CN113582803B

CN113582803B - 5-11 Bicyclo-sesquiterpene skeleton compound and preparation thereof

Info

Publication number: CN113582803B
Application number: CN202110709524.8A
Authority: CN
Inventors: 刘雪婷; 蒋岚; 杨欢婷; 张立新; 黎晓莹; 吕康杰; 张雪; 朱国良; 王芷馨
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: CN113582803A

Abstract

The invention provides a 5-11 bicyclo-sesterterpene skeleton compound and a preparation method thereof, wherein the structural formula of NIGTETRAENE is shown as the specification, the amino acid sequence of synthetase NnNS is shown as SEQ ID NO.3, the synthetic gene is cloned from CS12199 strain (Nectria nigrescens 12199) genome, and the polynucleotide sequence is shown as SEQ ID NO. 1. NnNS protein has the functions of catalyzing chain length extension and structural cyclization of a substrate, and can assist the synthesis of NIGTETRAENE compound parent nucleus. The invention provides a new resource for biosynthesis of 5-11 bicyclo-sesquiterpene compounds and provides a choice for synthesis of the compound types.

Description

5-11 Bicyclo-sesquiterpene skeleton compound and preparation thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a 5-11 bicyclo-sesquiterpene skeleton compound NIGTETRAENE synthetic gene NnNS obtained by cloning NECTRIA NIGRESCENS 12199 and application thereof.

Background

Terpenoid is a small molecule compound formed by combining isoprene or isopentane in various ways, and is also the largest class of compounds in natural products, and more than 8 million ¹ have been found from plants, animals and microorganisms to date. Terpenes can be generally classified into monoterpenes (C ₁₀), sesquiterpenes (C ₁₅), diterpenes (C ₂₀), sesterterpenes (C ₂₅), diterpenes (C ₁₀), and combinations thereof according to the number of isoprene units contained, Triterpene (C ₃₀), etc. Of these, the number of sesterterpenes is less than 2% of the total number of terpenes, which is particularly interesting ². The vast majority of sesterterpenes are currently isolated from marine sponges, but are also found in lichens, fungi, insect secretions, plants. The sesterterpene compound has broad-spectrum physiological activity, for example, the ophiobolins sesterterpene compound ophiobolins A is an important calmodulin inhibitor ³, and also has antimalarial and antiglucoma activities, and the derivative 3-anhydro-6-hydroxyophiobolin A can promote the degradation of PC12 cell alpha synaptoprotein and has potential application ⁴ in the treatment of parkinsonism. the terretonins class of sesterterpene compounds terretonins E and terretonins F have the function ⁵ of inhibiting the mitochondrial respiratory chain. In addition, it has antibacterial ⁶ and receptor ⁷ inhibiting activities. 70 or more filamentous fungi-derived sesterterpene compounds have been discovered in 1965 so far, and the concise structure and the various biological activities lead the sesterterpene compounds to have good application prospects ⁸ in biomedical aspects and the like.

Terpenes originate from the two C ₅ units of the isomeric dimethylallyl pyrophosphates (DIMETHYLALLYL PYROPHOSPHATE, DMAPP) and isopentenyl diphosphate (Isopentenyl pyrophosphate, IPP), form a chain-like polyisopentenyl pyrophosphate precursor by catalytic chain extension with isopentenyl transferase (PRENYLTRANSFERASE, PTs), then cyclize to form a terpenoid skeleton under the catalysis of terpene cyclase (TERPENE CYCLASE, TCs) and form end products with different cyclized structures under the catalysis of various post-modification enzymes. The fungi-derived sesterterpenes are mostly produced by a special bifunctional terpene synthase (Bifunctional TERPENE SYNTHASES, abbreviated as BFTSs), which has both prenyl transferase and terpene cyclase, and catalyzes the extension and cyclization of the polyisoprenyl pyrophosphate chain, respectively. Since the discovery of bifunctional terpene synthases PaFS ⁹ from plant pathogenic fungi Phomopsis amygdali by the Sassa team in Japan in 2007, a total of 22 cases BFTSs have been reported.

The terpene compound obtained by the traditional chemical synthesis method has the defects of complicated operation steps, unstable reaction conditions, unfriendly environment and the like, and is most critical to have certain limitation in obtaining a new terpene skeleton. However, in recent years, with the newer iteration of genomic sequencing technology and the rapid development of bioinformatics, microorganisms have been found to have great potential in the production of terpenoids. Therefore, the biosynthesis gene cluster is reconstructed in a heterologous host which is easy to operate and has clear genetic background for heterologous expression, and a large number of novel skeleton sesterterpene compounds and derivatives thereof can be obtained. This not only enriches the natural product resource pool, but also provides more options for the discovery of new drugs. Heterologous expression hosts currently in common use for fungal secondary metabolites include prokaryotic expression systems, yeast expression systems, and filamentous fungal expression systems ¹⁰. Prokaryotic expression systems such as E.coli can not cleave introns in eukaryotic genes, and can be used for overexpression and functional identification of partial enzymes in biosynthetic pathways. Yeast expression systems such as Saccharomyces cerevisiae, whose secretory pathway is capable of correct protein processing and post-translational modification, are useful for the resolution of fungal natural product biosynthesis mechanisms. Filamentous fungal expression systems such as Aspergillus oryzae, which spontaneously accomplish correct cleavage of introns in genes, are useful for biosynthetic pathway resolution and heterologous production of fungal natural products. The Oikawa professor group successfully expressed diterpenoid compounds pleuromutilin ¹¹, sesquiterpene compounds (-) -TERPESTACIN ¹², and other natural terpenoid products in Aspergillus oryzae.

Disclosure of Invention

The invention provides 5-11 bicyclo-sesquiterpene 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 5-11 bicyclo-sesquiterpenoids which are metabolic products of the digging strain N.nigrocens 12199, the functional analysis of the gene proves that the NnNS gene is related to the terpene synthesis, and the NnNS gene is involved in the biosynthesis of 5-11 bicyclo-sesquiterpenoids. Further, the in vitro enzymatic reaction is carried out by obtaining NnNS protein through heterologous expression NnNS gene, and the NnNS gene is verified to be a synthetic gene of 5-11 bicyclo-sesquiterpene compound NIGTETRAENE.

The primary object of the present invention is to provide a structure of 5-11 bicyclo-sesquiterpene 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 5-11 bicyclic diterpene scaffold 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 5-11 bicyclo-sesquiterpene skeleton compound NIGTETRAENE having the formula C ₂₅H₄₀ and the structural formula is as follows:

In a second aspect of the present invention, a synthetase NnNS of the 5-11 bicyclo-sesquiterpene skeleton compound is provided, the amino acid sequence of the synthetase is shown as SEQ ID No.3, and the N-terminal and the C-terminal of the synthetase are respectively responsible for terpene cyclization and isopentenyl transfer functions.

Further, the synthase contains two conserved domains: the terpene cyclase domain contains two characteristic conserved motifs DDVIE and NDYFSWDKE that recognize Mg ²⁺ and substrates, and the E-IPPS domain also contains two characteristic conserved motifs DDVED and DDYLN that have similar functions.

In a second aspect of the invention, there is provided a gene encoding the above-described synthetase, cloned from the genome of a CS12199 strain (N.nigrescens 12199), the polynucleotide sequence of which is shown in SEQ ID NO. 1. The CS12199 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.21943. The gene contains 7 introns, the cDNA size is 2205bp, 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 5-11 bicyclo-sesterterpene skeleton compound, the recombinant expression vector being a eukaryotic or prokaryotic expression vector carrying the above-described synthetase or gene, such as E.coli, yeast system and filamentous fungus.

In a fourth aspect of the invention, a recombinant expression host cell of a 5-11 bicyclo-sesterterpene 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 5-11 bicyclo-sesterterpene framework compounds.

In a sixth aspect, the present invention provides a method for the heterologous expression of a 5-11 bicyclic diterpene skeleton compound, comprising the steps of:

A. construction of NnNS Gene heterologous expression vector

Amplifying by using an N.nigrocens 12199 genome as a template through a PCR technology to obtain a NnNS-containing gene sequence, 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:

NnNS-F：cgGAATTCGAGCTCGATGGCTCCATTGTCGATCAT；

NnNS-R：actacaGATCCCCGGTCATCGAGCTTCAATTTCCAGCCG。

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

B. protoplast transformation

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

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

Inoculating heterologous expression strain AO-NnNS, 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 NnNS gene for synthesizing the 5-11 bicyclo-sesquiterpene skeleton compound NIGTETRAENE, and the coded NnNS protein can assist the synthesis of NIGTETRAENE compound parent nucleus. The invention provides a new resource for the biosynthesis of 5-11 bicyclo-sesquiterpene compounds and provides a choice for the synthesis of the compounds.

The NnNS gene found by the invention catalyzes and generates a novel 5-11 bicyclo-diterpene 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 NIGTETRAENE of the present invention.

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

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

FIG. 4 is a ¹H-¹ H COSY spectrum of a compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆.

FIG. 5 is a spectrum of HSQC of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆.

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

FIG. 7 is a NOESY spectrum of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆.

FIG. 8 is a diagram showing the amino acid sequence alignment of the NnNS gene encoded protein in NECTRIA NIGRESCENS 12199 with the reported protein.

Strain preservation information: CS12199 (NECTRIA NIGRESCENS 12199) is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), the preservation address is North Star Xiyu No. 1, 3 in the Korean area of Beijing, the preservation date is 2021, 04 month 02, and the preservation number is CGMCC No.21943.

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 5-11 bicyclo-sesterterpene compound NIGTETRAENE provided by the invention is cloned from NECTRIA NIGRESCENS 12199 and named as NnNS gene, and the gene sequence of the synthetic gene is shown as SEQ ID NO. 1. The NnNS gene contains 7 introns, the cDNA size is 2205bp, and the sequence is shown as SEQ ID NO. 2. The protein coded by NnNS gene is named NnNS protein, and its amino acid sequence is shown in SEQ ID NO. 3.

NnNS protein belongs to chimeric terpene synthases, and the N end and the C end of the NnNS protein are respectively responsible for terpene cyclization and isopentenyl transfer functions. NnNS protein contains two conserved domains, wherein the terpene cyclase domain contains two conserved motifs DDVIE and NDYFSWDKE characteristic of Mg ²⁺ and the substrate, and the E-IPPS domain also contains two conserved motifs DDVED and DDYLN characteristic of similar function (fig. 8).

Example 1: heterologous expression of 5-11 bicyclo-sesquiterpene compound NIGTETRAENE synthetic gene and structural identification of 5-11 bicyclo-sesquiterpene skeleton compound

By utilizing a heterologous expression method, nnNS genes in NECTRIA NIGRESCENS 12199 thalli are transferred into host aspergillus oryzae by constructing expression plasmids, and the production condition of a heterologous expression strain product is detected. The medium formulations used are shown in Table 1.

Table 1 Medium formulation used in the examples

Construction of NnNS Gene-derived expression vector

(1) The gene sequence containing NnNS was amplified by PCR technique using the n.nigrocens 12199 genome as template. Primer sequences used for amplification:

NnNS-F：cgGAATTCGAGCTCGATGGCTCCATTGTCGATCAT(SEQ ID NO.4)；

NnNS-R：actacaGATCCCCGGTCATCGAGCTTCAATTTCCAGCCG(SEQ ID NO.5)。

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

(3) The ligation product was transformed into E.coli DH10B and positive transformants were selected by ampicillin. And (3) culturing the positive transformant in a liquid mode, extracting plasmids, and carrying out PCR verification to obtain pUARA-NnNS 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 0.5ml to 2ml of protoplasts can be collected according to the growth of the cell.

(8) 200. Mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, 10. Mu.g of expression plasmid pUARA-NnNS 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 NnNS heterologous expression strain AO-NnNS.

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

(1) The heterologous expression strain AO-NnNS 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 is 50 ℃, the sample injection temperature is 270 ℃, the sample injection mode is not split (splitness), the carrier gas is helium, the flow rate control mode is linear, and the total flow rate is 10mL/min. 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-NnNS sesterterpene skeleton product

AO-NnNS 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 in total, 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 samples into a weighed sample bottle, weighing the samples 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; phase B acetonitrile, the flow rate is 4mL/min, the isocratic degree of 95% acetonitrile formic acid, 10 mu L of initial sample injection is carried out, the sample injection amount 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 isolated sesterterpene skeletons 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 carried out first, the determination is compared with the data in a database, and if the determination is of a new structure, the spectrum resolution of the HSQC, COSY, HMBC spectrum is completed again to determine a specific structure.

5. Identifying the sesterterpene framework compound NIGTETRAENE.

Identifying the diterpene skeleton compound NIGTETRAENE 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 NIGTETRAENE of the present invention dissolved in Benzene-d ₆.

FIG. 2 is a ¹³ C-NMR spectrum of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. FIG. 3 is a ¹³ C-DEPT 135 spectrum of a compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. FIG. 4 is a ¹H-¹ H COSY spectrum of a compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. FIG. 5 is a spectrum of HSQC of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. FIG. 6 is a chart showing the HMBC pattern of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. FIG. 7 is a NOESY spectrum of compound NIGTETRAENE of the present invention dissolved in Benzene-d ₆. The nuclear magnetic resonance spectrum of the compound NIGTETRAENE 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 fusaoxyspeneA

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

1 Mitsuhashi,T.&Abe,I.Chimeric Terpene Synthases Possessing both Terpene Cyclization and Prenyltransfer Activities.Chembiochem 19,1106-1114(2018).

2 Wang,L.,Yang,B.,Lin,X.-P.,Zhou,X.-F.&Liu,Y.Sesterterpenoids.Natural product reports 30,455-473(2013).

3 Guan,Z.et al.Metabolic engineering of Bacillus subtilis for terpenoid production. Applied Microbiology biotechnology 99,9395-9406(2015).

4 Xue,D.et al.3-Anhydro-6-hydroxy-ophiobolin A,a fungal sesterterpene from Bipolaris oryzae induced autophagy and promoted the degradation ofα-synuclein in PC12 cells.Bioorganic Medicinal Chemistry Letters 25,1464-1470(2015).

5 López-Gresa,M.P.et al.Terretonins E and F,inhibitors of the mitochondrial respiratory chain from the marine-derived fungus Aspergillus insuetus.Journal of natural products 72,1348-1351(2009).

6 Amagata,T.et al.Unusual C25 Steroids Produced by a Sponge-Derived Penicillium c itrinum.Organic letters 5,4393-4396(2003).

7 Hensens,O.D.et al.Variecolin,a sesterterpenoid of novel skeleton from Aspergillus variecolor MF138.The Journal of Organic Chemistry 56,3399-3403(1991).

8 Yin,R.&Hong,K.Filamentous fungal sesterterpenoids and their synthases.Chinese Journal of Biotechnology 32,1631-1641(2016).

9 Toyomasu,T.et al.Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi.Proceedings of the national academy of sciences 104,3084-3088(2007).

10 Ma Zihui, li Wei & Yin Wenbing. Research on heterologous production of fungal natural products. Microbiology report 56, 429-440 (2016).

11 Nagamine,S.et al.Ascomycete Aspergillus oryzae is an efficient expression host for production of basidiomycete terpenes by using genomic DNA sequences.Applied environmental microbiology 85,e00409-00419(2019).

12 Narita,K.et al.Total biosynthesis of antiangiogenic agent(-)-terpestacin by artificial reconstitution of the biosynthetic machinery in Aspergillus oryzae.The Journal of organic chemistry 83,7042-7048(2018).

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

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gaagaatatt tcactatggt cgatggaagt tagtattgga cctctgctca gtcaccgcac 2040

agtcaacact aacgctctcg gtctagaaac cggtagtctt ttcattctta tcgccgaact 2100

gatgcgttct gaagctacga agcataaaac actcgacgct ggccttctca tgaagcttgt 2160

gggccgcttc ttccaagcac gagatgatta tctaaatctc cagagtgaag aggtttgtaa 2220

ttaaaatatt ctgcattcat tggttcagtg gtgctaattt tgcacgtgtc aacacagtat 2280

acccagaaga aaggcatcgc ggaagatatc aacgaaggga aattctcgct gccgctcatt 2340

cacgctttaa ggagcaagtc accgcaccgc gatcgccttt tgagcattct gcagcaacga 2400

aagaggtacg acgatctttc ccctgaaata cgcaagctcg ctctcgacga tatcaaagcc 2460

actggagggc tggagtatgc ggagaagacg gctatagagc tacaggaggc ggttagcgag 2520

acgcttacta cgtatgagga gagggtagga gagaagaatt ggctcctgag attggcgcag 2580

aagcggctgg aaattgaagc tcgatga 2607

<210> 2

<211> 2205

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

atggctccat tgtcgatcat ccgcaactct gtccctctcc caccgcatac atacgaaaca 60

gacgagtact tttgtcgatt caccccacgt atccatcgtg acgtccgcct tgctgatgcc 120

ggctcgtggc aatgccaggt cgactttctg ggatctagta cggccgctcg ggctggtgca 180

acgcgcaaca aggatgtttc tagttacgcc gtgggctgca ttaacccagt cgtcggcaac 240

ttcactgcgc tgtgtgcttg cgaagccttg agtgaccggc ttgccctgac tacttatatg 300

gttgagtatg cttatattca tgatgatgtg atcgagtatg ccgagaacaa agacgaatct 360

cagcttctgg agacaaacca gcagctcatt gaaggtctca gtctcgaaga agacgtcagc 420

gctggctcga aagaccatgt cagaagacga cagttgcaag ctaaaatggt catggagctt 480

attgaaacag acaagaagca ggcaaaagag tgcctccgct tatggaggga aatgtcacac 540

gtatttgtcc agatcagaga catgcagttc actgaactga atgactatct caagttccgt 600

gtggtagacg cgggatgccc ttggactatg agcctcttat gcttttccat ggacttcaca 660

ctgaattcca gtgaagagga gagagcttcc gccgtcaccc aggcagcgta cgacgcctgg 720

gttctagtca acgactactt ttcttgggat aaggagtgga acaaccacca atctcgtggt 780

ggcaccggtg tgattgccaa ttccatcttc ctcttcatga aatggtactc cgttgacgca 840

aaggagggca aaacgatgct gcgaaaggag attctggctc gcgaagagaa gtattgcaag 900

gccaaagaag atcttgaggc aaatggttcc atgtccgaca agataacaca gtggctcgag 960

ttgctcgatc ttgtaacagc aggtaacttt gcttggagca tgacaactgc ccgctatcgc 1020

cttggcgctg aagacgcata tatagcttta cggaatgcgt atacagagac ccctggttct 1080

ggtacaactg acagtcttgg gagccccatt tcgcaaaacg ctcttgcgat ggcagataaa 1140

atcgatatcg tgctgaagga tcggaggtac ctggatctta gcatccgcga gcgcaagata 1200

atcaaacccg ttgatagacc cattggccga caaacaactc atcctcccga ggatgcgaac 1260

ttcaagaagt cgcaggtcag ccaagtttgg tctctccacc aatacgaaga gatgattcta 1320

caacctccaa agtacttgga gatgatgcca tcaaaggaag tgaggaacgc tgttatagac 1380

ggcctagaga cttggtacca tgtttcagag aagtcactcg cagccattcg agaaattgta 1440

aacttattgc atagctcttc cctcatgcta gacgatgttg aagataactc gcggctcaga 1500

cgaggatttc cagcaaccca cattatattt ggagtcagcc agactataaa ttcagccaac 1560

ctactcatca tgaaggctct taaagcagcg gaaaccctct cgcctctcgc cgtgcgcatc 1620

ctcatcgaaa gactcattga cgggcatatt ggtcaagggc tggatctcta ctggacacac 1680

cacactcaga cacccaccga agaagaatat ttcactatgg tcgatggaaa aaccggtagt 1740

cttttcattc ttatcgccga actgatgcgt tctgaagcta cgaagcataa aacactcgac 1800

gctggccttc tcatgaagct tgtgggccgc ttcttccaag cacgagatga ttatctaaat 1860

ctccagagtg aagagtatac ccagaagaaa ggcatcgcgg aagatatcaa cgaagggaaa 1920

ttctcgctgc cgctcattca cgctttaagg agcaagtcac cgcaccgcga tcgccttttg 1980

agcattctgc agcaacgaaa gaggtacgac gatctttccc ctgaaatacg caagctcgct 2040

ctcgacgata tcaaagccac tggagggctg gagtatgcgg agaagacggc tatagagcta 2100

caggaggcgg ttagcgagac gcttactacg tatgaggaga gggtaggaga gaagaattgg 2160

ctcctgagat tggcgcagaa gcggctggaa attgaagctc gatga 2205

<210> 3

<211> 734

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Met Ala Pro Leu Ser Ile Ile Arg Asn Ser Val Pro Leu Pro Pro His

1 5 10 15

Thr Tyr Glu Thr Asp Glu Tyr Phe Cys Arg Phe Thr Pro Arg Ile His

20 25 30

Arg Asp Val Arg Leu Ala Asp Ala Gly Ser Trp Gln Cys Gln Val Asp

35 40 45

Phe Leu Gly Ser Ser Thr Ala Ala Arg Ala Gly Ala Thr Arg Asn Lys

50 55 60

Asp Val Ser Ser Tyr Ala Val Gly Cys Ile Asn Pro Val Val Gly Asn

65 70 75 80

Phe Thr Ala Leu Cys Ala Cys Glu Ala Leu Ser Asp Arg Leu Ala Leu

85 90 95

Thr Thr Tyr Met Val Glu Tyr Ala Tyr Ile His Asp Asp Val Ile Glu

100 105 110

Tyr Ala Glu Asn Lys Asp Glu Ser Gln Leu Leu Glu Thr Asn Gln Gln

115 120 125

Leu Ile Glu Gly Leu Ser Leu Glu Glu Asp Val Ser Ala Gly Ser Lys

130 135 140

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

145 150 155 160

Ile Glu Thr Asp Lys Lys Gln Ala Lys Glu Cys Leu Arg Leu Trp Arg

165 170 175

Glu Met Ser His Val Phe Val Gln Ile Arg Asp Met Gln Phe Thr Glu

180 185 190

Leu Asn Asp Tyr Leu Lys Phe Arg Val Val Asp Ala Gly Cys Pro Trp

195 200 205

Thr Met Ser Leu Leu Cys Phe Ser Met Asp Phe Thr Leu Asn Ser Ser

210 215 220

Glu Glu Glu Arg Ala Ser Ala Val Thr Gln Ala Ala Tyr Asp Ala Trp

225 230 235 240

Val Leu Val Asn Asp Tyr Phe Ser Trp Asp Lys Glu Trp Asn Asn His

245 250 255

Gln Ser Arg Gly Gly Thr Gly Val Ile Ala Asn Ser Ile Phe Leu Phe

260 265 270

Met Lys Trp Tyr Ser Val Asp Ala Lys Glu Gly Lys Thr Met Leu Arg

275 280 285

Lys Glu Ile Leu Ala Arg Glu Glu Lys Tyr Cys Lys Ala Lys Glu Asp

290 295 300

Leu Glu Ala Asn Gly Ser Met Ser Asp Lys Ile Thr Gln Trp Leu Glu

305 310 315 320

Leu Leu Asp Leu Val Thr Ala Gly Asn Phe Ala Trp Ser Met Thr Thr

325 330 335

Ala Arg Tyr Arg Leu Gly Ala Glu Asp Ala Tyr Ile Ala Leu Arg Asn

340 345 350

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

355 360 365

Pro Ile Ser Gln Asn Ala Leu Ala Met Ala Asp Lys Ile Asp Ile Val

370 375 380

Leu Lys Asp Arg Arg Tyr Leu Asp Leu Ser Ile Arg Glu Arg Lys Ile

385 390 395 400

Ile Lys Pro Val Asp Arg Pro Ile Gly Arg Gln Thr Thr His Pro Pro

405 410 415

Glu Asp Ala Asn Phe Lys Lys Ser Gln Val Ser Gln Val Trp Ser Leu

420 425 430

His Gln Tyr Glu Glu Met Ile Leu Gln Pro Pro Lys Tyr Leu Glu Met

435 440 445

Met Pro Ser Lys Glu Val Arg Asn Ala Val Ile Asp Gly Leu Glu Thr

450 455 460

Trp Tyr His Val Ser Glu Lys Ser Leu Ala Ala Ile Arg Glu Ile Val

465 470 475 480

Asn Leu Leu His Ser Ser Ser Leu Met Leu Asp Asp Val Glu Asp Asn

485 490 495

Ser Arg Leu Arg Arg Gly Phe Pro Ala Thr His Ile Ile Phe Gly Val

500 505 510

Ser Gln Thr Ile Asn Ser Ala Asn Leu Leu Ile Met Lys Ala Leu Lys

515 520 525

Ala Ala Glu Thr Leu Ser Pro Leu Ala Val Arg Ile Leu Ile Glu Arg

530 535 540

Leu Ile Asp Gly His Ile Gly Gln Gly Leu Asp Leu Tyr Trp Thr His

545 550 555 560

His Thr Gln Thr Pro Thr Glu Glu Glu Tyr Phe Thr Met Val Asp Gly

565 570 575

Lys Thr Gly Ser Leu Phe Ile Leu Ile Ala Glu Leu Met Arg Ser Glu

580 585 590

Ala Thr Lys His Lys Thr Leu Asp Ala Gly Leu Leu Met Lys Leu Val

595 600 605

Gly Arg Phe Phe Gln Ala Arg Asp Asp Tyr Leu Asn Leu Gln Ser Glu

610 615 620

Glu Tyr Thr Gln Lys Lys Gly Ile Ala Glu Asp Ile Asn Glu Gly Lys

625 630 635 640

Phe Ser Leu Pro Leu Ile His Ala Leu Arg Ser Lys Ser Pro His Arg

645 650 655

Asp Arg Leu Leu Ser Ile Leu Gln Gln Arg Lys Arg Tyr Asp Asp Leu

660 665 670

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

675 680 685

Gly Leu Glu Tyr Ala Glu Lys Thr Ala Ile Glu Leu Gln Glu Ala Val

690 695 700

Ser Glu Thr Leu Thr Thr Tyr Glu Glu Arg Val Gly Glu Lys Asn Trp

705 710 715 720

Leu Leu Arg Leu Ala Gln Lys Arg Leu Glu Ile Glu Ala Arg

725 730

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

cggaattcga gctcgatggc tccattgtcg atcat 35

<210> 5

<211> 39

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

actacagatc cccggtcatc gagcttcaat ttccagccg 39

Claims

1. Application of CS12199 strain (Nectria nigrocens 12199) in preparation of 5-11 bicyclo-sesquiterpene skeleton compound NIGTETRAENE;

wherein, the synthetase NnNS of the 5-11 bicyclo-sesquiterpene skeleton compound NIGTETRAENE is prepared, the amino acid sequence of the synthetase is shown as SEQ ID NO.3, and the N end and the C end of the synthetase are respectively responsible for terpene cyclization and isopentenyl transfer functions;

The gene for encoding the synthetase NnNS is cloned from the genome of CS12199 strain (Nectria nigrocens 12199), and the polynucleotide sequence of the gene is shown as SEQ ID NO. 1;

the CS12199 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21943.

2. The use of claim 1, wherein the synthetase NnNS comprises two conserved domains: the terpene cyclase domain contains two characteristic conserved motifs DDVIE and NDYFSWDKE that recognize Mg ²⁺ and substrates, and the E-IPPS domain also contains two characteristic conserved motifs DDVED and DDYLN that have similar functions.

3. The use according to claim 1, wherein the gene encoding the synthetase NnNS comprises 7 introns and the cDNA size is 2205bp and the sequence is shown in SEQ ID NO. 2.