CN112142585B

CN112142585B - Mangicols sesterterpene compounds, synthetic method, gene cluster, nucleic acid molecule, construct and application thereof

Info

Publication number: CN112142585B
Application number: CN202010936790.XA
Authority: CN
Inventors: 刘天罡; 苑玉杰; 卞光凯; 闫盼; 马正宁; 陈蓉
Original assignee: Wuhan University WHU
Current assignee: Wuhan Hesheng Technology Co ltd
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2021-09-14
Anticipated expiration: 2040-09-08
Also published as: CN112142585A

Abstract

The invention discloses a Mangicols sesterterpene compound, a synthesis method, a gene cluster, a nucleic acid molecule, a construct and application thereof, which aims at the research of 5 new Mangicols sesterterpene compounds, biosynthesis gene clusters and pharmacological activity thereof, wherein the structure of the sesterterpene compounds is shown as formulas I-V. The mangicols compound biosynthesis pathway is reported for the first time. The synthetase involved in the biosynthetic pathway has the amino acid sequence of SEQ ID NO:1 to 7 or a pharmaceutically acceptable salt thereof. Meanwhile, the invention discloses the anti-inflammatory activity of 5 mangicols compounds. The invention lays an important foundation for realizing the high-efficiency synthesis of mangicols sesterterpene compounds and provides a new solution for obtaining the compounds.

Description

Mangicols sesterterpene compounds, synthetic method, gene cluster, nucleic acid molecule, construct and application thereof

Technical Field

The invention relates to the technical field of natural product biosynthesis, in particular to a Mangicols sesterterpene compound, a synthesis method, a gene cluster, a nucleic acid molecule, a construct and application thereof.

Background

Terpenoids are a generic term for compounds containing isoprene units. It is widely found in nature, and up to now, more than 8 thousands of terpenoids are found in animals, plants and microorganisms. The compounds have a plurality of physiological activities and are widely applied to the industries of food, cosmetics and medicines.

The sesterterpene compounds are a generic name of terpenoids with a core skeleton composed of 5 isoprene units, and are unique and complex structures synthesized by using geranyl farnesyl pyrophosphate (GFPP) with a longer carbon chain as a substrate through a series of steps of cyclization, rearrangement and the like, wherein a large number of novel product synthesis mechanisms are involved. Filamentous fungi are used as an important source of terpenoids, so far, researches on sesterterpene compounds in vivo and biosynthesis pathways thereof are few, more than 70 sesterterpene compounds are found so far, and account for 7% of the total amount of known sesterterpenes, even if the sesterterpene compounds derived from filamentous fungi show highly complex structural diversity, and some sesterterpene products have multiple potential medicinal values, such as ophiobolin family compounds with multiple biological activities, aspenterenols A and B with acetylcholinesterase inhibition activity, fusaproniferin with cytotoxic activity and the like, and show good application prospects in medicine development. Therefore, the sesterterpene compounds with potential application value and the biosynthesis pathways thereof, which are stored in filamentous fungi, are researched, and an important foundation is laid for the efficient synthesis and subsequent physiological activity evaluation of the compounds.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a Mangicols sesterterpene compound, a synthesis method, a gene cluster, a nucleic acid molecule, a construct and application thereof, in particular to 5 new sesterterpene compounds, namely Mangicols compounds, relating to the biosynthesis gene cluster and the pharmacological activity thereof and simultaneously relating to the biosynthesis enzyme, the nucleic acid molecule, the construct and the biosynthesis method and the anti-inflammatory activity of the Mangicols sesterterpene compounds.

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

in a first aspect, the present invention provides a Mangicols sesterterpene compound, which is characterized in that: the structure is shown in formulas I-V:

in a second aspect, the present invention provides a method for synthesizing the above Mangicols sesterterpene compounds, which is characterized in that: comprises the following steps:

1) constructing bacterial mutant strains of biosynthetic gene clusters of heterologously synthesized sesterterpene compounds;

2) preparing a fermentation culture of the bacterial mutant strains AO-mgcDE and AO-mgcCDEF;

3) extracting and separating fermentation culture and purifying the product;

the bacteria is any one of Aspergillus oryzae, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Alternaria alternata or Fusarium.

In a third aspect, the present invention provides a method for preparing a sesterterpene product, which is characterized in that:

respectively extracting the fermentation culture of AO-mgcDE and AO-mgcCDEF with ethyl acetate, combining ethyl acetate extract liquid, and concentrating to obtain crude extracts A and B; carrying out reverse silica gel column chromatography on the crude extract A, carrying out gradient elution on an n-hexane-ethyl acetate system, and collecting fractions with the volume ratio of n-hexane to ethyl acetate of 5: 1; subjecting the fraction to semi-preparative HPLC purification by eluting with pure acetonitrile and ultrapure water at a volume ratio of 75:25 to 90:10 as mobile phases, and collecting the fraction to obtain sesterterpene compounds represented by formulae I to III as claimed in claim 1; alternatively, the first and second electrodes may be,

subjecting the crude extract B to reverse silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate system, and collecting the fraction with petroleum ether/ethyl acetate volume ratio of 20: 1; carrying out gradient elution on the fraction by a chloroform-acetone system, and collecting the fraction with the chloroform/acetone volume ratio of 15: 1; purifying the fraction by HPLC, eluting with acetonitrile/water at a volume ratio of 45:55 as mobile phase, and collecting the fraction to obtain sesterterpene compounds represented by formula IV and formula V as described in claim 1;

the sesterterpene compound is prepared and separated from a fermentation culture of heterologous expression host bacteria;

In a fourth aspect, the present invention provides a sesterterpene biosynthesis gene cluster, which is characterized in that: a bacterial mutant strain constructed as described in claim 2 containing the heterologously synthesized sesterterpene compound biosynthetic gene cluster; the sesterterpene biosynthetic gene cluster contains 7 biosynthetic enzymes having the amino acid sequence shown in SEQ ID NO:1 to 7 or a pharmaceutically acceptable salt thereof.

In a fifth aspect, the present invention provides a nucleic acid molecule characterized in that: encoding the biosynthetic enzyme of claim 4; the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:8 to 14 in any one of the nucleotide sequences shown in the specification.

In a sixth aspect, the present invention provides a construct characterized in that: comprising the nucleic acid molecule of claim 5.

In a seventh aspect, the invention provides an application of the Mangicols sesterterpene compound in preparing anti-inflammatory drugs.

Specifically, the 5 mangicols sesterterpene compounds have the structure shown in the formulas I-V as shown in the specification, and are named as mangicol H-L respectively.

The biosynthetic gene cluster of the sesterterpene compounds is prepared by expressing terpene synthases in the biosynthetic gene cluster in Aspergillus oryzae (Aspergillus oryzae) by a heterologous expression method, constructing a mutant strain, and extracting and separating the target compound from a mutant strain fermentation culture. According to the embodiment of the invention, the terpene synthase contained in the gene cluster has the sequence shown in SEQ ID NO:1 to 7 or a pharmaceutically acceptable salt thereof.

The nucleic acid molecule of the present invention. According to an embodiment of the invention, the above-mentioned nucleic acid molecule encodes a terpene synthase as described above. Thus, the nucleic acid molecules according to embodiments of the invention can efficiently encode terpene synthases, thereby catalyzing the synthesis of the target compound. The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 8-14. Thus, the nucleic acid molecules according to embodiments of the invention are capable of efficiently encoding terpene synthases, thereby catalyzing a series of cascade reactions in order to obtain terpenoids with post-modified structures.

The construct of the present invention. The above construct contains the above nucleic acid molecule. Thus, constructs according to embodiments of the invention may catalyze the synthesis of a compound of interest by expressing a nucleic acid molecule encoding a synthetic terpene synthase.

The biosynthetic pathway of the above compounds of the present invention. The biosynthesis pathway contains the participation of at least one of the following terpene synthases: terpene synthase (mgcD), P450 enzymes (mgcE and mgcF), epoxide hydrolase (mgcC). Under the catalytic action of the enzyme, the target product is synthesized through two branch paths.

The anti-inflammatory activity of the above compounds is also proposed in the present invention. The compounds show anti-inflammatory activity at the cellular level and in experimental mouse models.

The invention has the following advantages and beneficial effects:

according to the invention, preferably Aspergillus oryzae is used as a dominant expression host, mgc gene clusters are efficiently heterogeneously reconstructed in vivo through rational design, and 5 novel mangicols sesterterpene compounds with anti-inflammatory activity are separated and identified, so that a solid foundation is laid for obtaining and further deeply exploring the products.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is bioinformatics information of the mangicols compound biosynthesis gene cluster according to one embodiment of the present invention.

FIG. 2 is a schematic diagram of a plasmid structure according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of an Aspergillus oryzae mutant strain according to one embodiment of the present invention.

FIG. 4 is a GC/MS detection profile of one embodiment of the invention.

FIG. 5 is a HR-LCMS detection profile according to one embodiment of the present invention.

FIG. 6 is a schematic structural diagram of Compound (1) according to one embodiment of the present invention;

FIG. 7 shows a compound (1) of the present invention¹H NMR spectrum;

FIG. 8 shows a compound (1) of the present invention¹³C NMR spectrum;

FIG. 9 shows a compound (1) of the present invention¹H-¹H COSY spectrogram;

FIG. 10 shows HSQC spectra of compound (1) of the present invention;

FIG. 11 shows a HMBC spectrum of the compound (1) of the present invention;

FIG. 12 shows a ROESY spectrum of the compound (1) of the present invention.

FIG. 13 is a schematic structural diagram of Compound (2) according to one embodiment of the present invention;

FIG. 14 shows a scheme for preparing a compound (2) of the present invention¹H NMR spectrum;

FIG. 15 shows a scheme for preparing a compound (2) of the present invention¹³C NMR spectrum;

FIG. 16 shows a scheme for preparing a compound (2) of the present invention¹H-¹H COSY spectrogram;

FIG. 17 is an HSQC spectrum of compound (2) of the present invention;

FIG. 18 is an HMBC spectrum of compound (2) of the present invention;

FIG. 19 is a ROESY spectrum of the compound (2) of the present invention.

FIG. 20 is a schematic structural view of Compound (3) according to an embodiment of the present invention;

FIG. 21 shows a scheme for preparing a compound (3) of the present invention¹H NMR spectrum;

FIG. 22 shows a scheme for preparing a compound (3) of the present invention¹³C NMR spectrum;

FIG. 23 shows a scheme for preparing a compound (3) of the present invention¹H-¹H COSY spectrogram;

FIG. 24 is an HSQC spectrum of compound (3) of the present invention;

FIG. 25 is an HMBC spectrum of compound (3) of the present invention;

FIG. 26 is a ROESY spectrum of the compound (3) of the present invention.

FIG. 27 is a schematic structural view of Compound (4) according to an embodiment of the present invention;

FIG. 28 shows a scheme for preparing a compound (4) of the present invention¹H NMR spectrum;

FIG. 29 shows production of Compound (4) of the present invention¹³C NMR spectrum;

FIG. 30 shows a scheme for preparing a compound (4) of the present invention¹H-¹H COSY spectrogram;

FIG. 31 is an HSQC spectrum of compound (4) of the present invention;

FIG. 32 is an HMBC spectrum of compound (4) of the present invention;

FIG. 33 is a ROESY spectrum of the compound (4) of the present invention.

FIG. 34 is a schematic structural view of Compound (5) according to an embodiment of the present invention;

FIG. 35 shows production of Compound (5) of the present invention¹H NMR spectrum;

FIG. 36Is of the Compound (5) of the present invention¹³C NMR spectrum;

FIG. 37 shows production of Compound (5) of the present invention¹H-¹H COSY spectrogram;

FIG. 38 is an HSQC spectrum of compound (5) of the present invention;

FIG. 39 is an HMBC spectrum of compound (5) of the present invention;

FIG. 40 is a ROESY spectrum of the compound (5) of the present invention.

FIG. 41 is a graph I showing the results of measurement of anti-inflammatory activity of compounds (1) to (5) in the examples of the present invention.

FIG. 42 is a graph II showing the results of measurement of anti-inflammatory activities of compounds (1) to (5) in examples of the present invention.

FIG. 43 shows the structural formula of the Mangicols sesterterpene compounds of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

Terpene synthases

In one aspect of the invention, the invention provides the biosynthesis gene cluster of the terpenoid for the first time. According to the embodiment of the invention, the synthetic enzyme contained in the terpene biosynthesis gene cluster has an amino acid sequence shown as SEQ ID NO. 1-7 in a sequence table.

Nucleic acid molecules

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the above terpene synthase. Thus, nucleic acid molecules according to embodiments of the invention can efficiently encode the above-described terpene synthases to catalyze a multi-step cascade of reactions to obtain a target terpenoid. According to the embodiment of the invention, the nucleic acid molecule has a nucleotide sequence shown as SEQ ID NO 8-14 in a sequence table.

Construct

In yet another aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct comprises a nucleic acid molecule as described above. Thus, constructs according to embodiments of the invention may catalyze a multistep cascade of reactions by expressing a nucleic acid molecule encoding a synthetic terpene synthase to obtain a target terpenoid.

Method for synthesizing terpenoid

In yet another aspect, the present invention provides a method for synthesizing the terpenoids described above. According to an embodiment of the invention, the method comprises: culturing the former construct under conditions suitable for expression of the terpene synthase so as to obtain a culture product; and isolating the terpenoid from the culture product. Thereby obtaining the target terpenoid.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 analysis of Bioinformation of Mangicols Compound biosynthesis Gene Cluster

Bioinformatics analysis of the mangicols compound biosynthesis gene cluster in the filamentous fungus Fusarium graminearum J1-012 strain (Fusarium graminearum J1-012) was performed based on the AntiSMASH software, and it was found that the gene cluster contained seven genes, 2 regulatory genes (mgcA and mgcB), 1 epoxide hydrolase gene (mgcC), 2 cytochrome P450 genes (mgcE and mgcF) associated with product oxidation and a monooxygenase gene (mgcG) of FMO type (FIG. 1).

Example 2 construction of expression vector

All genes are obtained by PCR amplification, and the primers are shown as SEQ ID NO. 15-78 in the sequence table.

Two methods, the Gibson method and Yeast association method, were mainly used for plasmid construction. The specific construction method comprises the following steps:

construction of pYJ152 plasmid: based on the predicted coding region information for mgcD, each exon fragment was amplified separately from the f.graminearum J1-012 genome and assembled into mgcD gene (i.e., cDNA fragment) containing the entire coding region in vitro, which was subsequently amplified with primers P1/P2. The complete sequence containing ura gene and corresponding promoter and terminator WAs amplified with primer P61/P62 using pYH-WA-pyrG-KI plasmid as template. Using pTAex3 plasmid as a template, the vector sequence was amplified using two pairs of primers, P3/P64 and P4/P63. The 4 amplified fragments were assembled by the Yeast assembly method to obtain pYJ152 plasmid (FIG. 2).

Construction of pYJ153 plasmid: based on the predicted coding region information for mgcE, each exon fragment was amplified separately from the f.graminearum J1-012 genome and assembled into mgcE gene (i.e., cDNA fragment) containing the entire coding region in vitro, which was subsequently amplified with primers P5/P6. The complete sequence containing ura gene and corresponding promoter and terminator WAs amplified with primer P61/P62 using pYH-WA-pyrG-KI plasmid as template. Using the pUSA plasmid as a template, the vector sequence was amplified using two pairs of primers P7/P63 and P8/P64. The 4 amplified fragments were assembled by the Yeast assembly method to obtain the pYJ153 plasmid (FIG. 2).

Construction of pYJ154 plasmid: based on the predicted coding region information for mgcF, each exon fragment was amplified separately from the f.graminearum J1-012 genome and assembled into mgcF gene (i.e., cDNA fragment) containing the entire coding region in vitro, which was subsequently amplified with primers P9/P10. The complete sequence containing ura gene and corresponding promoter and terminator WAs amplified with primer P61/P62 using pYH-WA-pyrG-KI plasmid as template. Using the pUSA plasmid as a template, the vector sequence was amplified using two pairs of primers P11/P63 and P12/P64. The 4 fragments amplified above were assembled by the Yeast assembly method to obtain the pYJ154 plasmid (FIG. 2).

Construction of pYJ174 plasmid: in order to replace the methionine selection marker with that of Ade, the sC gene in pYJ154 was replaced with that of Ade. Ade gene fragments were amplified from the pAde plasmid using the P13/P14 primers. The vector fragment was amplified using pYJ154 as template and P15/P16 primer. The two fragments were assembled by the Gibson method to obtain plasmid pYJ174 (FIG. 2).

Construction of pYJ175 plasmid: the glaA promoter sequence was amplified from plasmid pGB98 using the P17/P18 primer. The mgcC gene fragment was amplified from the F.graminearum J1-012 genome using P19/P20 primer. The niaD terminator sequence was amplified from plasmid pGB127 using the P21/P22 primer. The vector fragment was amplified from the template pYJ174 using the P23/P24 primer. The above fragments were assembled by the Yeast assembly method to obtain pYJ175 plasmid (FIG. 2).

Construction of pYJ176 plasmid: the glaA promoter sequence was amplified from plasmid pGB98 using the P17/P25 primer. The mgcG gene fragment was amplified from the F.graminearum J1-012 genome using P26/P27 primer. The niaD terminator sequence was amplified from plasmid pGB127 using the P28/P22 primer. The vector fragment was amplified from the template pYJ174 using the P23/P24 primer. The above fragments were assembled by the Yeast assembly method to obtain pYJ176 plasmid (FIG. 2).

Construction of pYJ177 plasmid: the agdA terminator sequence was amplified from plasmid pGB127 using the P29/P30 primer. The mgcC gene fragment was amplified from the F.graminearum J1-012 genome using P31/P32 primer. hlyA promoter sequence was amplified from a. oryzae genome using P33/P34 primers. The glaA promoter sequence was amplified from template pGB98 using the P35/P25 primer. The above fragments were assembled by the Yeast assembly method to obtain pYJ176 plasmid. The mgcG gene fragment was amplified from the F.graminearum J1-012 genome using P26/P27 primer. The niaD terminator sequence was amplified from plasmid pGB127 using the P28/P22 primer. The vector fragment was amplified from the template pYJ174 using the P23/P36 primer. The 7 fragments were assembled by the Yeast assembly method to obtain pYJ177 plasmid (FIG. 2).

Construction of pYJ178 plasmid: the glaA promoter sequence was amplified from plasmid pGB98 using the P37/P38 primer. The mgcA gene fragment was amplified from the F.graminearum J1-012 genome using P39/P40 primers. The niaD terminator sequence was amplified from plasmid pGB127 using the P41/P22 primer. The vector fragment was amplified from the template pYJ153 using the P23/P42 primer. The 4 fragments were assembled by the Yeast assembly method to obtain the pYJ178 plasmid (FIG. 2).

Construction of pYJ179 plasmid: the glaA promoter sequence was amplified from plasmid pGB98 using the P37/P43 primer. The mgcB gene fragment was amplified from the F.graminearum J1-012 genome using P44/P45 primer. The niaD terminator sequence was amplified from plasmid pGB127 using the P46/P22 primer. The vector fragment was amplified from the template pYJ153 using the P23/P42 primer. The 4 fragments were assembled by the Yeast assembly method to obtain pYJ179 plasmid (FIG. 2).

Construction of pYJ180 plasmid: the agdA terminator sequence was amplified from plasmid pGB127 using the P47/P48 primer. The mgcA gene fragment was amplified from the F.graminearum J1-012 genome using P49/P50 primers. hlyA promoter sequence was amplified from a. oryzae genome using P51/P34 primers. The glaA promoter sequence was amplified from template pGB98 using the P35/P43 primer. The mgcB gene fragment was amplified from the F.graminearum J1-012 genome using P44/P45 primer. The niaD terminator sequence was amplified from plasmid pGB127 using the P46/P22 primer. The vector fragment was amplified from the template pYJ153 using the P23/P52 primer. The 7 fragments were assembled by the Yeast assembly method to obtain pYJ180 plasmid (FIG. 2).

Construction of pYJ215 plasmid: the mgcG gene fragment was amplified from the F.graminearum J1-012 genome using P53/P54 primer. The vector fragment was amplified from the template pYJ153 using the P55/P56 primer. The 2 fragments were assembled by the Gibson method to obtain the pYJ215 plasmid (FIG. 2).

Construction of pYJ220 plasmid: the mgcC gene fragment was amplified from the F.graminearum J1-012 genome using P57/P58 primer. The vector fragment was amplified from the template pYJ174 using the P59/P60 primer. The 2 fragments were assembled by the Gibson method to obtain the pYJ220 plasmid (FIG. 2).

EXAMPLE 3 Aspergillus oryzae mutant Strain construction for the Synthesis of terpenoids

In order to synthesize the target terpenoid and analyze the biosynthesis pathway of the terpenoid, the invention reasonably designs and constructs a series of Aspergillus oryzae mutant strains. In the construction of the strains, plasmids containing different genes are transformed into Aspergillus oryzae by protoplast transformation. After the transformants grow out, 3-4 transformants are selected to extract the genome, and PCR verification is carried out on the target gene. And (4) carrying out subculture expansion on the strains with positive PCR results for subsequent seed preservation and fermentation experiments.

The invention transforms plasmid pYJ152 into Aspergillus oryzae to obtain strain AO-mgcD containing terpenoid synthase gene mgcD. In order to identify the function of P450 enzyme catalyzing the first oxidation reaction in the mangicols compound biosynthesis, the invention expresses the predicted three enzymes mgcE, mgcF and mgcG with oxidation function and mgcD in Aspergillus oryzae in a heterologous way respectively. Co-transforming plasmids pYJ152 and pYJ153 into Aspergillus oryzae to obtain a strain AO-mgcDE containing terpene synthase gene mgcD and P450 enzyme gene mgcE; co-transforming plasmids pYJ152 and pYJ154 into Aspergillus oryzae to obtain a strain AO-mgcDF containing terpene synthase gene mgcD and P450 enzyme gene mgcF; plasmids pYJ152 and pYJ215 were co-transformed into Aspergillus oryzae to obtain a strain AO-mgcDG containing the terpene synthase gene mgcD and the FMO monooxygenase gene mgcG. On the basis, in order to further verify the functions of other enzymes, the invention also constructs a series of Aspergillus oryzae mutant strains: co-transforming plasmids pYJ152, pYJ153 and pYJ220 into Aspergillus oryzae to obtain a strain AO-mgcCDE containing terpene synthase gene mgcD, P450 enzyme gene mgcE and epoxide hydrolase gene mgcC; co-transforming pYJ152, pYJ153 and pYJ174 into Aspergillus oryzae to obtain strain AO-mgcDEF containing terpene synthase gene mgcD, P450 enzyme gene mgcE and P450 enzyme gene mgcF; co-transforming pYJ152, pYJ153 and pYJ175 into Aspergillus oryzae to obtain strain AO-mgcCDEF containing terpene synthase gene mgcD, P450 enzyme gene mgcE, epoxide hydrolase gene mgcC and P450 enzyme gene mgcF; co-transforming pYJ152, pYJ153 and pYJ176 into Aspergillus oryzae to obtain strain AO-mgcDEFG containing terpene synthase gene mgcD, P450 enzyme gene mgcE, FMO monooxygenase gene mgcG and P450 enzyme gene mgcF; co-transforming pYJ152, pYJ153 and pYJ177 into Aspergillus oryzae to obtain a strain AO-mgcCDEFG containing terpene synthase gene mgcD, P450 enzyme gene mgcE, FMO monooxygenase gene mgcG, epoxide hydrolase gene mgcC and P450 enzyme gene mgcF; plasmids pYJ152, pYJ180 and pYJ177 were co-transformed into Aspergillus oryzae to yield strain AO-mgcABCDEFG (FIG. 3) containing the FgMS entire biosynthetic gene cluster.

Example 4 Synthesis and detection of terpenoids

In order to obtain the target terpenoid, the constructed mutant strain is subjected to amplification culture on a screening plate, then a proper amount of hypha and spores are taken and inoculated into 200mL of DPY liquid culture medium, and 1% maltose is added for induction expression. Culturing at 140rpm and 30 ℃ for 7 days. Filtering cultured thallus with nylon cloth, discarding culture medium, mincing thallus with stirrer, adding equal volume of ethyl acetate, extracting for three times, and mixing the three upper layers. Spin-drying with rotary evaporator, adding chromatographic grade ethyl acetate or methanol, re-dissolving the sample, high speed centrifuging, collecting supernatant, and detecting with GCMS or HR-LCMS.

The invention respectively carries out fermentation culture on three mutant strains of AO-mgcDE, AO-mgcDF and AO-mgcDG. And (4) after re-dissolving the fermentation product by ethyl acetate, and detecting by GCMS. The results show that three oxidatively modified products (1, 2 and 3) were detected in the AO-mgcDE mutant, while no corresponding oxidation products were produced in the other two mutants (FIG. 4). Thus, the present inventors have found that mgcE is an enzyme catalyzing the first oxidation reaction.

In order to identify the function of the P450 enzyme mgcF, the AO-mgcDEF mutant strain is subjected to fermentation culture. The fermentation product was reconstituted with methanol and tested by HR-LCMS. From the results, it was found that two oxidized products (4 and 5) were produced in the AO-mgcDEF mutant strain fermentation product (FIG. 5). On the basis, in order to verify the functions of other enzymes, AO-mgcCDEF and AO-mgcCDEFG mutant strains are further fermented and subjected to HR-LCMS detection respectively. The results showed that no other products were detected except for the same products produced by the AO-mgcDEF mutant (FIG. 5). In addition, the fermentation test of AO-mgcABCDEFG, a strain expressing the entire gene cluster, revealed that

compounds

4 and 5 disappeared (FIG. 5), thereby indicating that mgcA and mgcB are regulatory genes and inhibit the expression of the entire gene cluster.

EXAMPLE 5 enrichment and purification of terpenoids

In order to obtain enough products for compound structure identification, the invention performs mass fermentation on Aspergillus oryzae mutant strains AO-mgcDE and AO-mgcDEF. Placing the rice culture medium (5-10kg) with thallus into 30 deg.C incubator, and standing for 20-25 d. Crushing the cultured thallus with a stirrer, adding equal volume of ethyl acetate for extraction for 3-4 times, combining the extracted organic layers, and concentrating with a rotary evaporator to obtain crude extracts A and B. Carrying out reverse silica gel column chromatography on the crude extract A, carrying out gradient elution on an n-hexane-ethyl acetate system, and collecting fractions with the volume ratio of n-hexane to ethyl acetate of 5: 1; and (3) carrying out semi-preparative HPLC purification on the fraction, eluting with pure acetonitrile and ultrapure water as mobile phases in a volume ratio of 75:25 to 90:10, and collecting the fraction to obtain the sesterterpene compounds shown in formulas I-III. Carrying out reverse silica gel column chromatography on the crude extract B, carrying out gradient elution on a petroleum ether-ethyl acetate system, and collecting fractions with the volume ratio of petroleum ether to ethyl acetate being 20: 1; carrying out gradient elution on the fraction by a chloroform-acetone system, and collecting the fraction with the chloroform/acetone volume ratio of 15: 1; purifying the fraction by HPLC, eluting with acetonitrile/water at a volume ratio of 45:55 as mobile phase, and collecting the fraction to obtain sesterterpene compounds shown in formula IV and formula V.

Example 6 terpenoid Structure identification

The present invention uses NMR and HR-LCMS to determine the structure of the compounds. By passing¹H、¹³C. And (3) collecting HMBC, HSQC, ROESY, COSY and DEPT spectra, and combining the molecular weight detected by HR-LCMS to finally determine the molecular structure.

Compound (1) was a white powder. HR-LCMS result shows that the compound 1 is C₂₅H₄₀O₂，m/z373.3088，[M+H]⁺(calculated value373.3101)。¹The H NMR data suggest that the compound (1) has 6 methyl signals delta_H(1.38,3H, overlap; 1.38,3H, overlap; 1.07,3H, s; 1.05,3H, brs; 0.82,3H, s; 0.80,3H, brs), 1 alkene hydrogen delta_H(5.38,1H, d, J ═ 1.7Hz) (table 1, fig. 7). According to¹³C NMR and HSQC spectra can confirm that the compound has 25 carbons, respectively 5 methines, and contains 1 olefinic carbon delta_C(130.0); 8 methylene groups; 6 methyl delta_C(30.3,26.6,26.6,22.4,21.8, 21.5); and 6 quaternary carbons including 1 vicinal quaternary carbon delta_C(76.3), 1 olefin quaternary carbon. delta_C(144.6) and 1 carbon radical of delta_C(215.0) (fig. 8 and 10). The above data suggest that compound (1) belongs to the Mangicol class of tetracyclic sesterterpene skeleton compounds.¹H-¹The coupling relation suggested by H COSY is as follows: H-1/H-2/H₂-3/H₂-4/H-5/H₃-23，H₂-7/H₂-8/H-9/H₃-24，H₂-13/H₂-14 and H₂-16/H₂-17 (fig. 9). In addition, the following key signals can be obtained according to the HMBC map: h-23 and C-6; h-2 and C-4, C-5, C-7, C-15; h-1 and C-3, C-16, C-25; h-24 and C-8, C-9, C-10; h-11 with C-1, C-6, C-9, C-13; h-2, H-13 and H-17 with C-15; h-20 and H-16 with C-18 (FIGS. 6 and 11). Thus, the planar structure of the compound (1) was determined to be a tetracyclic sesterterpene. The relative configuration of this compound can be determined from the ROESY spectra (fig. 6 and 12), first from the following key signals: the association of H-1 with Me-25, H-1 with H-16a and H-2 with Me-22 suggests that the relative configurations of H-2 and Me-22 are identical, and the configurations of H-1 and Me-25 are identical, and are defined as α -and β -orientations, respectively. In addition, the orientation of the relative configuration of Me-23 and Me-24 can be determined as β -orientation based on the correlation of Me-23 and Me-25 and the correlation of Me-23 and Me-24.

TABLE 1 preparation of Compound 1¹H and¹³C-NMR data attribution.

Compound (2) is a white solid. HR-LCMS result shows that the compound 2 is C₂₅H₃₈O₂，m/z371.2932，[M+H]⁺(calculated value371.2944)。¹The H NMR data suggest the presence of 5 methyl signals delta for compound (2)_H(1.37,3H, s; 1.09,3H, s; 1.07,3H, s; 0.83,3H, overlap; 0.82,3H, overlap), 1 alkene hydrogen delta_H(5.39,1H, d, J ═ 1.7Hz) (table 2, fig. 14). According to¹³C NMR and HSQC spectra can confirm that the compound has 25 carbons, respectively 5 methines, and contains 1 olefinic carbon delta_C(130.2); 9 methylene groups containing 1 vicinal oxymethylene group delta_C(62.3); 5 methyl delta_C(30.3,26.7,22.4,21.6, 21.2); and 6 quaternary carbons including 1 vicinal quaternary carbon delta_C(69.1), 1 olefin quaternary carbon. delta_C(144.8) and 1 carbon radical of delta_C(210.5) (fig. 15 and 17). The data indicate that the compound (2) belongs to the Mangicol tetracyclic sesterterpene skeleton compoundA compound (I) is provided.¹H-¹HCOSY suggests the coupling relationship: H-1/H-2/H₂-3，H₂-4/H-5/H₃-23，H₂-7/H₂-8/H-9/H₃-24，H₂-13/H₂-14 and H₂-16/H₂-17 (fig. 13 and 16). In addition, the following key signals can be obtained according to the HMBC map: h-23 and C-4, C-6; h-2 with C-4, C-5, C-7, C-15; h-1 and C-3, C-16, C-25; h-24 and C-8, C-9, C-10; h-11 with C-1, C-6, C-9, C-13; h-2, H-13 and H-17 with C-15; h-20 and C-18, C-21; h-16 and C-18 (FIGS. 13 and 18). Thus, the planar structure of compound (2) was determined to be a tetracyclic sesterterpene. The relative configuration of this compound can be determined from the ROESY spectra (fig. 13 and 19), first from the following key signals: the association of H-1 with Me-25, H-1 with H-16a and H-2 with Me-22 suggests that the relative configurations of H-2 and Me-22 are identical, and the configurations of H-1 and Me-25 are identical, and are defined as α -and β -orientations, respectively. In addition, the orientation of the relative configuration of Me-23 and Me-24 can be determined as β -orientation based on the correlation of Me-23 and Me-25 and the correlation of Me-23 and Me-24.

TABLE 2 preparation of Compound 2¹H and¹³C-NMR data attribution.

Compound (3) is a white solid. HR-LCMS result shows that compound 3 is C₂₅H₃₈O₃，m/z 387.2883，[M+H]⁺(calculated value387.2894)。¹The H NMR data suggest the presence of 5 methyl signals delta for compound (3)_H(1.57,3H, s; 1.10,3H, s; 1.06,3H, d, J-7.1 Hz; 0.98,3H, s; 0.81,3H, d, J-7.4 Hz), 3 concatenated hydrogen signals delta_H(4.26,1H, m; 2.94,1H, d, J ═ 4.9 Hz; 2.89,1H, d, J ═ 4.9Hz), and 1 alkene hydrogen δ_H(5.38,1H, d, J ═ 1.7Hz) (table 3, fig. 21). According to¹³C NMR and HSQC spectra can confirm that the compound has 25 carbons, respectively 6 methines, and contains 1 vicinal oxymethylene delta_C(70.6) and 1 olefinic carbon δ_C(130.0); 8 methylene groups containing 1 oxygen linkageMethylene delta_C(52.6); 5 methyl delta_C(30.7,23.8,21.9,21.6, 17.3); and 6 quaternary carbons including 1 vicinal quaternary carbon delta_C(58.7), 1 olefin quaternary carbon. delta_C(144.5) and 1 carbon radical of delta_C(211.1) (FIGS. 22 and 24). The above data suggest that compound (3) belongs to the Mangicol class of tetracyclic sesterterpene skeleton compounds.¹H-¹The coupling relation suggested by H COSY is as follows: h₂-7/H₂-8/H-9/H₃-24，H-5/H₃-23，H-1/H-2/H₂-3/H₂-4 and H ₂16/H-17 (FIGS. 20 and 23). In addition, the following key signals can be obtained according to the HMBC map: h-24 and C-8, C-9, C-10; h-11 with C-1, C-6, C-9, C-13; h-25 and C-1, C-11; h-1 and C-3, C-16; h-2 with C-4, C-5, C-7, C-15; h-17 and C-15; h-20 and H-16 are related to C-18 (FIGS. 20 and 25). Therefore, the planar structure of the compound (3) was determined to be a tetracyclic sesterterpene compound. The partial relative configuration of this compound can be confirmed by ROESY spectra (FIGS. 20 and 26), first by the NOE effect present in H-1 and Me-25, H-1 and H-16a, and H-2 and Me-22, indicating that the H-2 and Me-22 relative configurations are identical, and the H-1 and Me-25 configurations are identical, defined as α -and β -orientations, respectively. In addition, the relative configuration of Me-23 and Me-24 can be oriented in the beta-orientation based on the NOE effect of Me-23 and Me-25 and the NOE effect of Me-23 and Me-24. The NOE effect of H-17 and H-22 indicates that H-17 is in the alpha-orientation.

TABLE 3 preparation of Compound 3¹H and¹³C-NMR data attribution.

Compound (4) was a white solid. HR-LCMS result showed that compound 4 was, compound 4 was C₂₅H₄₀O₅，m/z 419.2796，[M-H]^-(calculated value 419.2803)。¹H NMR data suggest the presence of 5 methyl signals delta for Compound (4)_H(1.36,3H, s; 1.12,3H, s; 1.08,3H, d, J-7.1 Hz; 0.96,3H, s; 0.86,3H, d, J-7.3 Hz), 4 concatenated hydrogen signals delta_H(3.70,1H,dd, J ═ 11.2,6.4 Hz; 4.69,1H, t, J ═ 7.6 Hz; 3.95,1H, d, J ═ 11.0 Hz; 3.52,1H, d, J ═ 11.0Hz), and 1 alkene hydrogen δ_H(5.47,1H, d, J ═ 1.7Hz) (table 4, fig. 28). According to¹³C NMR and HSQC spectra can confirm that the compound has 25 carbons, which are respectively 7 methines, and 2 vicinal oxymethylene delta are contained_C(73.7,82.8) and 1 olefinic carbon δ_C(132.9); 7 methylene groups containing 1 vicinal oxymethylene group delta_C(68.6); 5 methyl delta_C(30.5,23.3,22.4,21.2, 20.9); and 6 quaternary carbons including 1 vicinal quaternary carbon delta_C(80.1), 1 olefin quaternary carbon. delta_C(141.4) and 1 carbon radical of delta_C(216.3). The above data suggest that compound (4) belongs to the Mangicol class of tetracyclic sesterterpene skeleton compounds (fig. 29 and 31).¹H-¹HCOSY suggests the coupling relationship: h₂-7/H₂-8/H-9/H₃-24，H₂-3/H₂-4/H-5/H₃-23, H-1/H-2 and H ₂16/H-17 (FIGS. 27 and 30). In addition, the following key correlation signals can be obtained from HMBC mapping (fig. 27 and 32): h₃-24 and C-8, C-9, C-10; h-11 with C-1, C-6, C-9, C-12, C-13; h-1 and C-3; h-2 with C-4, C-5, C-7, C-15; h-17 and H-16 with C-15; h-20 and C-18. Thus, the planar structure of compound (4) was determined to be a tetracyclic sesterterpene. The partial relative configuration of this compound can be determined by ROESY spectroscopy (FIGS. 27 and 33), first by the NOE effect present in H-1 and Me-25, H-1 and H-16a, H-7 and H-2, and H-2 and Me-22, indicating that the H-2, H-7 and Me-22 relative configurations are identical, and the H-1 and Me-25 configurations are identical, defined as the α -and β -orientations, respectively. In addition, the relative configuration of Me-23 and Me-24 can be oriented in the beta-orientation based on the NOE effect of Me-23 and Me-25 and the NOE effect of Me-23 and Me-24. The NOE effect of H-17 and H-22 and H-7 and H-2 indicates that H-7 and H-17 are in alpha-orientation.

TABLE 4 preparation of Compound 4¹H and¹³C-NMR data attribution.

Compound (5) is a white solid. HR-LCMS result showed Compound 5 to be C₂₅H₄₀O₅，m/z419.2796，[M-H]^-(calculated value419.2803)。¹The H NMR data suggest the presence of 5 methyl signals delta for compound (5)_H(1.38,3H, s; 1.03,3H, s; 0.82,3H, d, J ═ 7.3 Hz; 1.19,3H, d, J ═ 7.2 Hz; 1.11,3H, s), 4 concatenated hydrogen signals delta_H(4.72,1H, m; 3.97,1H, d, J ═ 10.8 Hz; 3.82,1H, m; 3.55,1H, d, J ═ 10.8Hz), and 1 alkene hydrogen δ_H(5.46,1H, s) (Table 5, FIG. 35). According to¹³C NMR and HSQC spectra (FIGS. 36 and 38) confirmed the presence of 25 carbons, 7 methines each, containing 2 vicinal oxymethylene δ groups_C(73.7,78.0) and 1 olefinic carbon δ_C(131.2); 7 methylene groups containing 1 vicinal oxymethylene group delta_C(68.6); 5 methyl delta_C(30.2,23.8,22.5,21.4, 18.5); and 6 quaternary carbons comprising 1 vicinal oxygen quaternary carbon, 1 olefinic quaternary carbon, and 1 carbyl delta_C(80.1,141.5,216.3). The combination of the above data suggests that compound (5) belongs to the Mangicol class of tetracyclic sesterterpene skeleton compounds.¹H-¹The signals suggested by H COSY are: h₂-7/H-8/H-9/H₃-24，H-5/H₃-23, H-1/H-2 and H-16/H-17 (FIGS. 34 and 37). In addition, the following key correlation signals can be obtained from HMBC mapping (fig. 34 and 39): h₃-24 and C-8, C-9, C-10; h-11 with C-1, C-6, C-9, C-13; h-1 and C-3, C-16, C-11; h-2 with C-4, C-5, C-7, C-15; h-16 and C-1, C-8; h-17 and H-16 with C-15; h-20 and C-18. Thus, the planar structure of the compound (5) was determined to be a tetracyclic sesterterpene. The partial relative configuration of this compound was confirmed by ROESY spectroscopy (FIGS. 34 and 40), first by NOE correlation with the presence of H-1 and Me-25, H-1 and H-16a, H-7 and H-2, and H-2 and Me-22, indicating that the H-2, H-7 and Me-22 relative configurations are identical, and the H-1 and Me-25 configurations are identical, defined as α -and β -orientations, respectively. In addition, the relative configuration of Me-23 and Me-24 can be determined to be oriented in the beta-orientation based on the NOE correlation existing in Me-23 and Me-25 and the NOE correlation existing in Me-23 and Me-24. Second, the NOE correlation between H-17 and H-22 indicates that H-17 is in the alpha-orientation, and the NOE correlation between H-8 and H-5 and H-2 indicatesH-8 is in the alpha-orientation.

TABLE 5 preparation of Compound 5¹H and¹³C-NMR data attribution.

Example 7 anti-inflammatory Activity assay of terpenoids

(1) In vitro anti-inflammatory Activity assay

In this example, Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 was used as an in vitro inflammatory cell model. RAW264.7 mouse macrophages are scaled up to 3.0X 10⁵Cells/well were seeded in 24-well plates at 37 ℃ with 5% CO₂After 24 hours of incubation in an incubator, LPS (1. mu.g/mL) was allowed to act on macrophages of RAW264.7 mice to mold. Respectively acting compounds 1-5 and positive control drugs (L-NMMA, indometacin and diclofenac) (the drug concentration is 10 mu M) on model cells, after 16h, taking the supernatant, detecting the NO level in the supernatant by a Griess reagent color development method, and evaluating the anti-inflammatory activity of the compounds by taking the NO release inhibition capacity of the tested drugs as a screening index. The inhibitory effect of compounds 1-5 and positive control drugs on NO in cell lines is shown in FIGS. 41-42. The results show that the compounds 1-5 have inhibition effect on the generation amount of NO in mouse macrophage RAW264.7 induced by LPS, wherein the inhibition effect of the compound 3 is most obvious, and the compounds have anti-inflammatory activity and can be used for preparing novel anti-inflammatory activity medicaments.

(2) In vivo anti-inflammatory Activity assay

In this example, phorbol ester (PMA) induced ear swelling in mice was used as an in vivo model of inflammation. PMA was dissolved in acetone (acetone), applied to the inner and outer sides of the right ear of a mouse at an amount of 2. mu.g/ear to induce inflammation, and the left ear of a mouse was applied with an equal amount of acetone as a control. 1h after induction, topical administration (100. mu.g/ear, 100. mu.g in 20uL DMSO). At 6h after the administration, the thickness of the mouse ear was measured with a caliper, and the result was calculated as right ear (drug-affected group) -left ear (negative control group). 4 biological replicates (n-4) were set per group, and variance and Student's t-test (p <0.05) statistical analysis were calculated. The results show that the compounds 1-5 have inhibition effect on mouse ear swelling induced by PMA, and show that the compounds have anti-inflammatory activity in vivo.

Sequence listing

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Gln Thr Lys Gly Glu Leu Thr Ser Thr Tyr Asp Pro Val Gln Ala Ala

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Lys Glu Ala Met Met Glu Ser Thr Arg Pro Glu Ile His Arg Leu Pro

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Thr Pro Asp Ser Pro Arg Lys Glu Ser Phe Ala Val Arg Pro Leu Val

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Asn Gly Ser Gly Gln Tyr Asn Gly Asn Asn His Ile Asn Gly Val Ser

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Asn Glu Val Asp Val Arg Pro Ser Ile Glu Arg His Ala Ser Thr Lys

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Arg Ala Thr Ser Ala Asp Asp Ile Asp Trp Thr Ala His Lys Lys Val

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Val Met Glu Pro Tyr Arg Tyr Leu Cys Ser Leu Pro Ser Lys Gly Val

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Arg Asn Lys Thr Ile Asp Ala Leu Asn Phe Trp Leu Lys Val Pro Ile

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Glu Asn Ala Asn Thr Ile Lys Ala Ile Thr Glu Ser Leu His Gly Ser

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Ser Leu Met Leu Asp Asp Ile Glu Asp His Ser Gln Leu Arg Arg Gly

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Lys Pro Ser Ala His Ala Val Phe Gly Glu Ala Gln Thr Ile Asn Ser

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Ala Thr Phe Gln Tyr Ile Gln Ser Val Ser Leu Ile Ser Gln Leu Arg

515 520 525

Ser Pro Lys Ala Leu Asn Ile Phe Val Asp Glu Ile Arg Gln Leu Phe

530 535 540

Ile Gly Gln Ala Tyr Glu Leu Gln Trp Thr Ser Asn Met Ile Cys Pro

545 550 555 560

Pro Leu Glu Glu Tyr Leu Arg Met Val Asp Gly Lys Thr Gly Gly Leu

565 570 575

Phe Arg Leu Leu Thr Arg Leu Met Ala Ala Glu Ser Thr Thr Glu Val

580 585 590

Asp Val Asp Phe Ser Arg Leu Cys Gln Leu Phe Gly Arg Tyr Phe Gln

595 600 605

Ile Arg Asp Asp Tyr Ala Asn Leu Lys Leu Ala Asp Tyr Thr Glu Gln

610 615 620

Lys Gly Phe Cys Glu Asp Leu Asp Glu Gly Lys Phe Ser Leu Pro Leu

625 630 635 640

Ile Ile Ala Phe Asn Glu Asn Asn Lys Ala Pro Lys Ala Val Ala Gln

645 650 655

Leu Arg Gly Leu Met Met Gln Arg Cys Val Asn Gly Gly Leu Thr Phe

660 665 670

Glu Gln Lys Val Leu Ala Leu Asn Leu Ile Glu Glu Ala Gly Gly Ile

675 680 685

Ser Gly Thr Glu Lys Val Leu His Ser Leu Tyr Gly Glu Met Glu Ala

690 695 700

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

705 710 715 720

Glu Leu Ile Leu Glu Met Leu Arg Ile Asp

725 730

<210> 5

<211> 534

<212> PRT

<213> Fusarium (Fusarium guttiferme)

<400> 5

Met Glu Arg Leu Lys Met Asp Ser Leu Asn Ile Asn Ile Asn Asp Trp

1 5 10 15

Phe Glu Lys Asp Leu Pro Ala Thr Ala Asp Trp Lys Leu Phe Ala Leu

20 25 30

Ala Ser Val Val Phe Val Val Leu Arg Phe Thr Cys Ile Val Ile Tyr

35 40 45

Arg Ile Tyr Phe Ser Pro Leu Ser Lys Phe Pro Gly Pro Lys Leu Ala

50 55 60

Ala Ala Thr His Leu Tyr Glu Ser Tyr Tyr Asp Phe Trp Lys Lys Gly

65 70 75 80

Gln Tyr Tyr Lys Val Ile Gln Arg Met His Glu Val Tyr Gly Pro Leu

85 90 95

Val Arg Val Thr Pro Asp Glu Leu Ser Ile Asn Asp Pro Asp Tyr Tyr

100 105 110

Asp Thr Val Tyr Val Asn Gly Asn Val Arg Arg Thr Glu Ser Phe Gly

115 120 125

His Ser Phe Gly Gly Gly Leu Gly Ile Glu Asp Thr Phe Phe Ala Ser

130 135 140

Gln Asp His Asp Leu His Arg Lys Arg Arg Lys Pro Ile Glu Pro Tyr

145 150 155 160

Phe Ser Arg Asn Gly Val Leu Lys Leu Glu Asn Leu Ile Gly Glu Arg

165 170 175

Val Glu Lys Leu Phe His Lys Phe His Glu Leu Ser Gly Thr Gly Val

180 185 190

Val Ala Arg Leu Asp Tyr Ala Phe Glu Ala Phe Thr Gly Asp Val Met

195 200 205

Gln His Ile Cys Ile Glu Lys Pro Glu Ser Leu Leu Asn Ser Asp Asp

210 215 220

Phe Ser Ser Glu Trp Phe Glu Met Leu Arg Asn Val Ser Leu Ser Val

225 230 235 240

Pro Leu Met Gly Met Ile Pro Trp Leu Val His Val Leu Lys Phe Ile

245 250 255

Pro Glu Ser Val Ile Met Trp Leu Ala Pro Ser Ala Ala His Phe Gln

260 265 270

Thr Phe Arg Val Gln Ala Gly Arg Gln Ile Glu Gln Ala Lys His Glu

275 280 285

Lys Val Glu Asn Asp Arg Lys Gly Ile Thr Thr Val Gly Gly Lys Pro

290 295 300

Thr Leu Phe Arg Phe Leu Val His Glu Ser Gly Leu Ala Pro Glu Asp

305 310 315 320

Leu Ser Thr Glu Arg Leu Gln Lys Glu Ala Met Val Leu Leu Gly Gly

325 330 335

Gly Thr Thr Thr Thr Ala Arg Thr Ala Thr Met Thr Cys Phe Trp Met

340 345 350

Leu Ser Met Pro Glu Lys Gly Gln Arg Leu Arg Asp Glu Leu Lys Asp

355 360 365

Ile Met Ala Glu Tyr Pro Lys Lys Lys Pro Ser Leu Thr Glu Leu Glu

370 375 380

Lys Leu Pro Tyr Leu Gly Ala Val Ile Gln Glu Ser Leu Arg Met Ala

385 390 395 400

Tyr Gly Ser Met Arg Arg Leu Pro Arg Thr Ser Pro Asp Val Ala Leu

405 410 415

Gln Phe Lys Asp Trp Val Ile Pro Pro Gly Thr Pro Val Gly Met Asn

420 425 430

Ala Tyr Tyr Leu His Thr Asp Pro Asn Ala Phe Pro Glu Pro Phe Glu

435 440 445

Tyr Lys Pro Glu Arg Trp Leu Gly Asn Val Thr Pro Ala Met Lys Arg

450 455 460

Ser Phe Val Pro Phe Ser Arg Gly Ser Arg Arg Cys Pro Gly Ser Ser

465 470 475 480

Leu Ala Leu Ala Asp Leu His Phe Val Leu Ala Ala Leu Phe Gly Pro

485 490 495

Thr Gly Pro Lys Phe Glu Leu Phe Glu Ser Asp Arg Ser Asp Val Asp

500 505 510

Ala Ile His Asp Tyr Leu Met Pro Leu Pro Arg Leu Asp Ser Lys Gly

515 520 525

Val Arg Val Thr Val Lys

530

<210> 6

<211> 512

<212> PRT

<213> Fusarium (Fusarium guttiferme)

<400> 6

Met Glu Leu Pro Ser Phe Ser Leu Lys Phe Asp His Glu Lys Leu Leu

1 5 10 15

Pro Leu Ala Ile Ile Ser Thr Gly Leu Leu Val Thr Ser Leu Val Ala

20 25 30

Ile Ser Ile Tyr Arg Ile Trp Phe His Pro Leu Ala Ile Phe Pro Gly

35 40 45

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

50 55 60

Ile Ser Gly Thr Trp Ile Trp Arg Val Ser Ser Leu His Arg Lys Tyr

65 70 75 80

Gly Pro Ile Val Arg Ile Gly Pro Asn Arg Leu Ala Val Asp Gly Ser

85 90 95

Ile Gly Trp Phe Gln Val Tyr Ala Met Arg Gly Lys Glu Asp Glu Phe

100 105 110

Pro Lys Tyr Pro Glu Tyr Ile Phe Pro Gly Asp Gly Leu Ser Ile Leu

115 120 125

Gly Ala Asn Gln Val Asn His Arg Arg His Arg Arg Gln Phe Trp Ser

130 135 140

Ala Phe Asn Asp Gln Ala Leu Val Glu Gln Glu Ile Val Ile Gln Pro

145 150 155 160

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

165 170 175

Lys Pro Ile Asn Ile Val Asp Trp Ile Asn Phe Leu Leu Phe Asp Ile

180 185 190

Ala Gly Glu Leu Val Phe Ser Ser Pro Phe Asp Cys Leu Asp Lys Gln

195 200 205

Glu Tyr His Pro Trp Val Ala Asn Phe Phe Arg Ala Val Lys Gly Asn

210 215 220

Ala Val Asn Arg Phe Val Thr His Tyr Pro Ile Thr Lys Pro Ile Val

225 230 235 240

Asn Phe Leu Phe Thr Gly Lys Glu Gln Ile Gln Arg Glu Ala Asp Gln

245 250 255

Arg Asn Met Thr Phe His His Ala Met Gln Arg Met Lys Leu Gly Glu

260 265 270

Gln Pro Thr Pro Gly Arg Arg Asp Phe Met Ser Phe Leu Met Arg Arg

275 280 285

Asn Arg Asp Gly Gly Gly Leu Ser Asp Thr Glu Ile Leu Val Asp Cys

290 295 300

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

305 310 315 320

Gly Phe Phe Phe Tyr Leu Gly Ile Ser Pro Gln Ala Tyr Lys Arg Leu

325 330 335

Val Glu Glu Val Arg Ser Ser Phe Lys Ser Glu Ser Glu Ile Asn Met

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Val Tyr Gln Trp Gly Thr Phe His Asn Pro Asp Asn Phe Ala Asp Pro

405 410 415

Asp Glu Phe Ile Pro Glu Arg Trp Leu Gln Pro Ser His Pro Leu His

420 425 430

Asn Pro Lys Tyr Asp Asn Asp Asn Arg Ser Val Tyr Arg Pro Phe Gly

435 440 445

Phe Gly Met Arg Asp Cys Leu Gly Lys Asn Leu Ala His Ala Glu Ile

450 455 460

Arg Val Val Val Ser Arg Ile Leu Tyr Arg Phe Asp Tyr Glu Leu Ala

465 470 475 480

Pro Asn Gln Glu Asn Trp His Ala Asn Gln Lys Cys Phe Met Ala Trp

485 490 495

Asp Lys Thr Pro Leu Ile Leu Thr Leu Lys Pro Arg Asp Phe Ala Pro

500 505 510

<210> 7

<211> 614

<212> PRT

<213> Fusarium (Fusarium guttiferme)

<400> 7

Met Arg Val Ala Val Val Gly Ala Gly Pro Gly Gly Leu Val Thr Leu

1 5 10 15

Lys Tyr Leu Lys Glu Ala Thr Lys Phe Phe Asp Val Asp Pro Ile Asp

20 25 30

Val Arg Leu Phe Glu Arg Glu Asp Glu Val Gly Gly Thr Phe Thr Lys

35 40 45

Arg Thr Tyr Glu Asp Ala Glu Leu Val Ser Ser Lys Tyr Leu Thr Cys

50 55 60

Phe Ser Asp Trp Arg Ala Asp Leu Glu Asp Pro Asp Phe Leu Ser Ala

65 70 75 80

Asp Arg Phe Ile Arg Tyr Leu Lys Glu Tyr Ala Asp Tyr Phe Asn Leu

85 90 95

Trp Pro Glu Ile Ser Leu Ser Thr Pro Val Thr Ser Ile Arg Arg Gly

100 105 110

Gln Ala Gly Gly His Ile Val His Tyr Arg Gly Pro Asp Gly Ile Asp

115 120 125

Lys Thr Trp Glu Cys Asp Ala Val Ala Val Cys Ser Gly Leu His Val

130 135 140

Thr Pro Asn Ile Pro Asp Val Pro Gly Ile Asp Lys Val Lys Ile Val

145 150 155 160

Lys His Ser Ser Gln Phe Lys Lys Arg Asp Glu Phe Pro Gln Gly Ser

165 170 175

Gln Val Val Val Leu Gly Thr Gly Glu Thr Gly Met Asp Ile Ala His

180 185 190

Leu Ala Val Thr Ser Pro Thr Lys Arg Val Val Leu Cys His Arg Gln

195 200 205

Gly Phe Leu Gly Ala Pro Lys Lys Ile Pro Asn Pro Ile Leu Phe Pro

210 215 220

Ile Leu Gly Asn Lys Pro Asn Pro Asn Ala Gln Glu Leu Pro Ile Asp

225 230 235 240

Val Ser Trp Gln Ala Pro Leu Leu Asp Ser Tyr Leu Pro Pro Phe Leu

245 250 255

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

260 265 270

Ala Asn Trp Leu Cys Ser Gly Thr Thr Ala Gly Val Asp Gln Trp Ile

275 280 285

Gly Gly Leu Asp Ala Asp Arg Phe His Thr Ser Gln Ser Phe Phe Asn

290 295 300

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

305 310 315 320

Thr Asn Pro Gly Leu Val Glu Arg Ile Arg Arg Ala Ile Val Thr Ile

325 330 335

Pro Val Lys Glu Val Pro Gly Asn Lys Tyr Ile Asp Leu Ala Pro Trp

340 345 350

Pro Thr His Ile Asp Asp Lys Gly Ile Met His Phe Lys Glu Asn Gly

355 360 365

Arg Pro Glu Ala Glu Arg Met Lys Thr Leu Gly Pro Val Lys Pro Asp

370 375 380

Met Ile Val Tyr Ala Thr Gly Tyr Arg Gln Glu Phe Gly Phe Phe Asp

385 390 395 400

Glu Ala Asn Lys Asn Gly Glu Asp Tyr Pro Thr Cys Ser Asp Ala Asp

405 410 415

Val Arg Cys Ile Trp Arg His Asn Asp Pro Thr Val Gly Phe Ile Gly

420 425 430

Phe Ile Arg Pro Gly Tyr Gly Ala Ile Pro Pro Leu Ala Glu Leu Gln

435 440 445

Ala Gln Leu Trp Leu Met Thr Leu Ile Lys Pro Glu Val Ala Lys Ser

450 455 460

Leu Ser Ser Arg Glu Glu Tyr His Phe Lys Leu His Gly His Lys Arg

465 470 475 480

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

485 490 495

Asp Met Asp Ala Val Pro Ser Phe Trp Asp Gly Val Arg Val Gly Trp

500 505 510

Asn Ala Gly Ala Lys His Pro Gly Leu Trp Trp Arg Leu Pro Val Leu

515 520 525

Trp Leu Thr Gly Ala Gln Phe Asn Thr Lys Phe Arg Val Val Gly Pro

530 535 540

Tyr Gln Trp Asp Gly Ala Val Asp Val Leu Gly Gly Glu Leu Trp Glu

545 550 555 560

Thr Ile Thr Arg Arg Glu Gly Leu Phe Gly Ala Phe Val Met Thr Ile

565 570 575

Val Pro Met Thr Met Val Gly Thr Ser Ser Ile Ile Met Trp Phe Val

580 585 590

Gly Leu Phe Thr Ala Leu Leu Ser Ala Ile Gly Ser Phe Ala Lys Gly

595 600 605

Thr Phe Trp Arg Val Val

610

<210> 8

<211> 1221

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 8

atgccgctga gatcgcagga gttgtttcac tacttttgtg gaaatgcttc agcgtttagt 60

gctttaccga aagaccacag aaacaacttt cttgcctaca caatctcaaa ccccgaggcg 120

cttagaagtg cagtactaat ggcgggaata catttcgctt tcaacattgg ccatctagac 180

aaatttgaac cgacatttct gtatcacaag attgagactg tacagcaagt aaggaagttg 240

atatctaggg gagacctcaa gctacttgct gggatcacca agcagatctc tactctggct 300

tatgcagagc tctgtcgagg tgatgtaaaa ttggcagaga ctcacctgag cgttatctac 360

gctctttcaa atcgactaca aggtcaacag aatgaccagt gcaaaactct cgatcaagag 420

ctttcagatc ggtattttct tctaacatcg acttttgtca acggtttaga aagcctcatc 480

aaaggcgttg cttgcaaaca aggcctgggc ggcagcgtga ccaccatgga gttgagcgag 540

acaatgaact tcctccacaa tttccacttg acctctggcc aattctcgca caagaacaca 600

gtcaaggccg ttcggctaat tccagccttc ttcgatgccc ctcatgatgg cgcacagtta 660

ctcgacattg actacaggcc tatacttgag tgtctccagg gattagacga gaatcctggc 720

ccgaacgaac aatatgactt ttggctgtac ggtcgggcct cgacattttg gaccaatatc 780

atcaacgcac atttgaactc tatctactac gagggcgata gtagtgagtc caacgctact 840

acgcccgaag actcgaggta catgacgccc tggtgtgctt tgctggcggc agtaaagttt 900

tatgtcgaac aggttgttat tatatggcga cccttgagaa gggagatatt cctccacgct 960

ttacgtatac tgcagcgtga tattgcggtt gctatgcaaa agcctgtgtc acttcaacta 1020

ccagagatga tattatggga gtcgtttctt gggttggtga gtatccgtgg gcatgagaag 1080

ttcggggata tggaccagga acctggtctg aggcccttct tcgaagagat taagccttca 1140

acaatgctgg ctgctgctgc tgctgctgct gcctcgccaa tcccgacctc gtgtctcaca 1200

atcaagttac ccctgttgtg a 1221

<210> 9

<211> 1473

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 9

atgagttctg tcccacctgg tagtatctca agacttctgt gggtgtcttt cgatggtacc 60

ctagccggcg gatttggcag ctgtcgcgac acagttgtct cagtcttgcc tgacctcata 120

tctcagagcg aaaacctcaa ccaagttttc gtcccaggcg taggatccgg cttcacccct 180

ttcactcgcg tttttggtgt cctctgcggt tggggaaccc agcacaacgt catcactgca 240

tacagaagca ttgccacagc ttatgttcct ggtgataaaa ttatcctgtg cggattttct 300

cgaggtgcct gggctgctcg atacctcgcc caaatcatca gcgttcttgg tctacctaaa 360

cggtgtgata acaatttttt ccatctcttg gataaacaat gcgacaagga tccaacgttc 420

caatctcccg tcgaccccaa gttatggaaa tatgacaggt ggaacaatgt tgagatcgag 480

gccttgtgtt gcttcgacac agtcggctcc ctcgggctgc cattgtacgg tatcgctaag 540

ccactgtcca tctttcgccg ggggccaaga aaagcggata ttgtctctac agtagcaagc 600

aacgtcaaga attccttcca ctgcctagct ctccacgagc aacgagaacc gttcagtcca 660

acatacatga gagggaagaa tgtccatcaa gttttcttcg tcggtaacca cggcgatatg 720

ggctggattg accgacgcaa agaaagcttt gtccatgccc ctctcgcctg gattgtccaa 780

caactcgact ggcactctgg tatcctattc gatgaagtga agctcaaaga gtacttccct 840

agctatggcc gcgatccagg taacgactta ccgtgcatag acggtcccat cgcccgtaca 900

agccgtatta cacgactttt catggggatt aaggagcgcc agccatggaa tgtcgcgaac 960

ttctcgcgta ctgctaacaa cggcgagaac gatggtcatg acaccacggg cgatactatc 1020

ctctctgatg ttcagataca cgtcagtgct cgttattacg aacacccaga acttgctgtc 1080

ccgggctaca cacagaacgc ccgtatcgaa gagaaatttc actggataca acagagcaat 1140

tcacgccaga acagctcgag cagctcttta tccctcaagc agaaaccatg ttctgttgcc 1200

aatcacaaga gaattgcctg tctaccaggc tccgagccca ccaacaatgg tggtcagacc 1260

agacaccgca tttacccggc actcgtgggc cctttggagg ccagactgct agcattacca 1320

ccagcggctg tgtcttcccg ggcttgctgt gcgccaccat cagagacacc acaagatgaa 1380

tccacagtcc cagactccgt accggctaaa gggggagggg tcagacaagc tttctccaag 1440

ttcggcaaga gctttagctt ctctcgacga tga 1473

<210> 10

<211> 1212

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 10

atgactgtcg acattaagcc atacacgatc aatgtttcag actcagaaat tgagctcttg 60

aaaacaaaac tagagcacgc aaggtttccc tttgaaggcg aagtctctga cgactggacc 120

tacggtgcta gtctcagtga tgtgaagcgt ctcgctgcgt attggaaaga tggattcgac 180

tggagagctc aagaagccaa gctgaaccag tatccgcaat tcactactag tgtatccgtg 240

gatggctttg gggatcttga tattcatttc ttgcatcaga agagcagcaa acctgatagc 300

attcctctgc tcttcgttca tggctggcct ggaagctttg ttgaagttct gaagattctt 360

cctcttttga ctgaacccaa agatggacct tcgttccaca ttgttgcccc ttctttgcct 420

aaccatgtct tctcggatgg tgtatcgaag agcggttttg ggattcctcg atacgctgaa 480

actctacaca aactcatgat caagcttggt tacaacaaat atgtcactca aggaggcgat 540

tggggttacg tcatcacccg tctcatcggt tctcaatatc ccgagcactg cctcgcatca 600

cacatgagta tgattccagc tgtcagccct cctaaccccc tgaagactcc gtggcagttc 660

cttcgcttct ggctgtctcc tttcactcct ctggaaaagc aaggcattaa gcaaatgaag 720

catttctaca acgaaggtct tgcttacaac ctgataatga gcagcaagcc ctctactata 780

gggttcggtc ttgctgactc accagtggcc ttgctctcgt ggacttacga gaagctccat 840

gactggactg atgactacaa atggacagac gatgagatcc tgacatgggt ttccctctat 900

cagttttcca aagcagggcc ggctgcaagc tgcaggatct actatgagag tagacatgcg 960

gaccaggatc tcacgaagaa ggtcaacgac tgggttccga atgtccccct tgggctatcg 1020

tacttcccca aggacattgt ctttgtcccg aggacgtggg gaaggacttt gggacccatc 1080

gcgtttgaga agattcatac tagtggtgga cattttgctt caattgagag acctgaagag 1140

cttgtggagg acttgagaga gatgtttagt gagagtgaat tgggcaagca ggtcgctgag 1200

aagctgcgat aa 1212

<210> 11

<211> 2193

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 11

atggacttca cataccgcta ttcgttcgag cctacggact atgacactga cggtctctgt 60

gatggtgttc cggtccgtat gcacaagggt gcagacttgg acgaggttgc catcttcaaa 120

gctcagtatg actgggagaa gcatgttggt cctaagctgc cctttcgggg tgcattgggg 180

ccaagacaca acttcatctg tcttactctg ccagagtgct tgcctgagag actagagatt 240

gtgtcttacg ccaatgagtt tgccttcctt cacgatgata ttactgatgt cgagtcagct 300

gagacggttg ccgctgagaa cgatgagttc cttgatgccc ttcaacaagg tgttagagaa 360

ggtgacatcc agagccgtga gtccggaaag cgtcatctcc aggcttggat cttcaagtcc 420

atggtggcca ttgaccgtga tagagctgtg gccgctatga acgcttgggc cacctttatc 480

aacacaggtg caggatgcgc tcacgataca aacttcaagt cacttgatga gtatcttcac 540

tacagggcta cagatgtcgg ctacatgttc tggcacgctc ttatcatctt cggatgcgcc 600

atcaccattc ctgaacatga gattgagcta tgccatcaac tcgctcttcc agccatcatg 660

tccgtgactt tgacaaacga catctggtca tatggcaaag aagcagaggc agctgagaaa 720

tccggcaagc ccggagattt tgtcaacgct ctcgttgttc tgatgagaga gcacaactgc 780

tccattgaag aagccgagcg tctctgcaga gcgcgaaaca aaatcgaggt agccaagtgt 840

cttcaagtca caaaagagac acgagagcga aaagatgttt cacaagatct caaagattac 900

ctctaccata tgctgtttgg tgtcagtgga aatgcgatct ggagcactca gtgccgaaga 960

tatgacatga cagcgcctta caacgaaaga cagcaggcca gactcaagca gaccaagggt 1020

gagcttactt ccacatatga tcctgttcag gctgccaagg aggccatgat ggagtctact 1080

cgtcctgaga tccacagact gcctactccc gatagtccca ggaaggagag ctttgctgtt 1140

cgtcctttgg tgaatggcag tggacaatac aatggcaaca atcacatcaa tggagtctcc 1200

aatgaagttg acgtgcgtcc ttctattgag agacatgcct caaccaagcg agctacttca 1260

gctgatgaca tcgactggac ggcacataag aaggtcgtca tggaaccata ccgatatctg 1320

tgttctcttc cctcaaaggg agttagaaac aagactattg acgctcttaa cttctggctc 1380

aaggttccta ttgaaaatgc aaacaccatc aaggccatca ctgaaagcct tcacggatca 1440

tcactcatgc ttgatgatat cgaggaccat tcacaactgc gacgtggcaa gccttcggcc 1500

cacgctgttt ttggtgaggc acagaccatc aactctgcaa cattccagta cattcagtct 1560

gttagcctga ttagccagct tagaagccct aaggctttga acatctttgt tgatgagatt 1620

cgacaacttt tcatcggtca ggcttacgag ctccagtgga cctctaacat gatttgccca 1680

cctttggagg agtatttgcg aatggttgac ggaaaaactg gcgggttatt ccgccttctc 1740

actcgtctca tggctgctga gtccactact gaggtagatg ttgactttag ccgtctgtgc 1800

cagctttttg gtcgctactt ccagatccga gacgattacg ccaacctcaa gctcgcagac 1860

tacaccgaac aaaagggttt ctgtgaagac ctcgacgagg gcaagttctc actccctctc 1920

atcattgcct tcaacgagaa caacaaggcc cccaaagccg tagctcaact gcgcggcctc 1980

atgatgcagc gctgtgtcaa cggcggcctc acctttgaac agaaggtgct agcactgaat 2040

ctcattgagg aggctggtgg aatttcgggc acggagaagg tgctgcactc actttatggt 2100

gagatggagg ctgagctgga aaggttggct ggtgtctttg gggcggagaa tcatcagctt 2160

gagcttattc tggagatgct gcgtatagat tag 2193

<210> 12

<211> 1605

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 12

atggaaaggc tcaagatgga tagcctcaac atcaacatca atgactggtt cgagaaagac 60

ctgccagcca ctgcagactg gaagctcttt gctctagcca gtgttgtctt tgttgttcta 120

cgatttactt gcattgtcat ctatcgtatc tacttttctc ccctttccaa gttccctggt 180

cccaagctcg ctgctgcgac gcatctttat gagtcttact atgacttttg gaagaaaggg 240

cagtactaca aagtgattca gcgtatgcac gaggtctacg gaccgcttgt tcgtgtcacg 300

cctgatgaac tttcgatcaa cgaccctgac tattacgaca ctgtctatgt caacggtaat 360

gttcgacgta ctgagtcctt cggccattcc tttggtggcg gacttggtat tgaagacacc 420

ttcttcgcct ctcaggacca tgacctccac cgcaagagaa gaaaacccat cgagccttac 480

ttctctcgca atggtgtctt gaagctcgag aatcttatcg gtgaacgtgt tgagaaactg 540

ttccacaagt tccacgagct gtctggtact ggtgttgttg cccgtcttga ctatgccttt 600

gaggccttca ctggcgatgt catgcagcat atttgcattg agaagcctga atcactactc 660

aacagcgatg acttttcttc tgagtggttt gagatgcttc gcaatgtctc cttgtccgta 720

cctcttatgg gaatgatccc ttggcttgtc cacgtactga agttcatccc cgagagtgtc 780

atcatgtggc tcgcgccctc agctgcccac ttccagacct tccgtgttca agctggtcgt 840

cagattgagc aagccaagca cgagaaagtg gagaatgatc gcaaaggtat cactactgtc 900

ggcggcaagc ccaccctctt ccgcttcctt gtccacgaga gtggtctcgc accggaagac 960

ctgagcaccg agagactcca gaaggaggca atggttctac ttggcggtgg cactacaact 1020

actgcgcgta ctgcgaccat gacttgcttc tggatgctca gcatgcctga gaagggccaa 1080

cgtcttcgcg acgagctcaa ggacatcatg gccgagtacc ccaagaagaa gccttctttg 1140

accgagcttg agaagctgcc ttatttggga gctgtgatcc aagagagttt gagaatggcg 1200

tacggatcca tgcgtcgact tcctcgcact tctcccgatg tagctctgca gttcaaagat 1260

tgggtgatcc ctcctgggac tcctgttggc atgaatgctt attatcttca cactgatcct 1320

aatgcgttcc ccgagccatt tgagtacaag cccgaacgat ggcttggaaa tgtcacaccc 1380

gcgatgaagc gtagttttgt gcccttttcg cgcggttcgc gccgatgccc cggatctagc 1440

ttggctcttg ccgatctcca tttcgttctt gcagcattgt tcggaccaac tggacccaaa 1500

tttgagttgt tcgaatcgga caggtctgac gtggatgcca ttcatgacta cctgatgccg 1560

ctgcctcggt tggattccaa gggtgttcgc gtcactgtca agtag 1605

<210> 13

<211> 1539

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 13

atggaactac cgagcttctc cctcaagttc gatcatgaga agctcttgcc ccttgctatt 60

atcagcacag gccttctggt cacatctctt gtggccatat ccatctaccg catttggttt 120

catcctctcg ccatcttccc cggtcccaaa tggctcgtca tatccaacgt ccctgagcga 180

tacatgagta acatctcagg tacatggatt tggagagtct cgagtttaca tcgcaagtat 240

ggccctattg ttcgcattgg tccaaaccgt cttgccgttg atggaagtat tggatggttc 300

caagtctatg caatgagagg caaggaagat gagttcccca agtatcctga gtacattttc 360

cctggcgatg gcttgagtat ccttggtgcc aaccaggtca accatcgacg ccacaggcgc 420

cagttttggt ctgcattcaa cgatcaagcc cttgttgagc aggaaatcgt tatccagcct 480

tacaccgaca tgcttctcca gcgtctcagt gagcaagcca agatcggaaa gcccatcaac 540

atcgttgact ggatcaactt tctcctcttt gacattgcag gcgagctagt gttttcctcg 600

ccctttgatt gcctcgacaa gcaggaatac cacccatggg tagccaactt cttcagagct 660

gtcaagggta atgccgtgaa tcgcttcgtc acgcactacc ccatcaccaa acccattgtc 720

aactttctct tcactggcaa ggagcagatc cagcgtgagg ctgatcagcg aaacatgacg 780

ttccatcacg ccatgcagcg catgaagctt ggggagcagc caaccccagg tcgtcgtgat 840

tttatgagct tcttgatgcg gcgcaaccgt gatggcggtg gtctctcaga cactgagatc 900

cttgtcgact gtcccgttct gattggagcg tcgagtgaga ccaccaccac tgctctgtcg 960

ggcttcttct tctatcttgg tatctcccca caggcttaca agagacttgt ggaagaggtg 1020

cgatcttcat tcaaaagtga gagtgagatc aacatgaaga ctacgaagca gcttgagtac 1080

ctcaacgcca ccgttgacga agcattgcga gtttatcccc ctgctgccga gtcgcctccc 1140

agaattagcc ccggcgccga gattgatggc aaatatcttc ccaaaggagt cgttgtttcg 1200

gtctaccaat ggggaacctt ccacaacccc gacaactttg ccgacccgga tgagtttatc 1260

ccggagcgat ggctccagcc ctctcaccct ctgcataacc ccaagtatga caacgataat 1320

cgctcagtat accgcccgtt tgggtttggc atgagagatt gtcttggcaa gaacttggct 1380

catgcagaaa tccgggtcgt tgtcagtcgt atcctgtaca gattcgacta tgagttggct 1440

ccaaaccagg agaattggca tgccaaccag aaatgcttca tggcctggga taagacgccg 1500

cttatcttga ctttgaagcc aagagacttt gcaccttga 1539

<210> 14

<211> 1845

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 14

atgcgtgttg ctgtcgttgg cgctggacct ggaggtcttg ttacgctcaa gtacctcaaa 60

gaggctacca agttctttga cgttgacccc atcgatgtcc gtctctttga gcgtgaagac 120

gaagttggtg gaacttttac aaagcgtacc tatgaggatg cagagcttgt atcttccaag 180

tatctcacat gcttctctga ctggcgtgcc gaccttgaag atccagactt tctgagcgct 240

gacaggttta ttcgatactt gaaagagtac gcagactact tcaacctctg gcccgagatc 300

tcgctctcaa cgcctgtcac ctccattagg agaggtcagg ccggtggtca catcgttcat 360

taccgtggac ctgacggcat tgacaagact tgggaatgtg atgctgtcgc tgtatgcagc 420

ggccttcatg ttacacccaa tatccctgat gttcctggca tcgacaaggt gaagattgtc 480

aagcactctt cccagttcaa gaagagagat gagtttcctc agggcagcca agttgtcgtt 540

cttggtactg gtgagacggg tatggacatt gcccatcttg ctgttacatc acctacaaag 600

cgtgttgtcc tttgccacag acagggtttc cttggagcac caaagaaaat ccccaaccct 660

atcttgtttc ccatcttggg caacaagcct aaccccaacg ctcaggagct gcccatcgat 720

gtcagttggc aagctcctct gcttgactct tacttgccac ccttcttgag agatcgtctt 780

ttcacgtgga gattccaaga catcaacatc aagttggcta actggctttg ttctggtact 840

accgccggag tcgaccagtg gatcggaggt cttgatgctg accgattcca tacatctcag 900

agttttttca ataaggctgt ttggagatgc cttcactaca tcagtgagcc atatcgacct 960

accaacccag gtcttgtaga gcgcatccgc cgtgcaatcg tcacgattcc tgtcaaggag 1020

gtgccaggca acaagtacat cgacttggct ccttggccaa ctcatatcga tgacaaaggc 1080

atcatgcatt tcaaagagaa cggtcgccct gaagcagaac gcatgaagac cctcggccct 1140

gtgaagccgg acatgattgt ctacgctacg ggttaccgtc aggagtttgg cttctttgac 1200

gaagccaata agaatggtga agattaccca acatgctctg atgcggatgt acgctgtatc 1260

tggcgtcaca acgatcccac tgttgggttt atcggcttca tccgacctgg ctacggtgcc 1320

attcctccac ttgcagagct tcaagcccag ctttggctca tgactctcat caagcctgag 1380

gttgccaagt ctctcagctc tagggaggag tatcacttca agctccatgg tcacaagcgt 1440

atcgactatg gtgttcacca cgagtcttat gcctaccagc tagctcttga tatggatgct 1500

gttcccagct tctgggacgg cgtccgagtt ggttggaacg caggtgctaa gcatcccggt 1560

ctctggtggc gtctgcctgt tttgtggttg actggtgctc aattcaacac caagttccgc 1620

gtggttggac cttatcagtg ggatggcgct gtggacgttc ttggtggtga actttgggag 1680

actatcacca ggcgcgaggg cttatttggc gcctttgtta tgacgatcgt tcccatgact 1740

atggttggta cgagcagcat catcatgtgg ttcgttggat tgtttacggc gctgctgagt 1800

gcaattggaa gctttgccaa gggcaccttc tggagagttg tgtaa 1845

<210> 15

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 15

cagcaagctc cgaattcgcc accatggact tcacataccg ctattcg 47

<210> 16

<211> 44

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 16

gatccccggg taccgagctc ctaatctata cgcagcatct ccag 44

<210> 17

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 17

cggtatgtga agtccatggt ggcgaattcg gagcttgctg tggggtttat tg 52

<210> 18

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 18

agatgctgcg tatagattag gagctcggta cccggggatc tgtag 45

<210> 19

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 19

cagcaagctc cgaattcgcc accatggaaa ggctcaagat ggatagc 47

<210> 20

<211> 44

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 20

gatccccggg taccgagctc ctacttgaca gtgacgcgaa cacc 44

<210> 21

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 21

atcttgagcc tttccatggt ggcgaattcg gagcttgctg tggggtttat tg 52

<210> 22

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 22

ttcgcgtcac tgtcaagtag gagctcggta cccggggatc tgtag 45

<210> 23

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 23

cagcaagctc cgaattcgcc accatggaac taccgagctt ctccctc 47

<210> 24

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 24

gatccccggg taccgagctc tcaaggtgca aagtctcttg gcttc 45

<210> 25

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 25

aagctcggta gttccatggt ggcgaattcg gagcttgctg tggggtttat tg 52

<210> 26

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 26

caagagactt tgcaccttga gagctcggta cccggggatc tgtag 45

<210> 27

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 27

gccaagcttg catgcctgca actagtccgt catgtccagg aagataggtc ag 52

<210> 28

<211> 57

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 28

cacgctagtc tattatagga aaggcgcgcc gcgcaacagc atacgagtcc acaggag 57

<210> 29

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 29

tatgctgttg cgcggcgcgc ctttcctata atagactagc gtgcttggc 49

<210> 30

<211> 53

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 30

ctatcttcct ggacatgacg gactagttgc aggcatgcaa gcttggcact ggc 53

<210> 31

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 31

gtggactcgt atgctgttgc gcggatccga actccaaccg gggggag 47

<210> 32

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 32

ggcttaatgt cgacagtcat ggtggccagc ccctcacgga accccgacca ac 52

<210> 33

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 33

ggttccgtga ggggctggcc accatgactg tcgacattaa gccatacac 49

<210> 34

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 34

cctatgcgaa cccgtctaga ttatcgcagc ttctcagcga cctgc 45

<210> 35

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 35

gtcgctgaga agctgcgata atctagacgg gttcgcatag gtttgg 46

<210> 36

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 36

ctattatagg aaaggcgcgc cctctagtct acagtggccg ccttg 45

<210> 37

<211> 54

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 37

gcggccactg tagactagag ggcgcgcctt tcctataata gactagcgtg cttg 54

<210> 38

<211> 43

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 38

ccggttggag ttcggatccg cgcaacagca tacgagtcca cag 43

<210> 39

<211> 50

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 39

aacgacagca acacgcatgg tggccagccc ctcacggaac cccgaccaac 50

<210> 40

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 40

ggttccgtga ggggctggcc accatgcgtg ttgctgtcgt tggcgctg 48

<210> 41

<211> 44

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 41

cctatgcgaa cccgtctaga ttacacaact ctccagaagg tgcc 44

<210> 42

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 42

accttctgga gagttgtgta atctagacgg gttcgcatag gtttgg 46

<210> 43

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 43

tggactcgta tgctgttgcg cgatcttcct acacatatac aggac 45

<210> 44

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 44

gtcgctgaga agctgcgata attgtattgg tattaccaga acgtc 45

<210> 45

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 45

ttctggtaat accaatacaa ttatcgcagc ttctcagcga cctgc 45

<210> 46

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 46

cttcacacca caacaccgcc accatgactg tcgacattaa gccatacac 49

<210> 47

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 47

ttaatgtcga cagtcatggt ggcggtgttg tggtgtgaag ggtgattg 48

<210> 48

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 48

cccggttgga gttcggatcc tgagtgaagt tccggtcgga gcaatcag 48

<210> 49

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 49

ctccgaccgg aacttcactc aggatccgaa ctccaaccgg ggggag 46

<210> 50

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 50

tgtatatgtg taggaagatc gcgcaacagc atacgagtcc acaggag 47

<210> 51

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 51

tggattttta tatccaagat ggatccgaac tccaaccggg gggag 45

<210> 52

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 52

tgcgatctca gcggcatggt ggccagcccc tcacggaacc ccgaccaac 49

<210> 53

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 53

ggttccgtga ggggctggcc accatgccgc tgagatcgca ggagttg 47

<210> 54

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 54

acctatgcga acccgtctag atcacaacag gggtaacttg attgtg 46

<210> 55

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 55

tcaagttacc cctgttgtga tctagacggg ttcgcatagg tttgg 45

<210> 56

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 56

cccggttgga gttcggatcc atcttggata taaaaatcca aaatatgg 48

<210> 57

<211> 50

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 57

aggtgggaca gaactcatgg tggccagccc ctcacggaac cccgaccaac 50

<210> 58

<211> 50

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 58

ggttccgtga ggggctggcc accatgagtt ctgtcccacc tggtagtatc 50

<210> 59

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 59

acctatgcga acccgtctag atcatcgtcg agagaagcta aagctc 46

<210> 60

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 60

ttagcttctc tcgacgatga tctagacggg ttcgcatagg tttgg 45

<210> 61

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 61

tggattttta tatccaagat gatcttccta cacatataca ggacatg 47

<210> 62

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 62

tcaagttacc cctgttgtga ttgtattggt attaccagaa cgtcac 46

<210> 63

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 63

gttctggtaa taccaataca atcacaacag gggtaacttg attgtg 46

<210> 64

<211> 47

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 64

cttcacacca caacaccgcc accatgccgc tgagatcgca ggagttg 47

<210> 65

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 65

tgcgatctca gcggcatggt ggcggtgttg tggtgtgaag ggtgattg 48

<210> 66

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 66

ctgtatatgt gtaggaagat catcttggat ataaaaatcc aaaatatgg 49

<210> 67

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 67

cagcaagctc cgaattcgcc accatgcgtg ttgctgtcgt tggcgctgg 49

<210> 68

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 68

gagctactac agatccccgg gtaccttaca caactctcca gaaggtgcc 49

<210> 69

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 69

acgacagcaa cacgcatggt ggcgaattcg gagcttgctg tggggtttat tg 52

<210> 70

<211> 43

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 70

tggagagttg tgtaaggtac ccggggatct gtagtagctc gtg 43

<210> 71

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 71

cagcaagctc cgaattcgcc accatgactg tcgacattaa gccatacac 49

<210> 72

<211> 59

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 72

gtacatacat aaacatacgc gcacaaaagc agagattagg atttaatgca ggtgacgga 59

<210> 73

<211> 52

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 73

ttaatgtcga cagtcatggt ggcgaattcg gagcttgctg tggggtttat tg 52

<210> 74

<211> 43

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 74

gagaagctgc gataaggtac ccggggatct gtagtagctc gtg 43

<210> 75

<211> 46

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 75

aaacaaatag gggttccgcg ttcaattcat catttttttt ttattc 46

<210> 76

<211> 45

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 76

ggcacttttc ggggaaatgt gcatgattac gaattcatca cgtgc 45

<210> 77

<211> 49

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 77

aaaaaaaaaa tgatgaattg aacgcggaac ccctatttgt ttatttttc 49

<210> 78

<211> 48

<212> DNA

<213> Fusarium (Fusarium guttiferme)

<400> 78

gtgatgaatt cgtaatcatg cacatttccc cgaaaagtgc cacctgac 48

Claims

1. A method for synthesizing Mangicols sesterterpene compounds is characterized in that: the structure of the Mangicols sesterterpene compound is shown as formulas I-V:

comprises the following steps:

1) constructing a microbial mutant strain for heterogeneously synthesizing a sesterterpene compound biosynthetic gene cluster; the sesterterpene biosynthetic gene cluster contains 7 biosynthetic enzymes having the amino acid sequence shown in SEQ ID NO:1 to 7, or a pharmaceutically acceptable salt thereof;

2) the preparation of the fermentation culture of the microbial mutant strains AO-mgcDE and AO-mgcCDEF comprises the following steps:

the enzymes mgcE, mgcF, mgcG were obtained by the following method:

based on AntiSMASH software, bioinformatics analysis is carried out on a mangicols compound biosynthesis gene cluster in a strain of fusarium graminearum J1-012, and the gene cluster is found to contain 2 cytochrome P450 genes related to product oxidation, namely mgcE and mgcF, and a monooxygenase gene of FMO type, namely mgcG;

plasmids pYJ152, 153, 175 were constructed by the following method:

construction of pYJ152 plasmid: based on the predicted coding region information of mgcD, each exon fragment is amplified separately from the f.graminearum J1-012 genome and assembled into a mgcD gene, i.e., cDNA fragment, containing the entire coding region in vitro, which is then amplified with primers P1/P2; using pYH-WA-pyrG-KI plasmid as template, amplifying complete sequence containing ura gene and corresponding promoter and terminator by primer P61/P62; using pTAex3 plasmid as template, and using two pairs of primers P3/P64 and P4/P63 to amplify vector sequence; assembling the amplified 4 fragments by a Yeast assembly method to obtain a pYJ152 plasmid;

construction of pYJ153 plasmid: based on the predicted coding region information of mgcE, each exon fragment is amplified separately from the f.graminearum J1-012 genome and assembled into a mgcE gene, i.e., cDNA fragment, containing the entire coding region in vitro, which is then amplified with primers P5/P6; using pYH-WA-pyrG-KI plasmid as template, amplifying complete sequence containing ura gene and corresponding promoter and terminator by primer P61/P62; using pUSA plasmid as template, using two pairs of primers P7/P63 and P8/P64 to amplify vector sequence; assembling the amplified 4 fragments by a Yeast assembly method to obtain a pYJ153 plasmid;

construction of pYJ175 plasmid: amplifying the glaA promoter sequence from plasmid pGB98 with P17/P18 primer; amplifying a mgcC gene segment from a F.graminearum J1-012 genome by using a P19/P20 primer; amplification of the niaD terminator sequence from plasmid pGB127 with the P21/P22 primer; amplifying a vector fragment from the template pYJ174 by using a P23/P24 primer; assembling the fragments by a Yeast assembly method to obtain a pYJ175 plasmid;

the microbial mutants AO-mgcDE and AO-mgcCDEF are constructed by the following method:

transforming plasmid pYJ152 into Aspergillus oryzae to obtain strain AO-mgcD containing terpene synthase gene mgcD; the three predicted enzymes mgcE, mgcF and mgcG with oxidation function and mgcD are respectively expressed in aspergillus oryzae in a heterologous way; co-transforming plasmids pYJ152 and pYJ153 into Aspergillus oryzae to obtain a strain AO-mgcDE containing terpene synthase gene mgcD and P450 enzyme gene mgcE;

co-transforming pYJ152, pYJ153 and pYJ175 into Aspergillus oryzae to obtain strain AO-mgcCDEF containing terpene synthase gene mgcD, P450 enzyme gene mgcE, epoxide hydrolase gene mgcC and P450 enzyme gene mgcF;

3) extracting and separating fermentation culture and purifying the product;

the microorganism is Aspergillus oryzae.

2. A method for preparing a sesterterpene product is characterized in that:

extracting the fermentation culture of AO-mgcDE and AO-mgcCDEF prepared in step 2) of claim 1 with ethyl acetate, respectively, combining ethyl acetate extracts, and concentrating to obtain crude extracts A and B; carrying out reverse silica gel column chromatography on the crude extract A, carrying out gradient elution on an n-hexane-ethyl acetate system, and collecting fractions with the volume ratio of n-hexane to ethyl acetate of 5: 1; subjecting the fraction to semi-preparative HPLC purification by eluting with pure acetonitrile and ultrapure water at a volume ratio of 75:25 to 90:10 as mobile phases, and collecting the fraction to obtain sesterterpene compounds represented by formulae I to III as claimed in claim 1; alternatively, the first and second electrodes may be,

the sesterterpene compound is prepared and separated from a fermentation culture of a heterologous expression host microorganism;

the microorganism is Aspergillus oryzae.