene of the present invention dissolved in Benzene-d ₆.

FIG. 9 is a diagram showing the amino acid sequence alignment of the CsSS gene encoded protein of Cytospora schulzeri 12565,12565 with the reported protein.

Strain preservation information: CS12565 (Cytospora schulzeri 12565) is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), with preservation address of North Star Xili No. 1,3, and preservation date of 2021, 04 month 02, and preservation number of CGMCC No.21944.

Detailed Description

The present invention will be described in detail with reference to the drawings and examples thereof, which are provided on the premise of the technical solution of the present invention, and the detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The synthetic gene of the 5-12-5 tricyclic diterpene skeleton compound Schultriene is cloned from Cytospora schulzeri 12565 and named as CcCS gene, and the gene sequence is shown in SEQ ID NO. 1. The CcCS gene contains 3 introns, the cDNA size is 2280bp, and the sequence is shown as SEQ ID NO. 2. The protein coded by CcCS gene is named CcCS protein, and its amino acid sequence is shown in SEQ ID NO. 3.

CcCS protein belongs to chimeric terpene synthases, and the N end and the C end of the CcCS protein are respectively responsible for terpene cyclization and isopentenyl transfer functions. CcCS the protein contains two conserved domains, of which the terpene cyclase domain contains two conserved motifs DDACE and NDYWSWPRE characteristic of Mg ²⁺ and the substrate, and the E-IPPS domain also contains two conserved motifs DDIED and DDYMN characteristic of similar function (fig. 9).

Example 1: heterologous expression and structural identification of 5-12-5 tricyclic diterpene skeleton compound Schultriene synthetic gene

By utilizing a heterologous expression method, ccCS genes in Cytospora schulzeri 12565 thalli are transferred into a host Aspergillus oryzae by constructing an expression plasmid, and the production condition of a heterologous expression strain product is detected. The medium formulations used are shown in Table 1.

Table 1 Medium formulations used in the examples

Construction of CcCS Gene-derived expression vector

(1) The gene sequence containing CcCS was amplified by PCR technique using the C.schulzeri12565 genome as template. Primer sequences used for amplification:

CcCS-F：cgGAATTCGAGCTCGATGCTTGAAGCAAACGAGCT(SEQ ID NO.4)；

CcCS-R：tactacaGATCCCCGGTCAAACTCGCAAGGTTTCCACCAG(SEQ ID NO.5)。

(2) The amplified fragment is connected with pUARA carrier through homologous recombination to construct pUARA2-CcCS expression plasmid, and both sides of the carrier CcCS have homologous sequences consistent with pUARA carrier.

(3) The ligation product was transformed into E.coli DH10B and positive transformants were selected by ampicillin. Positive transformants were liquid cultured, and plasmid PCR was extracted for verification to obtain pUARA-CcCS plasmids.

2. Transformation of protoplasts

(1) Aspergillus oryzae Aspergillus oryzae NSAR1 was spread on PDA plates and incubated at 30deg.C for 7d.

(2) Spores were collected in 10mL of 0.1% Tween-80 (typically requiring collection of 1 plate of the arms) and counted using a hemocytometer. About 10 ⁷ spores were inoculated in 50mL of DPY and cultured at 30℃and 220rpm for 2-3d.

(3) 100Mg Yatalase was weighed, dissolved in solution 0, and 20ml was filter sterilized with a 0.22 μm filter and added to a 50ml centrifuge tube.

(4) And collecting the bacterial cells. Pouring 100ml of cultured mycelium into a P250 glass filter, removing the culture medium, washing with sterilized water (or 0.8M NaCl) for 3-5 times, squeezing out water with a sterilizing medicine spoon, and adding the squeezed mycelium into Yatalase solutions. Shake culturing at 30deg.C and 200rpm for 1-2 hr until the spherical mycelium disappears to make clear the dirt.

(5) The digested bacterial liquid was filtered through a Miracloth filter cloth, and protoplasts were collected and transferred to a new 50ml centrifuge tube and centrifuged at 4℃for 800g and 5 min.

(6) The supernatant was removed, washed by adding 20ml,0.8M NaCl, resuspended and centrifuged (washed twice) at 4℃under 800g for 5 min. The supernatant was removed and 10ml of 0.8M NaCl was added. The number of protoplasts was counted under a microscope with a bacterial counter. Number of protoplasts = total count/80 x 400ml x 10 ⁴ x dilution.

(7) The protoplast concentration was adjusted to 2X 10 ⁸ cells/ml. (sol 2/sol 3=4/1), and depending on the growth of the cells, 0.5ml to 2ml of protoplasts can be harvested.

(8) 200. Mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, 10. Mu.g of expression plasmid pUARA-CcCS was added and gently mixed. Standing on ice for 20min. The sterilized Top agar was incubated in a water bath at 50 ℃.

(9) 1Ml of sol 3 was added to the suspension of (8), and the mixture was gently mixed with a gun head. Standing at room temperature for 20min. 10ml of sol 2 was added and gently mixed.

(10) Centrifugation at 4 ℃,800g,10min, removal of supernatant, addition of 1ml sol 2, gentle suspension with a pipette, addition of 200 μl to pUARA plasmid screening of solid medium at the center (x 3 plate). 5ml of top agar incubated at 50℃was rapidly added around the dish and mixed rapidly. After the surface of the plate was sufficiently dried, it was wrapped with Parafilm, covered downward, and incubated at 30℃for 3-7 days.

(11) 2-3 Clones were picked per plate, 8 total. And carrying out PCR verification on the grown transformant, wherein the positive transformant is CcCS heterologous expression strain AO-CcCS.

3. Detection of expression product of heterologous expression strain AO-CcCS

(1) The heterologous expression strain AO-CcCS was inoculated on pUARA plasmid-screening liquid medium and cultured at 30℃for 3d.

(2) Centrifuging at 8000rpm for 10min to obtain fermentation thallus, adding 100ml of 80% acetone with equal volume, ultrasonic crushing for 20min, centrifuging at 8000rpm for 10min, and collecting supernatant.

(3) Extracted 1 time with 2 volumes of ethyl acetate, spin-dried with a rotary evaporator and then dissolved with 15mL of methanol (chromatographic grade).

(4) Taking 1mL of methanol solution, filtering the methanol solution by a 0.22 mu m filter membrane, and placing the methanol solution in a chromatographic bottle to obtain GC-MS and LC-MS samples.

(5) The samples were subjected to GC-MS detection: the initial temperature was raised to 310℃at a rate of 15℃per minute and then to 310℃at a rate of 5℃per minute using an Agilent-HP-5MS column, and maintained for 13 minutes. The GC-MS method parameters are as follows: sample module: the needle is washed 5 times before and after sample injection, the needle is washed 2 times for the sample, the viscosity compensation time is 0.2s, and the sample injection mode is normal. GC module: the column temperature was 50deg.C, the injection temperature was 270 deg.C, the injection mode was no split (splitness), the carrier gas was helium, the flow rate control mode was linear, the total flow rate was 10mL/min, and the column temperature control procedure was as shown in Table 3.5.MS module: the MS ion source temperature is 230 ℃, the interface temperature is 270 ℃, the solvent excision time is 2.5min, the acquisition time is 3min-60min, the acquisition mode is full scanning, EVENT TIME is set to be 0.3s, the scanning speed is 2000, and the scanning nuclear mass ratio is 40-600Da.

(7) LC-MS detection of samples: using Cholester chromatographic column, mobile phase A phase-0.1% formic acid water, B phase-acetonitrile, flow rate 1mL/min, mobile phase acetonitrile ratio in 30min rising from 5% to 100%, then maintaining for 6min, then mobile phase acetonitrile ratio in 10s falling to 5%, then maintaining for 4min 50s.

4. Separation, purification and identification of heterologous expression recombinant strain AO-CcCS sesterterpene skeleton product

AO-CcCS was co-fermented in 10L to give about 2g of crude extract. And separating and purifying the obtained fermentation crude extract by adopting a forward silica gel column chromatography method. And (3) loading the mixture on a column by adopting a dry method, performing isocratic elution by petroleum ether, collecting one tube per 10mL of effluent liquid, collecting 18 fractions, rapidly detecting each fraction by TLC, combining the same fractions of spots, concentrating under reduced pressure, steaming to dryness in a rotary manner, transferring the dried mixture into a weighed sample bottle, weighing the sample, and recording the weight. HPLC analysis of each fraction component accurately locates the target fraction. Optimizing preparation conditions, and preparing a target compound by adopting Cholester semi-preparative chromatographic columns, wherein the mobile phase is as follows: phase A-0.1% formic acid water; and B phase-acetonitrile, wherein the flow rate is 4mL/min, the isocratic of 95% acetonitrile formic acid is 10 mu L, the initial sample injection is gradually increased to 80 mu L on the basis of ensuring that the peak is unchanged, the peak of the target sesquiterpene compound appears about 20min, and the outflow solution is connected into a conical flask when the peak appears. Purity checking was performed on the prepared compound TLC and HPLC.

NMR testing of the isolated sesterterpene framework compounds employed Bruker 600MHz (¹H 600MHz;¹³ C150 MHz). The solvent of the sesterterpene skeleton compound is Benzene-d ₆, the resolution of an NMR spectrum instrument is 600MHz, the determination of ¹ H NMR and ¹³ C NMR is firstly carried out, the determination is compared with the data in a database, and if the structure is new, the specific structure is determined by complementing HSQC, COSY, HMBC spectrogram resolution.

5. Identifying the sesterterpene framework compound Schultriene.

Identifying the diterpene skeleton compound Schultriene obtained above:

(1) Appearance: is transparent and greasy.

(2) Solubility: is easily dissolved in methanol and is difficult to dissolve in water.

(3) Nuclear magnetic resonance spectroscopy: FIG. 1 is a ¹ H-NMR spectrum of compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 2 is a ¹³ C-NMR spectrum of compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 3 is a ¹³ C-DEPT 135 spectrum of a compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 4 is a ¹H-¹ H COSY spectrum of a compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 5 is a spectrum of HSQC of compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 6 is a chart showing the HMBC pattern of compound Schultriene of the present invention dissolved in Benzene-d ₆. FIG. 7 is a NOESY spectrum of compound Schultriene of the present invention dissolved in Benzene-d ₆. The nuclear magnetic resonance spectrum of the compound Schultriene of the present invention was studied and the signals of the 1D and 2D spectra were assigned as shown in table 2. And the final determined structure is as follows:

Table 2 assignment of peaks for 1D and 2D spectra of compound fusaoxyspene A

References referred to in the background of the invention are as follows:

[1]Guan,Z.et al.Metabolic engineering of Bacillus subtilis for terpenoid production.Applied Microbiology andbiotechnology 99,9395-9406(2015).

[2]Chang,M.C.&Keasling,J.D.Production of isoprenoid pharmaceuticals by engineered microbes.Nature chemical biology 2,674-681(2006).

[3]Leung,P.C.,Taylor,W.A.,Wang,J.H.&Tipton,C.L.Role of calmodulin inhibition in the mode ofaction ofophiobolinA.Plant physiology 77,303-308(1985).

[4]Nozoe,S.et al.The structure of ophiobolin,a C25 terpenoid having a novel skeleton.Journal ofthe American Chemical Society 87,4968-4970(1965).

[5]Peters,C.&Mayer,A.Ca 2+/calmodulin signals the completion ofdocking and triggers a late step ofvacuole fusion.Nature 396,575-580(1998).

[6]Jiang,N.,Yang,Q.,Fan,X.-L.&Tian,C.-M.Identification of six Cytospora species on Chinese chestnut in China.MycoKeys 62,1(2020).

While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Sequence listing

<110> University of Industy of Huadong

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tattggagtt cgcagtgtta ccgccacaat gactgggagc acatcccaga tcttcctggc 1020

gaggacgcac caaaacttca cgaacttgcc accctaggtc gacgattatt gctggacgaa 1080

gatctaccgc ccggggcttc gacctatgga caggatacgg atgcggacag cgcgaggttc 1140

actgaatgca agccatccgt gggtaccgtc tctggtgatg aaactcagtc actcccgggc 1200

aggagcacgt ccggcgaaga atcggcttct gggtacatgt actccatctc atcagccagt 1260

gccccaccaa gtccttccaa agagcaccaa tcctcatatc cgagcatccc ctaccagtcg 1320

tctgcgcacg tcgcgggtga tcttgactct ccagtcttga gggaaccgat caagtacatc 1380

aggaacatgc cgtccaagaa ccttcgaaca caactgatcg actgcttcaa catctggcta 1440

aatgcctctg ggcctgcgat ttcggtcatc aaagaagtca ttgactgcct gcaccattcc 1500

tcactgatcc tggatgacat cgaggacggt tcacatctac gacgcgggtt tccagccact 1560

catgtcgtct acggaacatg tcaggccgtc aacagcgcca cgtttctcta cgtccaagca 1620

gtggagagcg tccacgccgc cgcccggaac aaccccgaga tgatggacgt ctttttgaaa 1680

cacctgaggc agctgttcaa cggccaaagc tgggacctgt actggacgta ccaccgccaa 1740

tgccctaccg aggagcaata cctggacatg gtagatcaga aaactggcgc catgttgcaa 1800

ttgttagtgg gcttgatgca gacggcgcag ccgcagcatc cagggaaggt tggcggcgtt 1860

gtccatagtg aggtcctctt caggttcaca cagttgtttg gccgattctt ccaggtccgt 1920

gacgactata tgaacctcac gtcgacagac tacgctcggc agaagggctt tgctgaggac 1980

ctcgacgagc agaagttctc gtacatgata gtacacatgt accagagata ccccgaggcg 2040

aaggacaagg tcgagggtgt cttcagggcg atgcaacagg gtggcatctc acaggttgca 2100

gctgacacga gcaagaggta catcttgtcc atccttgacg agacaggctc aaccgcagct 2160

accaaggcac tgctattaaa gtggcatgac gagattacgg aggagattgg ggctttggag 2220

aggcattttg gggttgataa tgccttactt cgcttgctgg tggaaacctt gcgagtttga 2280

<210> 3

<211> 759

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Met Leu Glu Ala Asn Glu Leu Tyr Pro Tyr Ser Val Ala Val Asp Arg

1 5 10 15

Asp Glu Val Val Gln Ser Gly Ala Leu Thr Ser Leu Pro Val Arg Ile

20 25 30

His Arg Tyr Asn His Leu Ala Asp Ala Gly Ala Leu Cys Leu Thr Asn

35 40 45

Asp Trp Arg Cys Thr Met Lys Asp Gly Gln Asp Arg Lys Ser Asn Gly

50 55 60

Ser Pro Cys Val Val Gly Asn Trp Gly Ser Phe Ile Trp Pro Glu Ser

65 70 75 80

Arg Pro Glu Arg Leu Gly Leu Leu Cys Tyr Leu Leu Asp Ala Gly Cys

85 90 95

Phe His Asp Asp Ala Cys Glu Glu Met Pro Ile Ala Ala Ala His Gln

100 105 110

Glu His Leu Asp Leu Asp Ala Ala Met Asp Val Glu Asp Arg Arg Glu

115 120 125

Leu Ser Ser Asp Ser Arg Ser Leu Arg Thr Lys Gln Leu Ile Ser Asn

130 135 140

Ala Val Leu Glu Cys Ile Lys Val Asp Arg Val Gly Ala Met Arg Met

145 150 155 160

Leu Glu Ala Tyr Arg Lys Lys Trp Leu Arg Ile Met Glu Thr Tyr Asn

165 170 175

Thr Glu Glu Ile Asn Thr Val Glu Asp Tyr Phe Leu Ala Arg Ala Asn

180 185 190

Asn Gly Gly Met Gly Ala Tyr Tyr Ala Met Leu Glu Phe Ser Leu Gly

195 200 205

Ile Leu Val Thr Asp Glu Glu Tyr Glu Met Met Ala Glu Pro Ile Ala

210 215 220

His Val Glu Arg Cys Met Leu Leu Thr Asn Asp Tyr Trp Ser Trp Pro

225 230 235 240

Arg Glu Arg Lys Gln Ala Glu Tyr Gln Glu Ala Gly Lys Val Phe Asn

245 250 255

Ile Val Trp Phe Leu Lys Lys Ile Glu Leu Cys Thr Glu Glu Glu Ala

260 265 270

Val Ser Lys Val Arg Asp Met Val His Ala Glu Glu Arg Asn Trp Thr

275 280 285

Ala Ala Lys Thr Arg Leu Tyr Ser Gln Phe Gly Asn Leu Arg Gln Asp

290 295 300

Leu Val Lys Phe Leu Glu Asn Leu His Thr Ala Leu Ala Gly Asn Asp

305 310 315 320

Tyr Trp Ser Ser Gln Cys Tyr Arg His Asn Asp Trp Glu His Ile Pro

325 330 335

Asp Leu Pro Gly Glu Asp Ala Pro Lys Leu His Glu Leu Ala Thr Leu

340 345 350

Gly Arg Arg Leu Leu Leu Asp Glu Asp Leu Pro Pro Gly Ala Ser Thr

355 360 365

Tyr Gly Gln Asp Thr Asp Ala Asp Ser Ala Arg Phe Thr Glu Cys Lys

370 375 380

Pro Ser Val Gly Thr Val Ser Gly Asp Glu Thr Gln Ser Leu Pro Gly

385 390 395 400

Arg Ser Thr Ser Gly Glu Glu Ser Ala Ser Gly Tyr Met Tyr Ser Ile

405 410 415

Ser Ser Ala Ser Ala Pro Pro Ser Pro Ser Lys Glu His Gln Ser Ser

420 425 430

Tyr Pro Ser Ile Pro Tyr Gln Ser Ser Ala His Val Ala Gly Asp Leu

435 440 445

Asp Ser Pro Val Leu Arg Glu Pro Ile Lys Tyr Ile Arg Asn Met Pro

450 455 460

Ser Lys Asn Leu Arg Thr Gln Leu Ile Asp Cys Phe Asn Ile Trp Leu

465 470 475 480

Asn Ala Ser Gly Pro Ala Ile Ser Val Ile Lys Glu Val Ile Asp Cys

485 490 495

Leu His His Ser Ser Leu Ile Leu Asp Asp Ile Glu Asp Gly Ser His

500 505 510

Leu Arg Arg Gly Phe Pro Ala Thr His Val Val Tyr Gly Thr Cys Gln

515 520 525

Ala Val Asn Ser Ala Thr Phe Leu Tyr Val Gln Ala Val Glu Ser Val

530 535 540

His Ala Ala Ala Arg Asn Asn Pro Glu Met Met Asp Val Phe Leu Lys

545 550 555 560

His Leu Arg Gln Leu Phe Asn Gly Gln Ser Trp Asp Leu Tyr Trp Thr

565 570 575

Tyr His Arg Gln Cys Pro Thr Glu Glu Gln Tyr Leu Asp Met Val Asp

580 585 590

Gln Lys Thr Gly Ala Met Leu Gln Leu Leu Val Gly Leu Met Gln Thr

595 600 605

Ala Gln Pro Gln His Pro Gly Lys Val Gly Gly Val Val His Ser Glu

610 615 620

Val Leu Phe Arg Phe Thr Gln Leu Phe Gly Arg Phe Phe Gln Val Arg

625 630 635 640

Asp Asp Tyr Met Asn Leu Thr Ser Thr Asp Tyr Ala Arg Gln Lys Gly

645 650 655

Phe Ala Glu Asp Leu Asp Glu Gln Lys Phe Ser Tyr Met Ile Val His

660 665 670

Met Tyr Gln Arg Tyr Pro Glu Ala Lys Asp Lys Val Glu Gly Val Phe

675 680 685

Arg Ala Met Gln Gln Gly Gly Ile Ser Gln Val Ala Ala Asp Thr Ser

690 695 700

Lys Arg Tyr Ile Leu Ser Ile Leu Asp Glu Thr Gly Ser Thr Ala Ala

705 710 715 720

Thr Lys Ala Leu Leu Leu Lys Trp His Asp Glu Ile Thr Glu Glu Ile

725 730 735

Gly Ala Leu Glu Arg His Phe Gly Val Asp Asn Ala Leu Leu Arg Leu

740 745 750

Leu Val Glu Thr Leu Arg Val

755

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

cggaattcga gctcgatgct tgaagcaaac gagct 35

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

tactacagat ccccggtcaa actcgcaagg tttccaccag 40

Claims

1. Application of CS12565 strain (Cytospora schulzeri 12565) in preparation of 5-12-5 tricyclic diterpene skeleton compound Schultriene;

Wherein, the synthetase CcCS of 5-12-5 tricyclic diterpene skeleton compound Schultriene is prepared, the amino acid sequence is shown in SEQ ID NO.3, and the N end and the C end are respectively responsible for terpene cyclization and isopentenyl transfer functions;

The gene for encoding the synthetase CcCS is cloned from the genome of a CS12565 strain (Cytospora schulzeri 12565), and the polynucleotide sequence of the gene is shown as SEQ ID NO. 1;

the CS12565 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.21944.

2. The use of claim 1, wherein the synthetase CcCS comprises two conserved domains: the terpene cyclase domain contains two characteristic conserved motifs DDACE and NDYWSWPRE that recognize Mg ²⁺ and substrates, and the E-IPPS domain also contains two characteristic conserved motifs DDIED and DDYMN that have similar functions.

3. The use according to claim 1, wherein the gene encoding the synthetase CcCS comprises 3 introns, the cDNA size of which is 2280bp and the sequence of which is shown in SEQ ID NO. 2.