CN105985967B - Biological synthesis gene cluster of oosporins and application thereof - Google Patents

Abstract

The invention relates to an oosporine biosynthesis gene cluster, in particular to cloning, sequencing, analyzing and function research of an oosporine biosynthesis gene cluster which is generated by Beauveria bassiana (Beauveria bassiana) and has insecticidal and bacteriostatic functions and application thereof. The whole gene cluster contains 7 genes: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS 7. Genetic manipulation of the biosynthesis genes can block the biosynthesis of oosporine, or change the yield of oosporine, or produce novel compounds. The invention also discovers the biosynthesis route of the oosporine for the first time, and has great research significance.

Description

Biological synthesis gene cluster of oosporins and application thereof

Technical Field

The invention belongs to the field of microbial gene resources and genetic engineering, and particularly relates to cloning, analysis, functional research and application of a biosynthetic gene cluster of oosporine with insecticidal and bacteriostatic effects.

Background

Oosporin (oosporin) was first identified in the last 60 centuries as an extracellular red pigment produced by Beauveria bassiana (Beauveria bassiana) and Beauveria brockii (Beauveria brongniartii), and had certain insecticidal and bacteriostatic activity. The abnormal activity of monoamine oxidase (MAO) can cause various dysfunctions of organisms to form disease states, such as Parkinson's disease, senile dementia, depression which is popular in cities in recent years and the like, which are related to the abnormal activity of MAO, and the oosporine serving as a monoamine oxidase inhibitor can help to develop medicines related to diseases caused by the abnormal activity of monoamine oxidase to a certain extent. Although oosporin has been discovered and its structure has been analyzed (fig. 1), the biosynthesis pathway of oosporin has not been clarified.

Therefore, the method uses the oosporine from microorganisms as a target molecule, starts from cloning a biosynthesis gene cluster of the oosporine, adopts a method combining microbiology, molecular biology, biochemistry and organic chemistry to research the biosynthesis of the oosporine, explores the biosynthesis pathway and a regulation mechanism of the oosporine, and verifies the insecticidal and bacteriostatic effects of the oosporine.

Disclosure of Invention

The invention relates to cloning, analysis, functional research and application of a biosynthetic gene cluster of oosporine with insecticidal and bacteriostatic effects.

The first aspect of the invention provides an oosporine biosynthesis gene cluster, which comprises 7 oosporine synthesis related genes related to oosporine biosynthesis and comprises the following components: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS 7;

wherein OpS1 is located at 24759-32012 of the gene cluster nucleotide sequence, encodes polyketide synthase/glycosidic acid synthase and has a length of 2211 amino acids;

OpS2 is positioned at position 32992-34102 of the gene cluster nucleotide sequence, encodes a transport protein and has the length of 350 amino acids;

OpS3 is positioned at the 36584-38809 th site of the gene cluster nucleotide sequence, encodes a transcription factor and has the length of 741 amino acids;

OpS4 is located at 39149-40901 th site of the gene cluster nucleotide sequence, encodes hydroxylase, and has the length of 427 amino acids;

OpS5 is located at position 41885-44041 of the gene cluster nucleotide sequence and encodes laccase with the length of 590 amino acids;

OpS6 is located at 44430-45147 site of the gene cluster nucleotide sequence, encodes glutathione S transferase, and has length of 218 amino acids;

the OpS7 is located at the 45713-46768 th site of the gene cluster nucleotide sequence and codes the Cupin protein, and the length is 305 amino acids.

In another preferred embodiment, the sequence of the gene cluster is selected from the group consisting of positions 8391-54012 of SEQ ID NO. 1.

In another preferred embodiment, the gene cluster further comprises a combination of one or more (e.g. 2, 3, 4, 5, 6 or 7) genes of the 7 oosporin synthesis-related genes or a gene cluster or fragments thereof.

In a second aspect, the invention provides a protein related to the biosynthesis of oosporine, wherein the amino acid sequence of the protein is selected from the amino acid sequences shown in SEQ ID NO. 2-8.

In another preferred example, the biosynthesis-associated protein is SEQ ID No.:2 or a polyketide synthase/glycosidase synthase shown in figure 2.

In another preferred embodiment, the biosynthesis-related protein is a transporter protein shown in SEQ ID No. 3.

In another preferred example, the biosynthesis-related protein is a transcription factor shown in SEQ ID No. 4.

In another preferred example, the biosynthesis-related protein is hydroxylase shown in SEQ ID No. 5.

In another preferred embodiment, the biosynthesis-related protein is laccase as shown in SEQ ID No. 6.

In another preferred example, the biosynthesis-related protein is glutathione transferase shown in SEQ ID No. 7.

In another preferred example, the biosynthesis-related protein is a Cupin protein shown in SEQ ID No. 8.

In a third aspect, the present invention provides a gene related to the biosynthesis of oosporine, which encodes a protein related to the biosynthesis of oosporine according to the second aspect of the present invention.

In another preferred embodiment, the biosynthesis-related gene is selected from the group consisting of:

OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS7, or a combination thereof.

In another preferred embodiment, the information on the nucleotide sequence of the synthesis-related gene is as described above or in Table 1.

In a fourth aspect, the present invention provides an expression vector comprising the ovosporine biosynthesis gene cluster according to the first aspect of the present invention or a fragment thereof, or the ovosporine biosynthesis-related gene according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a recombinant host cell comprising the expression vector of the fourth aspect of the present invention, or a gene cluster for biosynthesis of the oosporine of the first aspect of the present invention or a gene involved in biosynthesis of the oosporine of the third aspect of the present invention, which is exogenously integrated into the chromosome of the host cell.

In another preferred embodiment, the recombinant host cell comprises a eukaryotic cell, such as beauveria bassiana, pichia pastoris.

The sixth aspect of the present invention provides Beauveria bassiana (Beauveria bassiana) in which one or more genes selected from the group consisting of the following genes in the biosynthetic gene cluster of oosporins are inactivated: OpS1, OpS3, OpS4, OpS5, OpS6, OpS7, so that no ovosporine is produced.

In another preferred embodiment, the inactivated gene is selected from the group consisting of: OpS1, OpS3, OpS4, OpS5, OpS6, and OpS 7.

In another preferred embodiment, the Ops2 gene is retained in Beauveria bassiana.

The seventh aspect of the present invention provides a Beauveria bassiana (Beauveria bassiana) in which one or more genes in an oosporine biosynthesis gene cluster are overexpressed, thereby increasing or restoring the oosporine yield,

wherein the overexpressed gene is selected from the group consisting of: OpS1, OpS3, OpS4, OpS5, OpS6 and OpS 7.

In another preferred embodiment, the Ops2 gene is knocked out or down-regulated in Beauveria bassiana.

In another preferred embodiment, the improvement refers to the ratio of the oosporin production of beauveria bassiana A1 to A0 in the overexpressed beauveria bassiana compared to the oosporin production of the original strain (beauveria bassiana A0) is greater than or equal to 1.5, preferably greater than or equal to 2.0, more preferably greater than or equal to 2.5, such as 1.5-10, preferably 2-5.

The eighth aspect of the invention provides an application of an oosporine biosynthesis gene or a protein thereof in synthesizing a precursor or an intermediate of oosporine, wherein the oosporine biosynthesis gene or the protein thereof is selected from the following group: OpS1, OpS4, OpS5, OpS 7.

In another preferred embodiment, the synthesis is in vitro enzymatic synthesis or artificial synthesis.

In another preferred embodiment, there is provided the use of the OpS1 gene or a protein thereof for the synthesis of a synthetic precursor of ovosporine.

In another preferred embodiment, the synthetic precursor of oosporine is a thioctic acid.

In another preferred embodiment, the OpS1 gene or protein thereof catalyzes the synthesis of the acid from acetyl-CoA as a starting material.

In another preferred embodiment, the structure of the glycosidic acid is shown in formula I:

in another preferred embodiment, there is provided a use of the OpS4 gene or a protein thereof, wherein the OpS4 gene or the protein thereof is used for catalyzing decarboxylation hydroxylation reaction of the compound of formula I, so as to generate the intermediate of formula II.

In another preferred embodiment, the intermediate is compound 2.

In another preferred embodiment, the compound 2 is trihydroxytoluene.

In another preferred embodiment, the structure of the compound 2 is shown as formula II:

in another preferred embodiment, the compound 2 is structurally unstable and will be partially converted into the compound 3 and the compound 4.

In another preferred embodiment, the compound 3 has a ketone structure represented by formula III:

in another preferred embodiment, the compound 4 is formed by oxidative polymerization of 2 compounds 2, and the structure of the compound is shown in formula IV:

in another preferred embodiment, there is provided a use of the OpS7 gene or a protein thereof, wherein the OpS7 gene or the protein thereof is used for catalyzing hydroxylation reaction of a compound of formula II to generate tetrahydroxytoluene.

In another preferred embodiment, the structure of the tetrahydroxytoluene is shown as formula V:

in another preferred embodiment, the tetrahydroxytoluene is structurally unstable and is partially converted to compound 1.

In another preferred embodiment, the compound 1 has a ketone structure represented by formula VI:

in another preferred embodiment, there is provided a use of the OpS5 gene or a protein thereof, wherein the OpS5 gene or the protein thereof is used for catalyzing oxidative polymerization of tetrahydroxytoluene to produce the oosporine.

In another preferred embodiment, the oosporine has the structure shown in formula VII:

the ninth aspect of the invention provides a method for preparing an oothecycin, which comprises the following steps:

(i) culturing an oothecin-producing host cell under conditions suitable for culturing and with the addition of a starting compound, thereby producing oothecin, wherein the starting compound is selected from the group consisting of: a glycoside acid (formula I), a compound 2 (formula II), tetrahydroxytoluene (formula V), or a combination thereof; and

(ii) separating or purifying the oothecin.

In another preferred embodiment, the host cell is beauveria bassiana.

In another preferred embodiment, the host cell is beauveria bassiana introduced with exogenous gene related to the synthesis of the oothecin, wherein the gene is selected from the group consisting of: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS7 genes.

In a tenth aspect, the present invention provides a method for modifying a host cell, comprising the steps of:

(a) determining the expression and/or activity of each of the related genes belonging to the family of oomycete mycin synthesis genes in said host cell;

(b) according to the determination result, introducing the related gene of the oothecin synthesis gene cluster into the host cell so as to improve/restore the ability of the host cell to produce oothecin,

wherein the related genes of the gene cluster comprise one or more of the following genes: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS 7.

In another preferred embodiment, the host cell is beauveria bassiana.

In another preferred embodiment, when the OpS1 gene of the gene cluster is inactivated or has reduced activity, the method further comprises the following steps:

adding a feedstock compound to the culture system, the feedstock compound selected from the group consisting of: the compound 2 (formula II), tetrahydroxytoluene (formula V), or a combination thereof to restore the formation of the oothecin.

In an eleventh aspect, the present invention provides a compound related to oothecin, selected from the group consisting of:

compound 2 (formula II), compound 3 (formula III), compound 4 (formula IV), and compound 1 (formula VI).

The twelfth aspect of the invention provides a method for improving the ability of beauveria bassiana to produce oothecycin, which comprises the following steps:

introducing genes related to an oothecycin synthesis gene cluster into the beauveria bassiana so as to improve or restore the capacity of the beauveria bassiana to produce the oothecycin, wherein the genes related to the gene cluster comprise one or more of the following genes: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS 7; or knocking out the OpS2 gene of the oothecycin synthesis gene cluster in the beauveria bassiana, thereby improving the capacity of the beauveria bassiana to produce the oothecycin.

In a thirteenth aspect, the invention provides use of an isolated OpS2 polypeptide or gene encoding same for inhibiting oosporin synthesis.

In another preferred embodiment, the OpS2 polypeptide is selected from the group consisting of: (i) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3;

(ii) (ii) a polypeptide derived from (i) which is formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO. 3 and has the function of inhibiting the synthesis of the oomycete.

In another preferred embodiment, the coding sequence for the OpS2 polypeptide is selected from the group consisting of seq id no:

(1) a polynucleotide sequence encoding the polypeptide of SEQ ID NO. 3;

(2) the polynucleotide sequence shown as SEQ ID NO. 10;

(3) the polynucleotide complementary to the polynucleotide sequence of (1) or (2).

In a fourteenth aspect, the invention provides a use of the oospore mycin biosynthesis gene cluster described in the first aspect of the invention or partial genes thereof, which is characterized in that partial genes in the gene cluster are used for carrying out heterologous complementation in a mutant strain in which a beauveria bassiana ARSEF2860 related gene is deleted, so that the production of oospore mycin is recovered; or part of genes in the gene cluster are subjected to heterologous expression in pichia pastoris, so that synthesis precursors and intermediates of the oosporine and oosporine analogues are generated.

In another preferred embodiment, the pichia pastoris is selected from GS 115.

In another preferred embodiment, the synthetic precursor of ovosporine is a glycosidic acid (formula I).

In another preferred embodiment, the synthesis intermediates of the oosporine are compound 2 (formula II), compound 3 (formula III), tetrahydroxytoluene (formula V) and compound 1 (formula VI).

In another preferred embodiment, the oosporine analogue is compound 4 (formula IV).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the chemical structure and color of the oosporine.

FIG. 2 shows the structure of the oosporin synthesis gene cluster.

Figure 3 shows OpS1 knock-out vector structure.

FIG. 4 shows the color change of fermentation broth of each knockout mutant and wild strain.

FIG. 5 shows the HPLC detection results of the fermentation broth of each mutant and wild type strain.

FIG. 6 shows the OpS3 overexpression vector structure.

FIG. 7 shows color comparisons of fermentation broths of wild-type, OpS3 knock-out and over-expression mutants.

FIG. 8 shows the HPLC detection results of the wild-type and each mutant fermentation broth.

FIG. 9 shows a comparison of oosporin production by wild type strains and various mutants.

FIG. 10 shows the RT-PCR results of the genes involved in the synthesis of oosporin of the wild type strain and each mutant.

FIG. 11 shows the domain composition of OpS1 in comparison to OrSA.

FIG. 12 shows the color comparison of wild type strains to the Δ OpS1 mutant glycosidic acid feeding experimental fermentation broths.

Figure 13 shows the HPLC detection results of wild type and Δ OpS1 mutant glycosidic acid feeding experiments.

FIG. 14 shows the HPLC assay results of the fermentation broth of the wild-type and the OpS1 heterologous expression strain and the isochromanoic acid standard (OrA).

FIG. 15 shows the color comparison of the fermentation broths of the wild type strain and each of the mutant strains.

FIG. 16 shows the HPLC detection results of the wild-type and each mutant fermentation broth.

FIG. 17 shows the HPLC detection results of the fermentation broth of OpS4 as well as the wild type GS 115.

FIG. 18 shows the "Compound 1" feed test HPLC assay results for wild type and mutant Δ OpS7: "OpS 3.

FIG. 19 shows the synthesis pathway of ovosporine.

Figure 20 shows the results of the galleria mellonella bioassay.

FIG. 21 shows hemolymph microscopy results of greater wax moth infected with Beauveria bassiana.

FIG. 22 shows a comparison of wild-type and mutant infected cadavers.

Fig. 23 shows an oosporin inhibition test.

Detailed Description

The inventor of the present invention has conducted extensive and intensive research, and has found that, taking oosporins derived from beauveria bassiana as target molecules, starting from cloning a biosynthetic gene cluster of the oosporins in beauveria bassiana ARSEF2860, the biosynthesis of the oosporins is researched by combining microbiology, molecular biology, biological analysis informatics, biochemistry and organic chemistry, and the biosynthetic gene cluster of the oosporins is identified for the first time, and specifically, the gene cluster includes 7 genes, which are respectively: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS 7. Wherein OpS1 encodes polyketide synthase/glycosidic acid synthase, OpS2 encodes transporter, OpS3 encodes transcription factor, OpS4 encodes hydroxylase, OpS5 encodes laccase, OpS6 encodes glutathione S transferase, OpS7 encodes Cupin protein; in addition, the inventor also discovers the biosynthesis pathway of the oosporine for the first time and verifies the insecticidal and bacteriostatic effects of the oosporine. On this basis, the present inventors have completed the present invention.

Related gene and protein for synthesis of oothecin

As used herein, the present invention discloses an ovomycin synthesis gene cluster (gene cluster) comprising: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS7 genes.

Wherein the OpS1 gene encodes a ketone synthase/glycosidic acid synthase and the OpS1 gene has at least one or more characteristics selected from the group consisting of:

(i) encoding the polypeptide as shown in SEQ ID NO: 2; (ii) encoding the polypeptide as shown in SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 9; (iv) has a sequence similar to that shown in SEQ ID NO: 9, or a polynucleotide whose sequence is complementary to the polynucleotide sequence shown in figure 9.

The OpS2 gene encodes a transporter protein, and the OpS2 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 3; (ii) encoding the polypeptide as shown in SEQ ID NO:3 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 10; (iv) has a sequence similar to SEQ ID NO:10, or a polynucleotide complementary to the polynucleotide sequence shown in figure 10.

The OpS3 gene encodes a transcription factor, and the OpS3 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 4; (ii) encoding the polypeptide as shown in SEQ ID NO: 4 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 11; (iv) has a sequence similar to SEQ ID NO: 11, or a polynucleotide whose sequence is complementary to the polynucleotide sequence shown in figure 11.

The OpS4 gene encodes hydroxylase; and the OpS4 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 5; (ii) encoding the polypeptide as shown in SEQ ID NO: 5 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 12; (iv) has a sequence similar to SEQ ID NO: 12, or a polynucleotide whose sequence is complementary to the polynucleotide sequence shown in figure 12.

The OpS5 gene encodes a laccase; and the OpS5 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 6; (ii) encoding the polypeptide as shown in SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 13; (iv) has a sequence similar to SEQ ID NO: 13, or a polynucleotide whose sequence is complementary to the polynucleotide sequence shown in figure 13.

The OpS6 gene encodes glutathione S transferase; and the OpS6 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 7; (ii) encoding the polypeptide as shown in SEQ ID NO: 7 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the structure shown as SEQID NO: 14; (iv) has a sequence similar to SEQ ID NO: 14, or a polynucleotide whose sequence is complementary to the polynucleotide sequence shown in fig. 14.

The OpS7 gene encodes a Cupin protein; and the OpS7 gene has at least one or more characteristics selected from the group consisting of: (i) encoding the polypeptide as shown in SEQ ID NO: 8; (ii) encoding the polypeptide as shown in SEQ ID NO: 8 by substitution, deletion or addition of one or more amino acid residues to form a polypeptide derived from (i); (iii) has the sequence shown in SEQ ID NO: 15; (iv) has a sequence similar to SEQ ID NO: 15, or a polynucleotide complementary to the polynucleotide sequence shown in figure 15.

When an amino acid fragment of an gene involved in the synthesis of oothecin is obtained, a nucleic acid sequence encoding the gene can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using commercially available Wen library or Wen library prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Determination of ovosporin biosynthesis Gene Cluster

The inventor finds a homologous Gene OpS1 of the Gene orsA in the beauveria bassiana and a Gene cluster (genecluster) thereof by using a Gene Blast method on the basis of completing genome sequencing of the beauveria bassiana ARSEF2860 strain, wherein the Gene cluster comprises the following genes: OpS1(BBA _08179), OpS2(BBA _08180), OpS3(BBA _08181), OpS4(BBA _08182), OpS5(BBA _08183), OpS6(BBA _08184), and OpS7(BBA _08185) (fig. 2).

Determination of relevance and completeness of oosporine biosynthesis gene cluster

Since biosynthesis genes of microbial secondary metabolites exist in linked clusters on chromosomes, the inventors performed knock-out experiments on each gene in the obtained sequence to verify the relevance and integrity of the obtained gene cluster and the biosynthesis of oosporine.

The gene knockout OpS1, OpS3, OpS4, OpS5, OpS6, or OpS7 completely interrupted the production of oosporangin and the gene overexpression OpS1, OpS3, OpS4, OpS5, or OpS7 was able to increase the production of oosporangin, proving that the screened gene cluster is indeed related to oosporangin biosynthesis; the gene knockout OpS2 increases the yield of the oosporine, which shows that OpS2 has the effect of inhibiting the synthesis of the oosporine; it was thus confirmed that the resulting gene cluster contained all the genes required for the biosynthesis of oosporin. In each gene knockout or overexpression mutant, some completely interrupt the production of oosporine, some have changed yield, some have no obvious influence, and some have new compounds.

Based on the above experimental results, and compared with the biosynthetic gene cluster of the same class of compounds, the present inventors determined that the biosynthetic gene cluster of oosporine contains 7 open reading frames from OpS1 to OpS7 (fig. 2), encompassing the region of chromosome 22 kb. In the whole gene cluster, OpS1 encodes polyketide Synthase/glycosidic acid Synthase (Polyletide Synthase/Orselinic Synthase), OpS2 encodes Transporter (Transporter), OpS3 encodes transcription factor (TranscriptionnFactor), OpS4 encodes Hydroxylase (Hydroxylase), OpS5 encodes Laccase (Lactase), OpS6 encodes Glutathione S transferase (Glutathione S-Transferases/GSTs), OpS7 encodes Cupin protein (Cupin domaining protein) (Table 1, FIG. 2).

TABLE 1

Wherein "-" means "does not generate"; "NA" means "no overexpression experiments have been performed".

Synthetic route to oosporin precursors

Polyketide synthases (PKSs) are a class of multifunctional enzymes consisting of a plurality of modules, each module incorporating a two-carbon unit in turn, starting with Acetyl-CoA as a starting substrate and Malonyl-CoA as a precursor. The PKS initiation module contains only AT-domain and ACP-domain, arranged in the order of "AT-ACP". The extension module of the protein consists of three Core domains (Core-domains), namely a ketoester acyl synthesis domain (KS-domain), an acyl transfer domain (AT-domain) and an acyl carrier protein domain (ATP-domain), which are arranged in the sequence of 'KS-AT-ACP'. The last module of PKS also contains a thioesterase domain (Te-domain), arranged in the order "KS-AT-ACP-Te". In addition to these core domains, PKSs often contain several modification domains (Tailoreng-domains), such as ketoacyl reductase Domain (KR-Domain), dehydrogenase Domain (DH-Domain), enoyl reductase Domain (ER-Domain), and methyltransferase Domain (MT-Domain). PKSs can be classified into 3 types according to structural differences, wherein the PKSs of fungi belong to type I iterative PKSs. Type I iterative PKS contains only one module, and different products are synthesized by recycling this module. The gene OpS1 was predicted to be the type I iterative PKS encoding gene and to be homologous to the glycosidic acid synthase gene (orsA) in aspergillus nidulans, and the domain composition of the PKS they encode is shown in fig. 11.

The precursor of ovosporine, the glycosidic acid, was experimentally shown to be synthesized by OpS1(PKS) under the responsibility. Specifically, the inventor conducts a glycoside acid back feeding test on the beauveria bassiana delta OpS1 mutant, and finds that the addition of the glycoside acid restores the capability of the mutant delta OpS1 to synthesize the oosporine, thereby indicating that the glycoside acid is really a precursor for synthesizing the oosporine and is responsible for synthesizing by OpS 1.

Biosynthetic pathway for oosporins

The inventors found through experiments that the biosynthesis of the ovamycin was mediated by OpS1, OpS4, OpS5 and OpS7 as shown in fig. 19.

Specifically, the biosynthetic pathway of oosporins is as follows:

(1) the polyketide synthase OpS1 takes acetyl coenzyme A as a raw material to catalyze the synthesis of the thioctic acid;

(2) the glycosidic acid is catalyzed by hydroxylase Ops4, and is subjected to decarboxylation and hydroxylation reaction to generate a compound 2 (trihydroxy toluene);

(3) "Compound 2" is unstable and will convert to its ketone structure, "Compound 3", and Compound 2 will oxidatively polymerize to form a small amount of "Compound 4" (5, 5' -dideoxyoomycin);

(4) "Compound 2" (trihydroxytoluene) is hydroxylated by Cupin protein OpS7 to produce tetrahydroxytoluene, and a small amount of tetrahydroxytoluene is converted into its ketone structure "Compound 1";

(5) tetrahydroxytoluene is catalyzed by laccase OpS5, and is subjected to oxidation and free radical dimerization reaction to generate the oosporine.

Carrier

The term "vector" as used herein includes cloning vectors and other vectors which enable the expression of an inserted gene of interest into a host cell. The expression vector can comprise a prokaryotic expression vector and a eukaryotic expression vector, and can be a plasmid, a cosmid, a phage or a virus, and the like. Typical expression vectors carry regulatory sequences which allow for gene expression and, in place, restriction enzyme sites into which foreign genes can be inserted.

In a specific embodiment, the expression vector of the present invention comprises an oosporin biosynthesis gene of the present invention, or comprises an oosporin biosynthesis gene cluster of the present invention.

Host cell

The term "host cell" as used herein has the meaning commonly understood by a person of ordinary skill in the art, i.e., a cell that contains and is capable of expressing an exogenous gene of interest. For example, the host cell can be a prokaryotic host cell (e.g., E.coli, Bacillus subtilis, Streptomyces, Micromonospora), a eukaryotic host cell (e.g., yeast), a plant cell, and the like.

In a particular embodiment, the host cell of the invention preferably comprises an expression vector of the invention, or has integrated on its chromosome one or more copies of an exogenous oosporin biosynthesis gene, or a single or multiple copies of an oosporin biosynthesis gene cluster.

In a preferred embodiment, the host cell of the invention comprises beauveria bassiana, pichia pastoris.

Preferably, the engineered bacterium of the present invention is obtained by genetic engineering of Beauveria bassiana ARSEF 2860.

Mutant beauveria bassiana with inactivated structural gene in oosporin biosynthesis gene cluster

The present invention also provides a mutant strain of beauveria bassiana in which one or more genes of the oosporine biosynthetic genes of the present invention are inactivated, such that the mutant strain does not produce oosporine.

In specific embodiments, the inactivated gene or genes are selected from the group consisting of genes encoding the genes set forth in SEQ ID No. 9, 11-15. More preferably, the inactivated gene is selected from OpS1, OpS3, OpS4, OpS5, OpS6 or OpS 7.

The beauveria bassiana mutant strain with one or more inactivated structural genes in the oosporine biosynthesis gene cluster can be used as a model or a reference strain for verifying the gene function in the oosporine gene cluster and/or a host cell for exogenously expressing oosporine.

Mutant beauveria bassiana or mutant pichia pastoris for structural gene overexpression in oosporin biosynthesis gene cluster

The invention also provides a mutant strain of beauveria bassiana, wherein one or more genes of the oosporin biosynthesis genes are overexpressed in the mutant strain, so that the mutant strain improves or restores the production of oosporine.

In specific embodiments, the overexpressed gene or genes are selected from the group consisting of genes encoding the genes set forth in SEQ ID No. 9, 11-15. More preferably, the inactivated gene is selected from OpS1, OpS3, OpS4, OpS5, OpS6 or OpS 7.

The beauveria bassiana mutant strain with one or more over-expressed structural genes in the oosporin biosynthesis gene cluster provided by the invention can be used as a model or a reference strain for verifying the gene function in the oosporin gene cluster and/or a host cell for exogenously expressing oosporin.

The main advantages of the invention include:

(1) the invention provides a biosynthesis way for the oothecin and explores a new biosynthesis mechanism.

(2) The nucleotide sequence provided by the invention or at least a part of the cloned gene of the nucleotide sequence can be expressed in an exogenous host by a suitable expression system to obtain the corresponding enzyme or other higher biological activity or yield. These foreign hosts include Streptomyces, Beauveria bassiana, Pseudomonas, Escherichia coli, Bacillus, yeast, plants, animals, and the like.

(3) The invention discloses the structure of an oothecin biosynthesis gene cluster for the first time, and the function of each gene is analyzed and researched.

(4) The invention provides a new method for searching a silent intermediate product, namely, the intermediate product is obtained by over-expressing transcription factors for controlling the expression of each gene in a gene cluster in a mutant.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.

The general method comprises the following steps:

1. transformation method of agrobacterium tumefaciens AGL-1

1.1 competent preparation of Agrobacterium tumefaciens AGL-1:

⑴ original strain AGL-1 is picked with toothpick, streaked on YEB plate (containing 50 mug/ml carbenicillin) for activation, ⑵ single colony on plate is picked, inoculated into 4ml YEB liquid culture medium (containing 50 mug/ml carbenicillin), 28 ℃, 200rpm, cultured overnight, ⑶ 2ml of the above bacterial liquid is taken to transfer into 50ml YEB liquid culture medium (containing 50 mug/ml carbenicillin), 28 ℃, 200rpm, cultured until bacterial liquid OD600 is 0.5 (generally about 8h), ⑷ is taken out, bacterial liquid is taken out, ice bath is 30min, 4 ℃, 8000rpm, 5min centrifugal collection thalli, ⑸ supernatant is abandoned, 10ml 20mM CaCl2 is added for precipitation, ⑹ 4 ℃, 8,000rpm, 5min, again centrifugal collection thalli, ⑺ supernatant is again collected, 2ml 20mM CaCl2 is added, sterile glycerol is added to the final concentration of 15-20%, the final concentration is added to resuspension in 1.5. heavy suspension tube, 100ml EP 80 ml liquid nitrogen is added, and then the thalli is quickly frozen in EP 80 ℃ quick freezing tube.

1.2 plasmid transformation of Agrobacterium tumefaciens AGL-1 competence:

⑴ the agrobacterium is frozen at-80 deg.C, placed on ice to melt for about 10min, ⑵ the liquid nitrogen is added into the competence for 0.5-1 mug of plasmid, mixed evenly and stood on ice for 30min, ⑶ the liquid nitrogen is added for 5min, water bath is carried out at 37 deg.C for 5min, after treatment, the liquid nitrogen is immediately placed on ice for 2min, ⑷ the liquid medium is added into 1ml of non-antibiotic YEB, 28 deg.C, 150rpm, 3h, ⑸ 8,000,000 rpm, 2min is centrifuged to collect the thallus, ⑹ the supernatant is discarded, 100 mug of non-antibiotic YEB is used to re-suspend the thallus and then evenly spread on YEB plate (containing 50 mug/ml carbenicillin and 50 mug/ml kanamycin), transformant is inverted and cultured at 28 deg.C for 2 days, ⑺ PCR is carried out to verify, positive transformant is picked up and shaken with 3ml YEB liquid (containing 50 mug/ml carbenicillin and 50 mug/ml kanamycin) kanamycin, 28 deg.C, 220rpm, ⑻ the bacterial liquid is taken up to the final concentration of 15 deg.C overnight.

2. Agrobacterium tumefaciens mediates genetic transformation of beauveria bassiana:

⑴ preparation of white muscardine fungus conidium suspension, selecting white muscardine fungus cultured on PDA plate of potato solid culture medium for two weeks, scraping appropriate amount of hypha to 1ml of sterile 0.05% Tween-20 water solution, separating hypha and conidium by vortex oscillation, filtering with cellosilk cotton or three-layer mirror paper to remove hypha, collecting filtrate, centrifuging at 12,000rpm for 1min, collecting spores, discarding supernatant, resuspending spores with 1ml of sterile water, removing residual nutrition, centrifuging at 12,000rpm for 1min, again collecting spores, repeating steps 4-5 once to completely remove residual nutrition on spore surface, discarding supernatant, resuspending spores with appropriate amount of sterile water, counting number of spores with blood cell counter, adjusting spore suspension concentration to 5 × 10⁵⑵ transformation of Beauveria bassiana by transferring AGL-1 frozen bacteria with successfully transformed corresponding vector to 3ml liquid YEB (containing 50 ug/ml carbenicillin and 50 ug/ml kanamycin), culturing at 28 deg.C and 220rpm overnight, culturing at 8,000rpm for 2min, centrifuging to collect bacteria, discarding supernatant, resuspending bacteria with appropriate amount of IM liquid, and adjusting to OD₆₅₀Is 0.15; induced at 28 ℃ and 150rpm to OD₆₅₀0.5-0.8, generally requiring 6 hours; respectively taking 100 mu l of induced agrobacterium liquid and freshly prepared beauveria bassiana spore suspension, and uniformly mixing in a 1.5ml EP tube; coating 100 mul of the mixed solution on an IM plate, and co-culturing for 48h at 28 ℃; cooling the sterilized M-100 solid culture medium to 60 deg.C, and adding corresponding antibiotics (Thielavia, glufosinate/benomyl); each co-cultured IM plate is covered by 15ml of the M-100 medium, and cultured at 25 ℃ for 3-5 days until single resistant colonies appear; picking the resistant colonies to a new M-100 resistant culture medium plate by using toothpicks, and carrying out secondary screening; carrying out shake culture on the resistant bacterial colony on the secondary screening plate by using an SDB liquid culture medium, and marking the corresponding position on the back surface of the M-100 plate; and (5) carrying out suction filtration on the SDB cultured mycelia, extracting a genome and carrying out PCR verification.

3. Agrobacterium-mediated fungal transformation solution and culture medium

2.5 XInduction Medium basal salt solution

KH2PO4	3.625g
		K2HPO4·3H2O	6.72g
MgSO4·7H2O	1.250g
		NaCl	0.375g
CaCl2	0.125g
		FeSO4·7H2O	0.0062g
(NH4)2SO4	1.250g
		Double distilled water	Final constant volume of 1L

M-100 trace element solution

H3BO3	30mg
		MnCl2·4H2O	70mg
ZnCl2	200mg
		Na2MoO4·2H2O	20mg
FeCl3·6H2O	50mg
		CuSO4·5H2O	200mg
Double distilled water	Final constant volume to 500ml

M-100 salt solution

KH2PO4	16g
		Na2SO4·10H2O	9.064g
KCl	8g
		MgSO4·7H2O	2g
CaCl2	1g
		M-100 trace element solution	8ml
Double distilled water	Final constant volume of 1L

Induction medium (liquid)

After sterilization, cooling to 50 ℃ and adding:

induction medium (solid)

After sterilization, cooling to 50 ℃ and adding:

M-100(For 1L)

m-100 saltSolutions of	62.5ml
		Glucose	10g
KNO3	3g
		Double distilled water	Final constant volume of 1L
Agar powder	15g

Preparing MES and AS mother liquor:

	stock solution concentration	pH (adjusted with 5M KOH)
			2- (morpholino) ethanesulfonic acid MES	1M	5.3
Acetosyringone AS	10mM	8

4. The method for separating and purifying the oosporin comprises the following steps:

4.1 extraction of ovosporine:

separating the white muscardine fungus hypha and the culture medium by suction filtration, and concentrating the separated fermentation liquor into one fourth of the original volume by vacuum rotary evaporation at 50 ℃. Trifluoroacetic acid was added to the concentrated broth and shaken until the broth turned from a purple-red color to an orange color (pH. apprxeq.2). The fermentation broth was extracted with three volumes of ethyl acetate and the organic and aqueous phases were separated. The ethyl acetate phase is evaporated to dryness by vacuum rotary evaporation and finally dissolved in a proper amount of methanol.

4.2 isolation of ovosporine:

the macroporous resin D101 is suspended in industrial alcohol and filled in a chromatographic column. The packed macroporous resin in the column is equilibrated with water of pH 2 (pH adjusted with trifluoroacetic acid) until the effluent eluent pH 2 (i.e., the macroporous resin is acidic). And uniformly dropwise adding the crude oosporine extract on the top of a chromatographic column to make the sample fully adsorbed on the macroporous resin. The 3 column volumes were eluted with water (pH 2) as mobile phase and the sample was eluted with aqueous potassium hydroxide (pH 12). The eluted purple solution was collected, adjusted to pH ≈ 2 with trifluoroacetic acid (solution becomes orange), and then concentrated to one tenth of the original volume by rotary evaporation under vacuum. The solution was re-extracted with ethyl acetate, concentrated to dryness and dissolved in a little methanol in an EP tube. After the EP tube was left to stand at-20 ℃ for overnight precipitation, it was centrifuged at 12000rpm for 1min to collect the precipitate. The precipitate is the separated oosporin crystal.

A standard curve for oosporine at UV 287nm was plotted.

The separated and purified oosporine is prepared into dimethyl sulfoxide (DMSO) solutions with the concentrations of 10mg/ml, 8mg/ml, 6mg/ml, 4mg/ml, 2mg/ml, 1mg/ml, 0.8mg/ml, 0.6mg/ml and 0.4mg/ml respectively. The sample was loaded in 3. mu.l by HPLC under UV 287 nm. And calculating the integral peak areas of the oosporins at different concentrations, and drawing an oosporin standard curve. The linearized equation is: y is 2E-07x-0.4486(y is the mass of the oosporine and x is the area of the oosporine peak).

6. Preparation of Beauveria bassiana RNA

6.1 extraction of Beauveria bassiana RNA:

(1) grinding and freezing beauveria bassiana hypha into powder by using liquid nitrogen, and putting 100mg of the powder into an EP tube; (2) immediately adding 1ml Trizol, blowing and uniformly mixing by using a pipette gun, counting 1/5 volumes (200 mu l) of chloroform, uniformly mixing, and placing on ice for 2-3 min; (3) after centrifugation at 12000rpm at 4 ℃ for 5min, 400. mu.l of the supernatant was aspirated into another EP tube, and 1/2 volumes (200. mu.l) of chloroform were added: isoamyl alcohol (24:1), and mixing uniformly; (4) centrifuging at 12000rpm and 4 deg.C for 5min, transferring the supernatant into another EP tube (repeatedly deproteinizing), adding 1/2 volume (200 μ l) isopropanol, mixing, and standing on ice for 10 min; (5) centrifuging at 12000rpm at 4 deg.C for 15min, discarding supernatant, and washing with 800 μ l 75% ethanol for 2 times; (6) centrifuging at 12000rpm at 4 deg.C for 5min, removing supernatant, and air drying at room temperature; (7) dissolving the RNA precipitate with 50. mu.l DEPC water; (8) the RNA concentration was determined.

6.2 removal of DNA impurities:

prepare 50 ul reaction system, degrade DNA:

10×buffer	5μl
		total RNA	45μl
RRI	1μl
		DNase I	1μl

The reaction was carried out at 37 ℃ for 1 h.

6.3 recovery of purified RNA:

(1) to 50. mu.l of the reaction system were added 50. mu.l of DEPC water, and 100. mu.l of chloroform: isoamyl alcohol (24:1), and mixing uniformly; (2)12000rpm, 4 ℃ centrifugation for 5min, supernatant transferred to another EP tube, added 100 u l chloroform: mixing isoamyl alcohol (24: 1); (3) centrifuging at 12000rpm at 4 deg.C for 5min, transferring the supernatant into another EP tube, adding 10 μ l 3M sodium acetate and 250 μ l anhydrous ethanol (ice bath), mixing, and standing at-80 deg.C for 20 min; (4) centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, and washing with 800 μ l 75% ethanol for 2 times; (5)12000 rpm. Centrifuging at 4 deg.C for 5min, collecting precipitate, air drying, and dissolving with 30 μ l DEPC water; (6) and (5) measuring the RNA concentration.

7. Preparation of Beauveria bassiana cDNA (TOYOBO RNA reverse kit)

7.1RNA denaturation:

prepare 12. mu.l of reaction system

After 5min at 65 ℃ the reaction mixture was immediately placed on ice.

7.2RNA inversion:

20. mu.l of reverse reaction system was prepared

Modified RNA	12μl
		5×RT buffer	4μl
dNTP Mixture	2μl
		RNase Inhibitor	1μl
Rever Tra Ace	1μl

Reaction procedure

30℃	10min
		42℃	20min
85℃	5min

4℃

5min

8. Transformation method of pichia pastoris GS115

8.1 preparation of Pichia pastoris electrotransformation competence:

(1) removing GS115 single colony from YPD plate, inoculating to 5ml conventional YPD medium, and shake culturing at 30 deg.C overnight; (2) inoculating 0.02-0.1ml of seed culture solution into 2 bottles of 50ml YPD medium (filled into 250ml triangular bottles), and culturing overnight to OD₆₀₀1.3-1.5; (3) subpackaging two bottles of bacteria liquid into 2 centrifugal tubes of 50ml, centrifuging at 1500g and 4 ℃ for 5min, and respectively rinsing and suspending the bacteria with sterile water in a 50ml ice bath; (4)1500g, centrifugating for 5min at 4 ℃, and respectively resuspending the thalli with sterile water in 25ml ice bath; (5) centrifuging again (same method as above), and resuspending the cells with 2ml of sorbitol (1M) in ice bath; (6) centrifuged (as above), resuspended separately with 0.1ml ice-bath 1M sorbitol and split-packed into precooled80. mu.l of each of the EP tubes of (1) was put on ice for use.

8.2 Pichia pastoris electrotransformation method:

(1) adding 5-10 μ g of linearized plasmid into GS115 electrotransformation competent bacteria solution, and mixing; (2) transferring the mixed liquid of the competent bacteria into a precooled electric rotating cup (2mm), and standing on ice for 5 min; (3) electric rotation (2 kv); (4) 1ml of ice-bath 1M sorbitol is rapidly added into the electric rotating cup, and the mixed solution is transferred into a 1.5ml EP tube; (5) standing the bacterial liquid at 30 ℃ and incubating for 1-2 h; (6) mixing the bacterial liquid, spreading 200 μ l bacterial liquid on YPDS plate of bleomycin/geneticin, and culturing at 30 deg.C for 2-3 days.

8.3 screening of Pichia mutants:

(1) picking out the mutant colonies on the screening plate by using a gun head, respectively inoculating the mutant colonies into 3ml of YPD culture medium (containing bleomycin/geneticin), and carrying out shake culture at 30 ℃ for 24 h; (2) respectively taking 1ml of bacterial liquid and putting the bacterial liquid into corresponding EP tubes; (3) centrifuging at 12000rpm for 1min, and discarding the supernatant; (4) resuspending the thallus with 200. mu.l of lysate, respectively, and adding 100mg of 0.5mm ceramic beads; (5) oscillating for 8 times on an oscillator, 30s each time; (6) centrifuging at 12000rpm for 1min, and transferring the supernatant into another EP tube; (7) the mutants were verified by PCR.

8.4 fermentation and inducible expression of Pichia pastoris:

(1) taking 100 mu l of pichia pastoris bacterial liquid in a conventional YPD culture medium, inoculating the pichia pastoris bacterial liquid in 50ml of MGY culture medium (filled in a 250ml triangular flask), and carrying out shake culture at 30 ℃ and 220rpm for 24 h; (2) centrifuging at 1500g for 5min, discarding supernatant, resuspending thallus with 1ml conventional MMH culture medium, transferring to 100ml MMH culture medium (in 500ml triangular flask), and adjusting to OD ₆₀₀1 is ═ 1; (3) 0.5% methanol (500. mu.l) was added to the MMH medium to induce gene expression, and the mixture was shake-cultured at 30 ℃ and 220rpm for 48 hours.

9. Culture medium for pichia pastoris transformation and expression

YPD + Agar Medium (For 100ml) (Geneticin 0.5mg/ml or bleomycin 0.1mg/ml)

Yeast extract	1g
		Peptone	2g
Glucose	2g
		Agar powder	1.5g
Double distilled water	100ml

YPD medium (For 100ml) (Geneticin 0.5mg/ml or bleomycin 0.1mg/ml)

Yeast extract	1g
		Peptone	2g
Glucose	2g
		Double distilled water	100ml

MGY medium (For 100ml)

YNB	1.34g
		Glycerol	1ml
Vitamin B	0.04mg
		Histidine	4mg
Double distilled water	Adding to 100ml

MMH medium (For 500ml)

1.34％YNB	6.7g
		Histidine	20mg

Vitamin B	0.2mg
		Methanol	2.5ml
Double distilled water	Adding to 500ml

Lysis solution

Example 1 identification of an oosporin synthetic Gene Cluster

Sequencing beauveria bassiana ARSEF2860 to obtain sequenced genome data, and performing comparative genome analysis on the data to find OpS1 in beauveria bassiana and a gene cluster in which the OpS1 is located, wherein the gene cluster comprises: OpS1(BBA _08179), OpS2(BBA _08180), OpS3(BBA _08181), OpS4(BBA _08182), OpS5(BBA _08183), OpS6(BBA _08184), and OpS7(BBA _08185) (fig. 2).

Bioinformatic analysis showed that these genes might encode the following proteins, respectively, including: OpS1 encodes polyketide synthase/glycosidase synthase, OpS2 encodes transporter, OpS3 encodes transcription factor, OpS4 encodes hydroxylase, OpS5 encodes laccase, OpS6 encodes glutathione S transferase, OpS7 encodes Cupin protein (table 1).

Example 2 identification of genes involved in the Synthesis of oosporine and their functions

OpS1(BBA _08179), OpS2(BBA _08180), OpS3(BBA _08181), OpS4(BBA _08182), OpS5(BBA _08183), OpS6(BBA _08184) and OpS7(BBA _08185) were subjected to knockdown and overexpression, respectively, and the fermentation broth of the knockdown mutant was examined by High Performance Liquid Chromatography (HPLC) to observe the effect of deletion or overexpression of a specific gene on synthesis of oosporins.

2.1 Effect of Each Gene in the OpS Gene Cluster on oosporin Synthesis

Taking the pDHt-ks of a binary vector pCAMBIA1300 commonly used in plant genetics as a framework, respectively introducing a glufosinate resistance gene Bar and a benomyl resistance gene Ben into the framework, and constructing the binary vectors pDHt-Bar and pDHt-Ben for fungal transformation; based on the vector pDHt-Bar, knock-out vectors for knocking out genes OpS1, OpS2, OpS3, OpS4, OpS5, OpS6 and OpS7 are respectively constructed.

The upstream and downstream homology arms of the knock-out vector were PCR amplified using primers OpS1UF/OpS1UR and OpS1DF/OpS1DR (see Table 2 for specific primer sequences), respectively, and the upstream and downstream homology arm fragments were ligated to the upstream and downstream sites of vector pDHt-Bar, respectively (FIG. 3). The constructed OpS1 knock-out plasmid is used for transforming Agrobacterium tumefaciens AGL-1 to obtain the Agrobacterium containing the knock-out vector, and the Agrobacterium containing the knock-out vector is used for transforming beauveria bassiana ARSEF 2860. OpS1 knockout mutants of Beauveria bassiana obtained by glufosinate-resistance plate screening (delta OpS1)

Sequentially knocking out according to the method to obtain knock-out mutants of delta OpS1, delta OpS2, delta OpS3, delta OpS4, delta OpS5, delta OpS6 and delta OpS7, respectively collecting spores of each mutant and wild strain WT with 0.05% Tween solution, and preparing into 10% concentration⁸Spore suspension of each spore/ml, and 40. mu.l of each spore suspension was inoculated into a triangular flask containing 20ml of SDB medium, and shake-cultured at 25 ℃ and 200rpm for 3 days as a seed medium.

Inoculating 1ml of seed culture medium into triangular flasks containing 50ml of SDB culture medium, placing in a 25 deg.C light incubator (light time is 12 h/day), and standing for 5 days. Finally, mycelium is filtered off, and fermentation liquor is collected.

As shown in FIG. 4, the fermentation broth of the wild type strain WT was red, and the fermentation broths of the knockout mutants of the other genes were colorless except that the red color of the fermentation broth of the. DELTA. OpS2 was darkened.

Extracting the fermentation liquor with ethyl acetate, concentrating the crude extract by vacuum rotary evaporation, eluting with methanol, and detecting with High Performance Liquid Chromatography (HPLC). HPLC detection Using C18 column (CNW Corp.) (

4.6 mm. times.250 mm, 5 μm). The elution conditions were: column temperature 40 ℃, water (containing 0.1% trifluoroacetic acid): acetonitrile 87:13, elution 35And (5) min. The ultraviolet detection wavelength is UV 287 nm.

The results are shown in FIG. 5, in which the peak of ovamycin is marked in red. The results show that the deletion of the genes OpS1, OpS3, OpS4, OpS5, OpS6 and OpS7 results in no synthesis of oosporin, whereas the deletion of the gene OpS2 instead increases the yield of oosporin.

TABLE 2

2.2 Regulation of the Synthesis of oosporins by the transcription factor OpS3

And (3) qualitative experiment:

an overexpression vector of the gene OpS3 is constructed on the basis of the vector pDHt-Ben, a promoter of beauveria bassiana 3-glyceraldehyde phosphate dehydrogenase gpdA (BBA _05480) is amplified by PCR by using a primer gpdA-F/gpdA-R (table 2) and is connected to a enzyme cutting site SpeI at the downstream of the vector pDHt-Ben, and the vector pDHt-Ben-gpdA is obtained. Then, the gene OpS3 was amplified with the primer OpS3-F/OpS3-R (Table 2) and ligated to the vector pDHt-Ben-gpdA at the downstream XbaI cleavage site. The ligation direction was verified by using the verification primer gpdA-YF/OpS3-YR (Table 2), and the structure of the resulting over-expression vector pDHt-ben-gpdA-OpS3 is shown in FIG. 6.

The constructed vector is used for transforming beauveria bassiana through the mediation of agrobacterium tumefaciens AGL-1, and an overexpression mutant WT of the beauveria bassiana OpS3 gene is obtained through screening of a resistance plate containing benomyl, wherein OpS3 is a mutant of the beauveria bassiana OpS3 gene.

Separately collecting wild strain WT, delta OpS3 mutant and over-expression mutant WT of Beauveria bassiana, preparing spores of OpS3, and making into 10% concentration⁸Spore suspension of spore/ml, fermenting each mutant and wild type strain, filtering to separate mycelium and fermentation liquid, as shown in FIG. 7, wild type strainThe fermentation broth of WT appeared red, the fermentation broth of the strain Δ OpS3 was colorless, while the fermentation broth of WT: OpS3 was darkened.

HPLC detection is carried out on the collected fermentation liquor, and the detection result shows that the over-expression of OpS3 can obviously improve the yield of the oosporine, while the deletion of OpS3 causes that the beauveria bassiana cannot synthesize the oosporine (figure 8, the peak of the oosporine is marked in red).

Quantitative experiments:

and drawing a standard curve of the oosporine under the condition that the HPLC ultraviolet detection wavelength is UV 287nm by using the separated and purified oosporine standard sample. The peak areas of the oosporins were compared to the standard curve to obtain oosporins yields of the wild type strain WT and each mutant, as shown in fig. 9.

The results show that the over-expression of the transcription factor gene OpS3 obviously improves the yield of the oosporine, and the yield is improved from less than 50 mu g/ml to about 150 mu g/ml.

Research on action mechanism:

mycelium RNA of wild WT, delta OpS3 mutant and overexpression mutant WT are respectively extracted and subjected to reverse transcription to obtain cDNA. Expression of other genes of the OpS gene cluster in wild type strain WT and OpS3 mutant was detected by semi-quantitative RT-PCR (fig. 10, tubulin gene as internal standard).

The results show that the deletion of the gene OpS3 leads to the silencing of other oosporin synthesis related genes compared with the wild strain, and the over-expression of OpS3 greatly improves the expression of the genes.

2.3 functional characterization of polyketide synthase OpS1

The beauveria bassiana delta OpS1 mutant was subjected to a complementary feeding test of the glycosidic acid (formula I). Seed media of beauveria bassiana wild type strain WT and the delta OpS1 mutant were inoculated into triangular flasks containing 50ml of SDB medium, respectively. Wild type WT was inoculated into 3 flasks and the. DELTA.OpS 1 mutant was inoculated into 6 flasks, wherein 300. mu.l (100mg/ml) of an ethanol solution of the thiocyanic acid (OrA) was added to 3 flasks and 300. mu.l of ethanol was added to the other 3 flasks as a control, and fermentation was carried out for 5 days to collect the fermentation broth (FIG. 12).

The results show that the fermentation broth of wild-type WT is red, the fermentation broth of Δ OpS1 mutant without addition of the glycosidic acid is colorless, and the fermentation broth of Δ OpS1 mutant with addition of the glycosidic acid is also red.

Meanwhile, the fermentation liquor of different treatments is detected by HPLC, and the ultraviolet detection wavelength is UV 254 nm. The results are shown in FIG. 13, where the peak of the acid is marked yellow and the peak of the ovamycin is marked red. As can be seen, the addition of the thioctic acid restored the ability to synthesize ovosporine in mutant Δ OpS1, indicating that the thioctic acid was indeed a precursor for the synthesis of ovosporine and was responsible for the synthesis by OpS 1.

Heterologous expression of pichia pastoris:

the gene OpS1(BBA _08179) and phosphopantetheinyl transferase (PPTase) gene BBA _06793 are amplified from beauveria bassiana cDNA (Table 2), and are respectively connected with vectors pPICZB and pPIC3.5K to obtain heterologous expression vectors pPICZB-OpS1 and pPIC3.5K-06793, and GS115 is sequentially transformed, and after the selection of bleomycin and geneticin, a heterologous expression mutant GS115 of the gene OpS1 in Pichia pastoris is obtained, namely OpS 1.

Wild type strain GS115 and OpS1 heterologously expressed mutant GS115: OpS1 was shake-cultured in MMH medium at 30 ℃ and 220rpm for 48 h. And centrifuging at 4000rpm to collect fermentation liquor, and respectively detecting the fermentation liquor of the wild type and the heterologous expression mutant and a glycoside acid standard sample (OrA) by HPLC, wherein the ultraviolet detection wavelength is UV 210 nm.

The result is shown in fig. 14, the glycoside is marked as yellow, no accumulation peak is generated in the fermentation broth of pichia pastoris wild strain GS115, and the accumulation peak of the glycoside is found in the fermentation broth of OpS1 heterologous expression strain GS115:: OpS1, and the molecular weight is MW 168. Experiments demonstrated that OpS1(PKS) is responsible for the synthesis of the glycosidic acid.

2.4 functional characterization of other related genes

As shown in fig. 5, the deletions of OpS4, OpS5, OpS6 and OpS7 all resulted in no synthesis of oosporin, but no accumulation of intermediate products was found in the fermentation broth thereof. Therefore, in order to find the accumulation peak of the oosporin synthesis intermediate in each knockout mutant, the inventors overexpressed the transcription factor OpS3 in each knockout mutant.

The overexpression vector pDHt-Ben-gpdA-OpS3 was transformed into knock-out mutants Δ OpS4, Δ OpS5, Δ OpS6 and Δ OpS7, respectively, resulting in transcription factor overexpression mutants Δ OpS4: OpS3, Δ OpS5: OpS3, Δ OpS6: OpS3 and Δ OpS7: OpS 3. Wild-type strains and mutants were fermented (Δ OpS1, Δ OpS2, Δ OpS3, Δ OpS4:: OpS3, Δ OpS5:: OpS3, Δ OpS6:: OpS3 and Δ OpS7:: OpS3) and the fermentation broths were collected.

The results show that, in addition to the red color of the wild-type WT and Δ OpS2 mutant fermentation broths, the originally colorless OpS5, OpS6, and OpS7 knockout mutants exhibited red and orange colors, respectively, upon overexpression of the transcription factor (fig. 15).

Subsequently, the fermentation liquid is detected by HPLC, and the ultraviolet detection wavelength is UV 254 nm.

The results show (fig. 16):

(1) in the fermentation broth of mutant Δ OpS 4:ops 3 there was an accumulation peak of the glycosidic acid (MW 168) marked yellow.

(2) In mutant Δ OpS5, there was an accumulated peak in the fermentation broth of OpS3, labeled blue, and compound molecular weight MW 154, named "compound 1".

(3) In the fermentation broth of mutant Δ OpS 7:ops 3 there was one accumulated peak with MW 140, labeled green and designated "compound 2", and in addition there was one accumulated peak with MW 138, labeled orange and designated "compound 3".

(4) Mutants Δ OpS5:: OpS3, Δ OpS6:: OpS3 and Δ OpS7:: OpS3 all have an accumulation peak, the molecular weight is MW 274, the marker is red, and the mutant is named as "compound 4"; mutant Δ OpS6: the peak of ovamycin (MW 306) was found in the fermentation broth of OpS3 and marked red.

It was concluded that the hydroxylase OpS4, laccase OpS5 and Cupin protein OpS7 were involved in catalyzing the synthesis of oosporine with a precursor of the glycoside chromoacid, and that the hydroxylase OpS4 directly catalyzes the reaction with the substrate of the glycoside chromoacid. And OpS6 as S-glutathione transferase does not directly participate in the synthesis of oosporine, but may play a role in eliminating free radicals generated in the synthesis process of oosporine so as to protect cells from being damaged by the free radicals. Hydroxylase OpS4 has high amino acid sequence homology with salicylic acid hydroxylase, which catalyzes decarboxylation and hydroxylation of salicylic acid to produce catechol. The inventors therefore speculated that hydroxylase OpS4 also catalyzes a similar reaction, converting the glycosidic acid to trihydroxytoluene.

2.4.1 functional characterization of the OpS4 Gene

In order to further study the function of hydroxylase OpS4 and the reaction catalyzed by the hydroxylase, the inventor amplifies the gene OpS4 from the cDNA of beauveria bassiana and connects the amplified gene with a vector pPICZB to construct a heterologous expression vector pPICZB-OpS4 of the gene OpS4, transforms Pichia pastoris GS115, and obtains a heterologous expression mutant GS115 of the gene OpS4 through bleomycin screening, wherein the gene OpS4 is a mutant strain.

The wild type strain GS115 and the mutant GS115 were fermented in MMH medium, OpS4, and 300. mu.l (100mg/ml) of each of the glycoside acid was added to the fermentation broth of the wild type and the mutant, respectively, and the mixture was shake-cultured at 220rpm at 30 ℃ for 24 hours. The fermentation broth was collected by centrifugation at 4000rpm and examined by HPLC at UV 254nm, as shown in FIG. 17, in which the peak of the acid is marked yellow.

The results show that OpS4 heterologously expressed the mutant GS 115:comparedto the fermentation broth of the wild type strain, as shown in fig. 17, the glycosidic acid in the fermentation broth of OpS4 was largely consumed and converted to "compound 2", with a molecular weight of MW 140, and marked green. "compound 2" is unstable and will convert to "compound 3" (MW. RTM. 138) and "compound 4" (MW. RTM. 274), labeled orange and magenta, respectively.

The structure of the compound 4 is shown in a formula IV after nuclear magnetic resonance identification.

NMR(CD₃OD,400MHz)：

Atom	δ
		H5/H5’	4.899
H7/H7’	1.875
		C1/C1’	140.479
C2/C2’	157.615
		C3/C3’	186.266
C4/C4’	138.260
		C5/C5’	107.432
C6/C6’	182.957
		C7/C7’	11.854

The compound 4 has a structure similar to oosporine, is short of two hydroxyl groups compared with oosporine, is polymerized by two trihydroxytoluenes, and is named as ' 5,5 ' -dideoxy oosporine '. Since "5, 5' -dideoxy oosporine" is a product formed by oxidizing "compound 2", and "compound 2" has a molecular weight of MW 140, it is inferred that "compound 2" is trihydroxytoluene, and "compound 3" (MW 138) has a ketone structure, which is shown in formulas II and III, respectively. It was therefore concluded that hydroxylase OpS4 catalyzes the decarboxylative hydroxylation reaction of the glycosidic acid to form a trihydroxytoluene.

2.4.2 functional identification of the OpS7, OpS5 genes

As shown in FIG. 16, there was an accumulation of "Compound 2" in the fermentation broth of mutant Δ OpS7: OpS3, i.e., "Compound 2" was the substrate of OpS 7. The mutant delta OpS5 is separated and purified, wherein the 'compound 1' (with the molecular weight of MW 154) in the OpS3 fermentation broth is identified as a quinone compound through nuclear magnetic resonance, and the structure is shown as a formula VI.

The inventors concluded that OpS7 is a monooxygenase catalyzing the hydroxylation of the methyl ortho carbon of compound 2 to produce a tetrahydroxytoluene (formula V). When it is not available, it is converted from the enol form to the more stable keto form, compound 1. Meanwhile, the laccase OpS5 is found to oxidize polyphenol compounds into quinone compounds, and the carbon-carbon single bond is formed by polymerization between two free radicals through free radical reaction, while the laccase OpS5 knockout mutant is accumulated with 'compound 1', so that the inventor infers that laccase OpS5 catalyzes the oxidative polymerization of the enol structure 'tetrahydroxytoluene' (formula V) of 'compound 1' to form the oomycetin (formula VII).

NMR(CD₃OD,400MHz):

Atom	δ
		H3	4.975
H7	1.877

And (3) verification experiment:

the mutant delta OpS7 was fed with the isolated "Compound 1" by the inventors, OpS3, and the fermentation broth was checked by HPLC, the results are shown in FIG. 18.

From the figure we can see that the addition of "Compound 1" to the mutant Δ OpS7: OpS3 restores the synthesis of ovosporine and thus "Compound 1" is catalytically produced by OpS 7.

In conjunction with FIG. 16, the knock-out of OpS5 blocked the synthesis of ovosporine and resulted in the accumulation of "Compound 1", and thus, laccase OpS5 synthesized ovosporine using "Compound 1" as a substrate.

Example 3 Synthesis route to Oovamycin

The inventors concluded that the synthetic pathway for oosporine is shown in FIG. 19. The method comprises the following specific steps:

(1) the polyketide synthase OpS1 takes acetyl coenzyme A as a raw material to catalyze the synthesis of the thioctic acid;

(2) the glycosidic acid is catalyzed by hydroxylase Ops4, and is subjected to decarboxylation and hydroxylation reaction to generate a compound 2 (trihydroxy toluene);

(3) "Compound 2" is unstable and will convert to its ketone structure, "Compound 3", and Compound 2 will oxidatively polymerize to form a small amount of "Compound 4" (5, 5' -dideoxyoomycin);

(4) "Compound 2" (trihydroxytoluene) is hydroxylated by Cupin protein OpS7 to produce tetrahydroxytoluene, and a small amount of tetrahydroxytoluene is converted into its ketone structure "Compound 1";

(5) tetrahydroxytoluene is catalyzed by laccase OpS5, and is subjected to oxidation and free radical dimerization reaction to generate the oosporine.

Example 4 oosporine has insecticidal and bacteriostatic effects

4.1 insecticidal experiments

The inventor takes the greater wax moth as an experimental object to carry out a beauveria bassiana insecticidal experiment.

Taking spore suspension (with the concentration of 10) of OpS3 as a beauveria bassiana wild type WT, a mutant delta OpS1 which can not synthesize the oosporin and an oosporin high-yield strain WT⁶Each spore/ml), injecting galleria mellonella larvae respectively, injecting 10 mu l of spore suspension into each larva, and injecting 50 larvae into each strain; an additional set of blanks was injected with 0.05% Tween solution. The injected larvae of the galleria mellonella are respectively cultured in different containers according to different injected spores and are placed at 25 ℃ for breeding. And recording the times every 12h, and observing the death condition of the greater wax moth. The results of the experiment are shown in FIG. 20.

The result shows that the insecticidal toxicity of OpS3 is obviously stronger than that of a mutant delta OpS1 which can not synthesize the oosporine, and the oosporine plays an important role in the insecticidal process of the beauveria bassiana.

4.2 experiments on suppression of insect immune System

The inventors performed microscopic examination of hemolymph infected with greater wax moth at 24h, 36h and 48h after spore injection, respectively, and the results are shown in FIG. 21.

As can be seen from the figure, after 24h of injection of beauveria bassiana spores, the wild type WT and the oosporin high-producing strain WT are that the spores of OpS3 break through the package of the blood cells of the greater wax moth to start germination, while the spores of the mutant delta OpS1 cannot break through the package of the blood cells; 36h after injection, when the spores of the WT and WT: OpS3 have begun to multiply greatly and form worm bodies, only a few of the delta OpS1 spores have just begun to break through the package germination. It follows that oosporins play an important role in suppressing the insect immune system.

Meanwhile, we also observed that the bodies of OpS 3-infected greater wax moth were red, while the bodies of Δ OpS 1-infected greater wax moth were not red, and the hyphae in the bodies grew slowly, as shown in FIG. 22.

In conclusion, oosporine serving as a main exocrine secondary metabolite of beauveria bassiana not only influences the insecticidal process of the beauveria bassiana, but also is important for the subsequent growth and reinfection of the beauveria bassiana.

4.3 bacteriostatic experiments

Compared with WT and WT in OpS3, the larvae of the greater wax moth infected by the mutant delta OpS1 are more easily infected with bacteria, so that the dead bodies are rotten and smelly. Therefore, the inventors concluded that oosporine has a bacteriostatic effect, helping beauveria to compete against other saprophytic microorganisms. Therefore, the inventor carries out bacteriostasis experiments of gram-positive bacteria bacillus subtilis on the oosporine.

The experimental method comprises the following steps:

preparing oosporine water solutions with different concentrations, infiltrating a thick filter paper sheet, sticking the thick filter paper sheet on a culture medium plate with bacillus subtilis, and culturing the culture medium at 37 ℃ for 12 h.

The results of the experiment are shown in FIG. 23:

obvious inhibition zones are formed around the filter paper sheets with the concentrations of the oosporine of 1mg/ml and 0.8mg/ml, which shows that the oosporine has good inhibition effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (15)

1. The use of an oosporin biosynthesis gene or protein thereof for synthesizing oosporin and precursors or intermediates thereof, wherein the oosporin biosynthesis gene or protein thereof is selected from the group consisting of: OpS1, OpS4, OpS5 and OpS7,

wherein OpS1 is located at 24759-32012 of the gene cluster nucleotide sequence, encodes polyketide synthase/glycosidic acid synthase and has a length of 2211 amino acids; and the amino acid sequence of the OpS1 protein is shown as SEQ ID No. 2;

OpS4 is located at 39149-40901 th site of the gene cluster nucleotide sequence, encodes hydroxylase, and has the length of 427 amino acids; and the amino acid sequence of the OpS4 protein is shown as SEQ ID No. 5;

OpS5 is located at position 41885-44041 of the gene cluster nucleotide sequence and encodes laccase with the length of 590 amino acids; and the amino acid sequence of the OpS5 protein is shown as SEQ ID No. 6;

the OpS7 is positioned at the 45713-46768 th site of the gene cluster nucleotide sequence and codes the Cupin protein, and the length of the protein is 305 amino acids; and the amino acid sequence of the OpS7 protein is shown as SEQ ID No. 8;

the structure of the oosporine is shown as a formula VII:

2. the use of claim 1, wherein the nucleotide sequence of the OpS4 gene is as set forth in SEQ ID No. 12.

3. The use of claim 1, wherein the nucleotide sequence of the OpS5 gene is as set forth in SEQ ID No. 13.

4. The use of claim 1, wherein the nucleotide sequence of the OpS7 gene is as set forth in SEQ ID No. 15.

5. The use of claim 1, wherein the OpS1 gene or the protein thereof is used for the synthesis of a synthetic precursor of ovosporine.

6. The use according to claim 5, wherein the synthetic precursor of ovosporine is a glycosidic acid.

7. The use according to claim 6, wherein the glycosidic acid has the structure according to formula I:

8. the use of claim 7, wherein the OpS4 gene or protein thereof is used to catalyze the decarboxylation hydroxylation reaction of a compound of formula I to produce trihydroxytoluene.

9. The use of claim 8, wherein the trihydroxytoluene has the structure of formula II:

10. the use of claim 9, wherein the OpS7 gene or protein thereof is used to catalyze a hydroxylation reaction of a compound of formula II to produce tetrahydroxytoluene.

11. The use according to claim 10, wherein the tetrahydroxytoluene has the structure of formula V:

12. the use of claim 11, wherein the OpS5 gene or protein thereof is used to catalyze the oxidative polymerization of tetrahydroxytoluene to produce oosporine.

13. The use according to claim 12, wherein the oosporine has the structure according to formula VII:

14. the use of claim 1, wherein the oosporin biosynthesis gene is heterologously expressed in pichia pastoris, thereby producing oosporin and its synthesis precursors, intermediates.

15. The use of claim 14, wherein the pichia pastoris is selected from GS 115.

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