CN113582803A - 5-11 bicyclic sesterterpene skeleton compounds and preparation thereof - Google Patents

Abstract

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

Description

5-11 bicyclic sesterterpene skeleton compounds and preparation thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a 5-11 bicyclic sesterterpene skeleton compound Nigtetraene synthetic gene NnNS cloned from Nectria nigrescens 12199 and application thereof.

Background

Terpenoids are a small molecule compound formed by combining isoprene or isopentane in various ways, and are the largest group of compounds in natural products, and more than 8 ten thousand compounds have been discovered from plants, animals and microorganisms to date¹. Terpenoids can generally be classified as monoterpenes (C) according to the number of isoprene units contained therein₁₀) Sesquiterpenes (C)₁₅) Diterpenes (C)₂₀) Sesterterpene (C)₂₅) Triterpene (C)₃₀) And the like. Wherein the amount of sesterterpene is less than 2% of total terpenes, especially attention is paid². At present, most sesterterpenes are extracted and separated from marine organism sponges, but also exist in lichens, fungi, insect secretions and plants. Sesterterpene compounds have a broad spectrum of physiological activities, and as an important calmodulin inhibitor, the sesterterpene compounds, such as ophiobolins A, are useful³And simultaneously has antimalarial and anti-glioma activities, the derivative 3-anhydro-6-hydroxyyophiobolin A can promote the degradation of alpha synapsin of PC12 cells, and has potential application in the treatment of Parkinson's disease⁴. Teretonins sesterterpene compoundsThe teretonins E and F have the function of inhibiting mitochondrial respiratory chain⁵. In addition, it also has antibacterial effect⁶Inhibiting receptors⁷And the like. In 1965, more than 70 sesterterpene compounds derived from filamentous fungi are discovered, and the application prospect in biomedicine and other aspects is good due to the simple structure and various biological activities⁸。

Terpenoids start with two C₅The unit isomers of Dimethylallyl pyrophosphate (DMAPP) and Isopentenyl diphosphate (IPP) are subjected to chain extension under the catalysis of isoamyltransferase (PTs) to form a chain polyprenyl pyrophosphate precursor, and then are subjected to cyclization under the catalysis of Terpene Cyclase (TCs) to form a Terpene skeleton, and end products with different cyclization structures are formed under the catalytic reaction of various post-modifying enzymes. Most of sesterterpenes from fungi are generated by catalysis of special Bifunctional terpene synthases (BFTSs), and the enzymes simultaneously have prenyl transferase and terpene cyclase and respectively catalyze chain extension and cyclization of polyprenyl pyrophosphate. The bifunctional terpene synthase PaFS was explored from the plant pathogenic fungus Phomopsis amygdali by the Sassa team in Japan from 2007⁹Thereafter, a total of 22 BFTSs have been reported up to now.

The process of obtaining the terpenoid by the traditional chemical synthesis method has the defects of complicated operation steps, unstable reaction conditions, unfriendliness to the environment and the like, and the most important point is that the process has certain limitation in obtaining a new terpenoid skeleton. However, in recent years, with the recent iteration of genome sequencing technology and the rapid development of bioinformatics, microorganisms have been found to have great potential in the production of terpenoids. Therefore, the biosynthetic gene cluster is reconstructed in a heterologous host which is easy to operate and has clear genetic background for heterologous expression, and a large amount of new framework sesterterpene compounds and derivatives thereof can be obtained. Not only is the natural product resource library enriched, but also more choices are provided for finding new drugs. Is commonly used at present for fungus gradeHeterologous expression hosts for metabolites including prokaryotic expression systems, yeast expression systems, and filamentous fungal expression systems¹⁰. Prokaryotic expression systems such as E.coli, because they cannot cut introns in eukaryotic genes, can be applied to overexpression and functional identification of some enzymes in biosynthetic pathways. Yeast expression systems, such as Saccharomyces cerevisiae, whose secretory pathways enable correct protein processing and post-translational modifications, can be used for the analysis of the biosynthetic machinery of fungal natural products. Filamentous fungal expression systems, such as Aspergillus oryzae, can spontaneously accomplish the correct splicing of introns in a gene and can be used for biosynthetic pathway resolution and heterologous production of fungal natural products. Subject group of professor Oikawa successfully expressed diterpene compound pleuromutilin in Aspergillus oryzae¹¹The sesquiterpene compound (-) -Terestacin¹²And the like natural terpene products.

Disclosure of Invention

The invention is carried out by the research, and provides 5-11 bicyclic sesterterpene skeleton compounds, synthetases thereof, coding genes of the synthetases and a heterologous expression method of the compounds.

The idea of the invention is as follows: in the biosynthesis process of the 5-11 bicyclic sesterterpene compounds serving as metabolites of the excavated strain N.nigrescens 12199, the functional analysis of gene genes shows that the NnNS gene is related to terpene synthesis, and the NnNS gene is involved in the biosynthesis of the 5-11 bicyclic sesterterpene compounds. Further obtains the NnNS protein through heterogeneously expressing the NnNS gene to carry out in vitro enzymatic reaction, and verifies that the NnNS gene is a synthetic gene of a 5-11 bicyclic sesterterpene compound Nigtetraene.

The invention aims at providing the structure of 5-11 bicyclic sesterterpene skeleton compounds; the second purpose is to provide an enzyme and a gene for synthesizing the compound; the third objective is to provide a method for heterologous expression of 5-11 bicyclic sesterterpene skeleton compounds.

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

in a first aspect of the invention, there is provided a 5-11 bicyclic sesterterpene skeleton compound, nitterlene,the molecular formula is C₂₅H₄₀The structural formula is as follows:

in a second aspect of the invention, the synthetase NnNS of the 5-11 bicyclic sesterterpene skeleton compound is provided, the amino acid sequence of the synthetase is shown in 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.

Further, the synthetase contains two conserved domains: the terpene cyclase domain contains two recognition domains for Mg²⁺And the characteristic conserved motifs of the substrates DDVIE and NDYFSWDKE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions DDVED and DDYLN.

In a second aspect of the present invention, there is provided a gene encoding the above-mentioned synthetase cloned from the genome of the CS12199 strain (N. nigrescens 12199), and the polynucleotide sequence thereof is shown in 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. The gene contains 7 introns, the cDNA size is 2205bp, and the sequence is shown in SEQ ID NO. 2.

In a third aspect of the present invention, there is provided a recombinant expression vector of 5-11 bicyclic sesterterpene skeleton compounds, wherein the recombinant expression vector is a eukaryotic or prokaryotic expression vector carrying the above-mentioned synthetase or gene, such as E.coli, yeast system and filamentous fungi.

The fourth aspect of the invention provides a recombinant expression host cell of 5-11 bicyclic sesterterpene skeleton compounds, which contains the recombinant expression vector to realize the heterologous expression of the compounds.

In a fifth aspect of the invention, the application of the above-mentioned synthetase or gene in the synthesis of terpenoid, in particular to the application in the synthesis of 5-11 bicyclic sesterterpene skeleton compounds is provided.

In a sixth aspect of the invention, a method for heterologous expression of a 5-11 bicyclic sesterterpene skeleton compound is provided, which comprises the following steps:

A. construction of NnNS gene heterologous expression vector

Amplifying by using a PCR technology and using a N.nigrescens 12199 genome as a template to obtain a gene sequence containing NnNS, wherein 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 a pUARA2 vector through homologous recombination to construct a pUARA2-NnNS expression plasmid, transforming the ligation product into Escherichia coli DH10B, screening positive transformants, extracting plasmid PCR verification after culture to obtain a pUARA2-NnNS plasmid,

B. protoplast transformation

Culturing Aspergillus oryzae NSAR1, collecting protoplast, mixing with pUARA2-NnNS plasmid, culturing, performing PCR verification on the grown transformant, wherein the positive transformant is the NnNS heterologous expression strain AO-NnNS,

C. culture of heterologous expression strain AO-NnNS and product separation

Inoculating a heterologous expression strain AO-NnNS, screening by a pUARA2 plasmid screening liquid culture medium, performing fermentation culture, separating and purifying the obtained fermentation crude extract by adopting a forward silica gel column chromatography method, detecting each flow part rapidly by TLC, combining the same flow parts with spots, concentrating under reduced pressure, performing rotary evaporation to dryness, transferring into a weighed sample bottle, weighing the sample and recording the weight.

The invention has the following beneficial effects:

the invention discovers the NnNS gene for synthesizing the 5-11 bicyclic sesterterpene skeleton compound Nigtetraene, and the encoded NnNS protein can assist the synthesis of the mother nucleus of the Nigtetraene compound. The invention provides a new resource for the biosynthesis of the 5-11 bicyclic sesterterpene compounds and provides a choice for the synthesis of the compounds.

The NnNS gene discovered by the invention catalyzes and generates a new 5-11 bicyclic sesterterpene skeleton compound, and provides a valuable lead compound resource for enriching a natural product compound library and discovering new antibiotics.

Drawings

FIG. 1 is a HR-EI-MS spectrum of the compound Nigtetraene of the present invention.

FIG. 2 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹H-NMR spectrum.

FIG. 3 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹³C-NMR spectrum.

FIG. 4 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹H-¹H COSY spectra.

FIG. 5 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆HSQC spectrum in (1).

FIG. 6 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆HMBC spectrum in (1).

FIG. 7 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆NOESY spectrum of (1).

FIG. 8 is a graph showing the alignment of the amino acid sequences of the protein encoded by the NnNS gene in Nectria nigrescens 12199 and the reported proteins.

Strain preservation information: CS12199(Nectria nigrescens 12199) is preserved in China general microbiological culture Collection center (CGMCC), with the preservation address of No.3, No. 04/02 at 2021 years and the preservation number of CGMCC No. 21943.

Detailed Description

The following embodiments are implemented on the premise of the technical scheme of the invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the invention is not limited to the following embodiments.

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

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

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

The NnNS protein belongs to a chimeric terpene synthetase, and the N end and the C end of the NnNS protein are respectively responsible for the functions of terpene cyclization and isopentenyl group transfer. The NnNS protein contains two conserved domains, wherein the terpene cyclase domain contains two domains for recognizing Mg²⁺And the characteristic conserved motifs of the substrate, DDVIE and NDYFSWDKE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions, DDVED and DDYLN (FIG. 8).

Example 1: heterologous expression of 5-11 bicyclic sesterterpene compound Nigtetraene synthetic gene and structural identification of 5-11 bicyclic bi-sesquiterpene skeleton compound

By using a heterologous expression method, the NnNS gene in the Nectria nigrescens 12199 thallus is 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 formulation used is shown in Table 1.

TABLE 1 culture Medium formulation used in the examples

Construction of NnNS Gene Source expression vector

(1) The gene sequence containing NnNS is obtained by PCR technology and amplification by using the N.nigrescens 12199 genome as a 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 a pUARA2 vector through homologous recombination to construct a pUARA2-NnNS expression plasmid, and the two sides of the vector NnNS have a homologous sequence which is consistent with the pUARA2 vector.

(3) The ligation product was transformed into E.coli DH10B and positive transformants were selected by ampicillin screening. Liquid culture of positive transformant, extraction of plasmid PCR verification, and obtaining of pUARA2-NnNS plasmid.

2. Transformation of protoplasts

(1) Aspergillus oryzae NSAR1 was spread on PDA plates and cultured at 30 ℃ for 7 days.

(2) Spores were collected in 10mL of 0.1% Tween-80 (1 plate of the brood is typically collected) and counted on a hemocytometer. Inoculation about 10⁷The spores were cultured in 50mL DPY at 30 ℃ and 220rpm for 2-3 days.

(3) 100mg of Yatalase was weighed, dissolved by addition of dissolution 0, 20ml was sterilized by filtration through a 0.22 μm filter and added to a 50ml centrifuge tube.

(4) And collecting the thallus. Pouring 100ml of cultured mycelia into a P250 glass filter, removing a culture medium, washing with sterile water (or 0.8M NaCl) for 3-5 times, squeezing out water with a sterile medicine spoon, and adding the pressed dry mycelia into a Yatalase solution. Culturing at 30 deg.C and 200rpm under shaking for 1-2 hr until the spherical mycelium disappears and the supernatant is clear and dirty.

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

(6) The supernatant was removed, 20ml of 0.8M NaCl was added for resuspension washing, and centrifugation was carried out at 4 ℃ at 800g for 5min (twice washing). The supernatant was removed and 10ml of 0.8M NaCl was added. The number of protoplasts was counted under a microscope using a bacteria counter. Protoplast count-total count/80X 400ml X10⁴X dilution factor.

(7) Adjusting the protoplast concentration to 2X 10⁸cell/ml. (sol 2/sol 3 ═ 4/1), according toThe protoplast of 0.5ml-2ml can be obtained according to the growth condition of the thallus.

(8) 200. mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, 10. mu.g of the expression plasmid pUARA2-NnNS was added, and gently mixed. Standing on ice for 20 min. During this time, the sterilized Top agar was incubated in a water bath at 50 ℃.

(9) To the suspension of (8) was added 1ml of sol 3 and gently mixed with a tip. Standing at room temperature for 20 min. Add 10ml of sol 2 and mix gently.

(10) Centrifugation was carried out at 4 ℃ and 800g for 10min to remove the supernatant, 1ml of sol 2 was added, the suspension was gently suspended by a pipette gun, and 200. mu.l of the suspension was added to the center of the pUARA2 plasmid selection solid medium (X3 plate). 5ml of top agar incubated at 50 ℃ was rapidly added around the petri dish and mixed rapidly. After the plate surface was sufficiently dried, it was wound with parafilm, and cultured at 30 ℃ for 3 to 7 days with the lid facing downward.

(11) Each plate was picked 2-3 clones, 8 in total. And carrying out PCR verification on the grown transformant, wherein a positive transformant is the NnNS heterologous expression strain AO-NnNS.

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

(1) Inoculating heterologous expression strain AO-NnNS into pUARA2 plasmid to screen liquid culture medium, and culturing at 30 deg.C for 3 d.

(2) Centrifuging at 8000rpm for 10min to collect fermented thallus, adding 100ml of 80% acetone with the same volume, ultrasonicating for 20min, centrifuging at 8000rpm for 10min, and collecting supernatant.

(3) The mixture was extracted 1 time with 2 volumes of ethyl acetate, dried by rotary evaporation and dissolved in 15mL of methanol (chromatographic grade).

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

(5) The samples were subjected to GC-MS detection: an Agilent-HP-5MS chromatographic column is adopted, the initial temperature is 60 ℃, the temperature is increased to 310 ℃ at the speed of 15 ℃/min, then the temperature is increased to 310 ℃ at the speed of 5 ℃/min, and the temperature is kept for 13 min. The GC-MS process parameters were as follows: a Sample module: the needle washing times before and after sample injection are 5 times, the needle washing times of the sample are 2 times, the viscosity compensation time is 0.2s, and the sample injection mode is normal. A GC module: the column temperature is 50 ℃, the sample injection temperature is 270 ℃, the sample injection mode is non-split flow (split), the carrier gas is helium, the flow rate control mode is linear, and the total flow rate is 10 mL/min. An MS module: the MS ion source temperature is 230 ℃, the interface temperature is 270 ℃, the solvent removal time is 2.5min, the acquisition time is 3min-60min, the acquisition mode is full scanning, the event time is set to be 0.3s, the scanning speed is 2000, and the scanning nuclear-to-mass ratio is 40-600 Da.

(7) Subjecting the sample to LC-MS detection: adopting a Cholester chromatographic column, and adopting mobile phase A-0.1% formic acid water and mobile phase B-acetonitrile with the flow rate of 1mL/min, wherein the proportion of the mobile phase acetonitrile in 30min is increased from 5% to 100%, then maintaining for 6min, then reducing the proportion of the mobile phase acetonitrile in 10s to 5%, and then maintaining for 4min and 50 s.

4. Separation, purification and identification of heterologously expressed recombinant strain AO-NnNS sesterterpene skeleton product

AO-NnNS co-ferments 10L, yielding about 2g of crude extract. The obtained crude fermentation extract is firstly separated and purified by adopting a forward silica gel column chromatography method. Loading the column by adopting a dry method, isocratic eluting by using petroleum ether, collecting one tube of effluent liquid every 10mL, collecting 18 flow parts, quickly detecting each flow part by TLC, combining the flow parts with the same spots, concentrating under reduced pressure, performing rotary evaporation to dryness, transferring the mixture into a weighed sample bottle, weighing the sample and recording the weight. HPLC is used for analyzing the components of each flow part and accurately positioning the target flow part. Optimizing the preparation conditions, selecting a Cholester semi-preparative chromatographic column to prepare a target compound, and carrying out mobile phase: phase a-0.1% formic acid water; and (3) phase B-acetonitrile, wherein the flow rate is 4mL/min, the acetonitrile formic acid with 95 percent is equal, the initial sample introduction amount is 10 mu L, the sample introduction amount is gradually increased to 80 mu L on the basis of ensuring that the peak type is not changed, the target sesterterpene compound peak appears in about 20min, and when the peak appears, the outflow solution is connected into a conical flask. The purity of the prepared compound was checked by TLC and HPLC.

NMR measurements of the isolated sesterterpene skeleton compounds were carried out using Bruker 600MHz (R) ((R))¹H 600MHz；¹³C150 MHz). The solvent of the sesterterpene skeleton compound is Benzene-d₆The resolution of NMR spectrometer is 600MHz, the first step is¹H NMR and¹³c NMR measurement, comparing with data in database, and supplementing if it is new structureAnd (4) performing full HSQC, COSY and HMBC spectrogram decomposition spectrum to determine a specific structure.

5. Identifying the sesterterpene skeleton compound Nigtetraene.

Identifying the sesterterpene skeleton compound Nigtetraene obtained by the method:

(1) appearance: is in the form of transparent grease.

(2) Solubility: is easily dissolved in methanol and hardly dissolved in water.

(3) Nuclear magnetic resonance spectroscopy: FIG. 1 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹H-NMR spectrum.

FIG. 2 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹³C-NMR spectrum. FIG. 3 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In¹³C-DEPT 135 Spectrum. FIG. 4 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆In (1)¹H-¹H COSY spectra. FIG. 5 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆HSQC spectrum in (1). FIG. 6 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆HMBC spectrum in (1). FIG. 7 shows the dissolution of the compound Nigtetraene of the present invention in Benzene-d₆NOESY spectrum of (1). The nuclear magnetic resonance spectrum of the compound Nigtetraene of the invention was studied and the signals of the 1D and 2D spectra were assigned, see Table 2. And finally the structure was determined as follows:

TABLE 2 assignment of peaks in the 1D and 2D spectra of the compound fusaoxyspeneA

The references involved 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 Malus floribunda, Li Wei & Yi England. research progress on heterologous production of natural products of fungi, microbiological report 56, 429-.

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 invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

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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 (9)

1. A5-11 bicyclic sesterterpene skeleton compound Nigtetraene is characterized in that: the molecular formula is C₂₅H₄₀The structural formula is as follows:

2. the 5-11 bicyclic sesterterpene skeleton compound synthase NnNS of claim 1, wherein the amino acid sequence of the synthase is shown in SEQ ID NO.3, and the N-terminal and the C-terminal of the synthase are respectively responsible for terpene cyclization and isopentenyl group transfer functions.

3. The synthetase according to claim 2, characterized in that the synthetase comprises two conserved domains: the terpene cyclase domain contains two recognition domains for Mg²⁺And the characteristic conserved motifs of the substrates DDVIE and NDYFSWDKE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions DDVED and DDYLN.

4. A gene encoding the synthetase according to claim 2, characterized in that: the polynucleotide sequence of the strain is shown as SEQ ID NO.1 in order to be cloned from the genome of a CS12199 strain (Nectria. nigrescens 12199), wherein the CS12199 strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21943.

5. The gene as claimed in claim 4, which contains 7 introns, and has a cDNA size of 2205bp and a sequence shown in SEQ ID NO. 2.

6. A recombinant expression vector of 5-11 bicyclic sesterterpene skeleton compound, wherein the recombinant expression vector is a eukaryotic or prokaryotic expression vector carrying the synthetase of claim 2 or 3 or carrying the gene of claim 3 or 4.

7. A recombinant expression host cell of a 5-11 bicyclic sesterterpene skeleton compound, characterized in that: comprising the recombinant expression vector of claim 6.

8. Use of the synthetase of claim 2 or 3 or the gene of claim 4 or 5 for the synthesis of 5-11 bicyclic sesterterpene skeleton compounds.

9. A method for heterologously expressing a 5-11 bicyclic sesterterpene skeleton compound, comprising the steps of:

A. construction of NnNS gene heterologous expression vector

Amplifying by using a PCR technology and using a N.nigrescens 12199 genome as a template to obtain a gene sequence containing NnNS, wherein primer sequences used for amplification are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

connecting the amplified fragment with a pUARA2 vector through homologous recombination to construct a pUARA2-NnNS expression plasmid, transforming the ligation product into Escherichia coli DH10B, screening positive transformants, extracting plasmid PCR verification after culture to obtain a pUARA2-NnNS plasmid,

B. protoplast transformation

Culturing Aspergillus oryzae NSAR1, collecting protoplast, mixing with pUARA2-NnNS plasmid, culturing, performing PCR verification on the grown transformant, wherein the positive transformant is the NnNS heterologous expression strain AO-NnNS,

C. culture of heterologous expression strain AO-NnNS and product separation

Inoculating a heterologous expression strain AO-NnNS, screening by a pUARA2 plasmid screening liquid culture medium, performing fermentation culture, separating and purifying the obtained fermentation crude extract by adopting a forward silica gel column chromatography method, detecting each flow part rapidly by TLC, combining the same flow parts with spots, concentrating under reduced pressure, performing rotary evaporation to dryness, transferring into a weighed sample bottle, weighing the sample and recording the weight.

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