CN105985967A - Biosynthetic gene cluster of oosporein and application of biosynthetic gene cluster - Google Patents

Abstract

The invention relates to a biosynthetic gene cluster of oosporein, in particular to cloning, sequencing, analysis and function studies of the oosporein biosynthetic gene cluster which is produced from beauveria bassiana and has insecticidal and antimicrobial functions as well as an application of the oosporein biosynthetic gene cluster. Totally, the entire gene cluster includes seven genes, namely OpS1, OpS2, OpS3, OpS4, OpS5, OpS6 and OpS7. By conducting a genetic operation the biosynthetic genes, the bio-synthesis of the oosporein can be blocked, or the yield of the oosporein can be changed or a new compound can be generated. The invention also discovers a bio-synthesis way of the oosporein for the first time, and the invention has an important research significance.

Description

The biological synthesis gene cluster of oosporein and application thereof

Technical field

The invention belongs to microbial gene resource and genetic engineering field, be specifically related to that there is parasite killing and antibacterial work The clone of biological synthesis gene cluster, analysis, functional study and the application thereof of oosporein.

Background technology

Oosporein (Oosporein) is initially the sixties in last century, and qualification is a kind of by beauveria bassiana (Beauveria bassiana) and muscardine (Beauveria brongniarti) produce outside born of the same parents Red pigments, there is certain insecticidal activity and bacteriostasis.The activity of monoamine oxidase, MAO (MAO) is different Often can cause body several functions obstacle, form morbid state, such as parkinson, senile dementia and near Depressions the most popular in city over Nian etc. are all relevant with MAO activity exception, and oosporein is as a class Oxidase inhibitor, has due to the abnormal disease related drugs caused of monoamine enzymatic activity possibly for research and development Certain help.Although oosporein early has been observed that at present, and has resolved its structure (Fig. 1), but The biosynthesis pathway of oosporein is the most unclear.

Therefore, we are with microbe-derived oosporein as target molecule, from the biology of clone's oosporein Synthetic gene bunch sets out, and uses microbiology, molecular biology, biochemistry and organic chemistry to combine Method study its biosynthesis, explore its biosynthesis pathway and regulatory mechanism, and verify oosporein Parasite killing and fungistatic effect.

Summary of the invention

The present invention relates to have the biological synthesis gene cluster of the oosporein of insecticidal and antifungal effect clone, point Analysis, functional study and application thereof.

A first aspect of the present invention provides the biological synthesis gene cluster of a kind of oosporein, and described gene cluster includes The coding 7 oosporein synthesis related gene: OpS1 involved by oosporein biosynthesis, OpS2, OpS3, OpS4、OpS5、OpS6、OpS7；

Wherein, OpS1 is positioned at gene cluster nucleotide sequence 24759-32012 position, encodes polyketide synthase/orsellinic acid Synthase, a length of 2211 aminoacid；

OpS2 is positioned at gene cluster nucleotide sequence 32992-34102 position, encodes transport protein, and a length of 350 Individual aminoacid；

OpS3 is positioned at gene cluster nucleotide sequence 36584-38809 position, encoding transcription factors, and a length of 741 Individual aminoacid；

OpS4 is positioned at gene cluster nucleotide sequence 39149-40901 position, encoding hydroxylase, a length of 427 ammonia Base acid；

OpS5 is positioned at gene cluster nucleotide sequence 41885-44041 position, encoding laccases, a length of 590 amino Acid；

OpS6 is positioned at gene cluster nucleotide sequence 44430-45147 position, coding for glutathion S transferring enzyme, length It is 218 aminoacid；

OpS7 is positioned at gene cluster nucleotide sequence 45713-46768 position, encodes Cupin albumen, and a length of 305 Individual aminoacid.

In another preference, the sequence of described gene cluster is 8391-54012 position in SEQ ID NO.1.

In another preference, described gene cluster also includes in described 7 oosporein synthesis related genes The base combining or being constituted that gene or multiple (such as 2,3,4,5,6 or 7) gene are constituted Cause bunch or its fragment.

Second aspect present invention provides the biosynthesis associated protein of a kind of oosporein, the amino of described albumen Acid sequence is selected from the aminoacid sequence as shown in SEQ ID NO.:2-8.

In another preference, described biosynthesis associated protein is the polyketide synthase shown in SEQ ID NO.:2 / orsellinic acid synthase.

In another preference, described biosynthesis associated protein is the transport protein shown in SEQ ID NO.:3.

In another preference, described biosynthesis associated protein is the transcription factor shown in SEQ ID NO.:4.

In another preference, described biosynthesis associated protein is the hydroxylase shown in SEQ ID NO.:5.

In another preference, described biosynthesis associated protein is the laccase shown in SEQ ID NO.:6.

In another preference, described biosynthesis associated protein is the glutathion shown in SEQ ID NO.:7 Transferring enzyme.

In another preference, described biosynthesis associated protein is the Cupin albumen shown in SEQ ID NO.:8.

Third aspect present invention provides the biosynthesis related genes of a kind of oosporein, and described gene code is originally The biosynthesis associated protein of oosporein described in invention second aspect.

In another preference, described biosynthesis related genes is selected from following gene:

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

In another preference, the information of the nucleotide sequence of described synthesis related gene as mentioned above or is shown in Table 1.

Fourth aspect present invention provides a kind of expression vector, and described expression vector contains first aspect present invention institute The biological synthesis gene cluster of the oosporein stated or its fragment, or the life of the oosporein described in third aspect present invention Thing synthesis related gene.

Fifth aspect present invention provides the host cell of a kind of restructuring, and described host cell contains the present invention the 4th Integrate on expression vector described in aspect, or its chromosome and have the oosporein described in first aspect present invention of external source Biological synthesis gene cluster or third aspect present invention described in the biosynthesis related genes of oosporein.

In another preference, the host cell of described restructuring includes eukaryotic cell, such as beauveria bassiana, finishes Red yeast.

Sixth aspect present invention provides a kind of beauveria bassiana (Beauveria bassiana), described In beauveria bassiana, being lost selected from lower group of one or more genes in the biological synthesis gene cluster of oosporein Live: OpS1, OpS3, OpS4, OpS5, OpS6, OpS7, thus do not produce oosporein.

In another preference, described it is deactivated the gene that gene is selected from lower group: OpS1, OpS3, OpS4, OpS5, OpS6, and OpS7.

In another preference, in described beauveria bassiana, Ops2 gene is retained.

Seventh aspect present invention provides a kind of beauveria bassiana (Beauveria bassiana), described ball In beauveria bassiana, in the biological synthesis gene cluster of oosporein, one or more genes are over-expressed, thus carry Height or the yield of recovery oosporein,

Wherein, the described gene that is over-expressed is selected from lower group: OpS1, OpS3, OpS4, OpS5, OpS6 And OpS7.

In another preference, in described beauveria bassiana, Ops2 gene is knocked or lowers.

In another preference, described raising refers to, produces with the oosporein of original strain (beauveria bassiana) Amount A0 compares, and oosporein yield A1 in the beauveria bassiana of described process LAN is >=1.5 with the ratio of A0, Preferably >=2.0, more preferably >=2.5, such as 1.5-10, preferably 2-5.

Eighth aspect present invention provides the purposes of a kind of oosporein biosynthesis gene or its albumen, is used for The precursor of synthesis oosporein or intermediate, wherein said oosporein biosynthesis gene or the choosing of its albumen From lower group: OpS1, OpS4, OpS5, OpS7.

In another preference, described synthesizes external enzyme' s catalysis or synthetic.

In another preference, it is provided that a kind of OpS1 gene or the purposes of its albumen, described OpS1 gene Or its albumen is for synthesizing the synthesis precursor of oosporein.

In another preference, the synthesis precursor of described oosporein is orsellinic acid.

In another preference, shown OpS1 gene or its albumen are with S-acetyl-coenzyme-A for catalytic material glycosides color The synthesis of acid.

In another preference, the structure of described orsellinic acid shown in formula I:

In another preference, it is provided that a kind of OpS4 gene or the purposes of its albumen, described OpS4 gene Or its albumen is for the decarboxylation hydroxylation reaction of catalysis type I, thus generate Formula II intermediate.

In another preference, described intermediate is compound 2.

In another preference, described compound 2 is trihydroxytoluene.

In another preference, the structure of described compound 2 is as shown in Formula II:

In another preference, described compound 2 structural instability, compound 3 can be partially converted into and change Compound 4.

In another preference, described compound 3 is ketone form structure, and its structure is as shown in formula III:

In another preference, shown compound 4 is formed by the oxidation polymerization of 2 compounds 2, and its structure is such as Shown in formula IV:

In another preference, it is provided that a kind of OpS7 gene or the purposes of its albumen, described OpS7 gene or Its albumen, for being catalyzed the hydroxylation reaction of Formula II compound, generates tetrahydroxy toluene.

In another preference, the structure of described tetrahydroxy toluene shown as a formula V:

In another preference, described tetrahydroxy toluene structural instability, compound 1 can be partially converted into.

In another preference, described compound 1 is ketone form structure, and its structure is as shown in Formula IV:

In another preference, it is provided that a kind of OpS5 gene or the purposes of its albumen, described OpS5 gene or Its albumen, for being catalyzed the oxidative polymerization of tetrahydroxy toluene, produces oosporein.

In another preference, the structure of described oosporein is as shown in Formula VII:

Ninth aspect present invention provides a kind of method preparing ooecium mycin, including step:

I (), in the condition of applicable cultivation and in the case of adding starting compound, is cultivated and is produced ooecium mycin Host cell, thus produce ooecium mycin, wherein said starting compound is selected from lower group: orsellinic acid (Formulas I), Compound 2 (Formula II), tetrahydroxy toluene (Formula V) or a combination thereof；With

(ii) isolated or purified goes out described ooecium mycin.

In another preference, described host cell is beauveria bassiana.

In another preference, described host cell is introduced into the ball spore of external source ooecium mycin synthesis related gene Muscardine, wherein said gene is selected from lower group: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS7 base Cause.

Tenth aspect present invention provides a kind of method transforming host cell, including step:

(a) measure each related gene belonging to ooecium mycin synthetic gene bunch in described host cell expression and/or Activity；

B (), according to measurement result, imports the related gene of ooecium mycin synthetic gene bunch in described host cell, Thus improve/recover described host cell and produce the ability of ooecium mycin,

Wherein, the related gene of described gene cluster include one or more following gene: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS7.

In another preference, described host cell is beauveria bassiana.

In another preference, when OpS1 gene inactivation or the activity decrease of described gene cluster, further comprise the steps of:

Adding starting compound in cultivating system, described starting compound is selected from lower group: orsellinic acid (Formulas I), Compound 2 (Formula II), tetrahydroxy toluene (Formula V) or a combination thereof, to recover the formation of ooecium mycin.

The present invention the 11st aspect provides a kind of compound relevant to ooecium mycin, and described compound is selected from down Group:

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

The twelfth aspect of the present invention provides a kind of method of ability improving beauveria bassiana generation ooecium mycin, bag Include step:

In described beauveria bassiana, import the related gene of ooecium mycin synthetic gene bunch, thus improve or recover Described beauveria bassiana produce ooecium mycin ability, wherein, the related gene of described gene cluster include one or Multiple following gene: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS7；Or Knock out the OpS2 gene of ooecium mycin synthetic gene bunch in described beauveria bassiana, thus improve described ball Beauveria bassiana produces the ability of ooecium mycin.

The present invention the 13rd aspect provides OpS2 polypeptide or the purposes of its encoding gene of a kind of separation, institute State OpS2 polypeptide or gene for suppressing the synthesis of oosporein.

In another preference, described OpS2 polypeptide is selected from lower group: (i) has amino shown in SEQ ID NO:3 The polypeptide of acid sequence；

(ii) by SEQ ID NO:3 aminoacid sequence through the replacement of one or several amino acid residue, disappearance Or add and formed and have suppression ooecium rhzomorph complex functionality the polypeptide derivative by (i).

In another preference, the coded sequence of described OpS2 polypeptide is selected from lower group:

(1) coding polynucleotide sequence of polypeptide as described in SEQ ID NO:3；

(2) polynucleotide sequence as shown in SEQ ID NO:10；

(3) polynucleotide that the polynucleotide sequence and described in (1) or (2) is complementary.

Fourteenth aspect of the present invention provides the biosynthesis base of oosporein described in a kind of first aspect present invention Cause bunch or the purposes of its portion gene, it is characterised in that in described gene cluster, portion gene is used at ball spore The mutant of muscardine ARSEF 2860 related gene disappearance carries out allos complementary, thus recover to produce ovum P0-357；Or portion gene in described gene cluster is carried out heterogenous expression in Pichia sp., thus produce ovum The synthesis precursor of p0-357, intermediate and oosporein analog.

In another preference, described Pichia sp. is selected from GS115.

In another preference, the synthesis precursor of described oosporein is orsellinic acid (Formulas I).

In another preference, the synthetic intermediate of described oosporein is compound 2 (Formula II), chemical combination Thing 3 (formula III), tetrahydroxy toluene (Formula V) and compound 1 (Formula IV).

In another preference, described oosporein analog is compound 4 (formula IV).

In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (as implemented Example) in can be combined with each other between each technical characteristic of specifically describing, thus constitute new or preferred skill Art scheme.As space is limited, the most tired at this state.

Accompanying drawing explanation

Fig. 1 shows chemical structural formula and the color of oosporein.

Fig. 2 shows oosporein synthetic gene clustering architecture.

Fig. 3 shows OpS1 knockout carrier structure.

Fig. 4 shows each knock out mutants body and the change of wild-type strain fermentation liquid color.

Fig. 5 shows each mutant and wild-type strain fermentation liquid HPLC testing result.

Fig. 6 shows OpS3 over-express vector structure.

Fig. 7 shows that wild type, OpS3 knock out and process LAN mutant fermentation liquid color contrast.

Fig. 8 shows wild type and each mutant fermentation liquid HPLC testing result.

Fig. 9 shows wild-type strain and each mutant oosporein yield comparison.

Figure 10 shows the RT-PCR knot of wild-type strain and each mutant oosporein synthesis related gene Really.

Figure 11 shows the domain composition contrast of OpS1 Yu OrSA.

Figure 12 shows that wild-type strain and Δ OpS1 mutant orsellinic acid feed experiment fermentation liquid color pair Ratio.

Figure 13 shows that wild type and Δ OpS1 mutant orsellinic acid feed the HPLC testing result of experiment.

Figure 14 shows wild type and OpS1 heterogenous expression bacterial strain fermentation liquor and orsellinic acid standard specimen (OrA) HPLC testing result.

Figure 15 shows wild-type strain and the color contrast of each mutants which had fermentation liquid.

Figure 16 shows wild type and each mutant fermentation liquid HPLC testing result.

Figure 17 shows wild type and mutant GS115::OpS4 fermentation liquid HPLC testing result.

Figure 18 shows " compound 1 " feeding trial HPLC of wild type and mutant Δ OpS7::OpS3 Testing result.

Figure 19 shows the route of synthesis of oosporein.

Figure 20 shows greater wax moth bioassay results.

Figure 21 shows the greater wax moth hemolymph microscopy result infected by muscardine.

Figure 22 shows that wild type and mutant infect the contrast of worm corpse.

Figure 23 shows oosporein bacteriostatic test.

Detailed description of the invention

The present inventor, through extensively in-depth study, divides with the oosporein in beauveria bassiana source for target Son, from cloning its biological synthesis gene cluster in beauveria bassiana ARSEF 2860, uses micro-life The technique study that thing, molecular biology, bioanalysis informatics, biochemistry and organic chemistry combine Its biosynthesis, the first identified biological synthesis gene cluster of oosporein, specifically, described gene cluster bag Include 7 genes, be respectively as follows: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS7.Wherein, OpS1 Coding polyketide synthase/orsellinic acid synthase, OpS2 encode transport protein, OpS3 encoding transcription factors, OpS4 Encoding hydroxylase, OpS5 encoding laccases, OpS6 coding for glutathion S transferring enzyme, OpS7 encode Cupin Albumen；Further, inventor is found that the biosynthesis pathway of oosporein first, and it is mould to demonstrate ovum spore The parasite killing of element and fungistatic effect.On this basis, the present inventor completes the present invention.

Ooecium mycin synthesis related gene and albumen

As used herein, the invention discloses a kind of ooecium mycin synthetic gene bunch (gene cluster), Described gene cluster includes: OpS1, OpS2, OpS3, OpS4, OpS5, OpS6, OpS7 gene.

Wherein, OpS1 gene code ketone synthase/orsellinic acid synthase, and OpS1 gene at least has selected from lower group One or more features:

(i) coding aminoacid sequence as shown in SEQ ID NO:2；(ii) coding is such as SEQ ID NO:2 Shown aminoacid sequence replace through one or several amino acid residue, lack or add and formed by (i) Derivative polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:9；(iv) have with such as The polynucleotide that polynucleotide sequence shown in SEQ ID NO:9 is complementary.

OpS2 gene code transport protein, and OpS2 gene at least has the one or more spies selected from lower group Levy: (i) coding aminoacid sequence as shown in SEQ ID NO:3；(ii) coding is such as SEQ ID NO:3 Shown aminoacid sequence replace through one or several amino acid residue, lack or add and formed by (i) Derivative polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:10；(iv) have with The polynucleotide that polynucleotide sequence shown in shown in SEQ ID NO:10 is complementary.

The OpS3 gene coding transcript factor, and OpS3 gene at least has the one or more spies selected from lower group Levy: (i) coding aminoacid sequence as shown in SEQ ID NO:4；(ii) coding is such as SEQ ID NO:4 Shown aminoacid sequence replace through one or several amino acid residue, lack or add and formed by (i) Derivative polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:11；(iv) have with The polynucleotide that polynucleotide sequence shown in SEQ ID NO:11 is complementary.

OpS4 gene code hydroxylase；And OpS4 gene at least has one or more features selected from lower group: (i) coding aminoacid sequence as shown in SEQ ID NO:5；(ii) coding is as shown in SEQ ID NO:5 Aminoacid sequence replace, lack or add through one or several amino acid residue and being spread out by (i) of being formed Raw polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:12；(iv) have and SEQ The polynucleotide that polynucleotide sequence shown in ID NO:12 is complementary.

OpS5 gene code laccase；And OpS5 gene at least has one or more features selected from lower group: (i) Coding aminoacid sequence as shown in SEQ ID NO:6；(ii) coding ammonia as shown in SEQ ID NO:6 It is derivative by (i) that base acid sequence replaces through one or several amino acid residue, lack or adds and formed Polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:13；(iv) have and SEQ ID NO: The polynucleotide that polynucleotide sequence shown in 13 is complementary.

OpS6 gene code glutathione s-transferase；And OpS6 gene at least has selected from one of lower group or Multiple features: (i) coding aminoacid sequence as shown in SEQ ID NO:7；(ii) coding is such as SEQ ID NO: Aminoacid sequence shown in 7 replace through one or several amino acid residue, lack or add and formed by I polypeptide that () is derivative；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:14；(iv) have The polynucleotide complementary with polynucleotide sequence shown in SEQ ID NO:14.

OpS7 gene code Cupin albumen；And OpS7 gene at least has the one or more spies selected from lower group Levy: (i) coding aminoacid sequence as shown in SEQ ID NO:8；(ii) coding is such as SEQ ID NO:8 Shown aminoacid sequence replace through one or several amino acid residue, lack or add and formed by (i) Derivative polypeptide；(iii) there is the polynucleotide sequence as shown in SEQ ID NO:15；(iv) have with The polynucleotide that polynucleotide sequence shown in SEQ ID NO:15 is complementary.

In the case of the amino acid fragment having obtained ooecium mycin synthesis related gene, can construct according to it Encode its nucleotide sequence, and design specific probe according to nucleotide sequence.Nucleotide full length sequence Or its fragment generally can use the method for PCR TRAP, recombination method or synthetic to obtain.PCR is expanded Increasing method, can design primer according to nucleotide sequence disclosed in this invention, especially open reading frame sequence, And with commercially available cloud tints storehouse or the cloud tints storehouse as prepared by conventional method well known by persons skilled in the art as template, Expand and obtain relevant sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly PCR amplification, then Again the fragment that each time amplifies is stitched together by proper order.

The determination of oosporein biological synthesis gene cluster

Inventor, on the basis of completing the order-checking of beauveria bassiana ARSEF 2860 strain gene group, passes through Gene The method of Blast, have found gene orsA homologous genes OpS1 in beauveria bassiana and place base thereof Because of bunch (gene cluster), including: OpS1 (BBA_08179), OpS2 (BBA_08180), OpS3(BBA_08181)、OpS4(BBA_08182)、OpS5(BBA_08183)、OpS6(BBA_08184) With OpS7 (BBA_08185) (Fig. 2).

Oosporein biological synthesis gene cluster dependency and the determination of integrity

Owing to the biosynthesis gene of microbial secondary metabolite is that linked in clusters exists on chromosome, The present inventor knocks out experiment to each gene obtained in sequence, with checking acquisition gene cluster and ovum The biosynthetic dependency of p0-357 and integrity thereof.

Gene knockout OpS1, OpS3, OpS4, OpS5, OpS6, or OpS7 have interrupted oosporein completely Generation and gene overexpression OpS1, OpS3, OpS4, OpS5, or OpS7 can increase the product of oosporein Amount, it was demonstrated that the gene cluster screened relevant to oosporein biosynthesis really；Gene knockout OpS2 Add the yield of oosporein on the contrary, illustrate that OpS2 has the effect of suppression oosporein synthesis；Thus demonstrate,prove Real, the gene cluster obtained contains all genes required for oosporein biosynthesis.At each clpp gene Removing or in process LAN mutant, the generation having interrupted oosporein completely having, some yield is varied from, Have has not significant impact, and the noval chemical compound that has having occurs.

Based on above experimental result, and compare with similar compound biological synthesis gene cluster, the present inventor Determine that the biological synthesis gene cluster of oosporein comprises 7 opening code-reading frames (Fig. 2) from OpS1 to OpS7, Contain the region of chromosome 22 kb.In whole gene cluster, OpS1 encodes polyketide synthase/orsellinic acid synthase (Polyletide Synthase/Orsellinic acid Synthase), OpS2 encode transport protein (Transporter), OpS3 encoding transcription factors (Transcription Factor), OpS4 coding Hydroxylase (Hydroxylase), OpS5 encoding laccases (Laccase), OpS6 coding for glutathion S Transferring enzyme (Glutathione S-Transferases/GSTs), OpS7 encode Cupin albumen (Cupin Domain containing protein) (table 1, Fig. 2).

Table 1

Wherein, " " refers to " not producing "；" NA " refers to " not doing process LAN experiment ".

The route of synthesis of oosporein precursor

Polyketide synthase (PKS) is a class multifunctional enzyme, is made up of multiple modules, with S-acetyl-coenzyme-A (Acetyl-CoA) be starting material, each module with malonyl coenzyme A (Malonyl-CoA) as precursor, Introduce two carbosilane units successively.PKS starting module comprises only AT-domain and ACP-domain, presses The order arrangement of " AT-ACP ".Its extension of module is made up of three Core domains (Core-domain), I.e. ketone ester acyl synthetic domain (KS-domain), acyl group transfer organization territory (AT-domain) and acyl group carries Body protein domain (ATP-domain), by the order arrangement of " KS-AT-ACP ".Last of PKS Individual module is possibly together with a thioesterase domain (Te-domain), by the order of " KS-AT-ACP-Te " Arrangement.Except these core textures are overseas, often possibly together with some modification structure territories in PKS (Tailoring-Domain), such as ketone ester acyl reductase domain (KR-domain), dehydrogenase structure Territory (DH-domain), enoyl reduction enzyme domains (ER-domain) and methyl transferase domains (MT-domain) etc..PKS can be divided into 3 classes according to structure difference, and wherein the PKS of fungus belongs to type I Iterative type PKS.Type I iterative type PKS comprises only a module, by the recycling to this module Synthesize different products.Gene OpS1 is predicted to be type I iterative type PKS encoding gene, and and structure Orsellinic acid synthase gene (orsA) homology in nest aspergillosis, the domain composition of the PKS that they are coded is right Ratio is as shown in figure 11.

The precursor orsellinic acid of oosporein is demonstrated experimentally is responsible for synthesis by OpS1 (PKS).Specifically, Inventor carries out orsellinic acid covering feeding trial and finds beauveria bassiana Δ OpS1 mutant, orsellinic acid Adding the ability making mutant Δ OpS1 recover synthesis oosporein, therefore explanation orsellinic acid is strictly ovum spore The precursor of mycin synthesis, and it is responsible for synthesis by OpS1.

The biosynthesis pathway of oosporein

Inventor is found through experiments, as shown in figure 19, the biosynthesis of oosporein by OpS1, OpS4, OpS5, OpS7 mediate.

Specifically, the biosynthesis pathway of oosporein is as follows:

(1) synthesis that polyketide synthase OpS1 is catalytic material orsellinic acid with S-acetyl-coenzyme-A；

(2) orsellinic acid is catalyzed by hydroxylase Ops4, generates " compound 2 " (three through decarboxylation hydroxylation reaction Hydroxy-methylbenzene)；

(3) " compound 2 " is unstable, can be converted into its ketone form structure " compound 3 ", and chemical combination Thing 2 oxidation polymerization can form a small amount of " compound 4 " (5,5 '-dideoxy oosporein)；

(4) " compound 2 " (trihydroxytoluene) generates tetrahydroxy through Cupin albumen OpS7 hydroxylating Toluene, a small amount of tetrahydroxy toluene can be converted into its ketone form structure " compound 1 "；

(5) tetrahydroxy toluene is catalyzed by laccase OpS5, and oxidized and free radical dimerization generates ovum spore Mycin.

Carrier

Term used herein " carrier " includes that the clone that insertion genes of interest can be made to enter host cell expression carries Body and other carriers.Expression vector can include prokaryotic expression carrier and carrier for expression of eukaryon, can be plasmid, stick Grain, phage or virus etc..Typical expression vector is with making the regulating and controlling sequence of gene expression, and in suitable position It is equipped with the restriction endonuclease sites that can be inserted into exogenous gene.

In a particular embodiment, expression vector of the present invention contains the oosporein biosynthesis gene of the present invention, Or the oosporein biological synthesis gene cluster containing the present invention.

Host cell

Term used herein " host cell " has the implication that those of ordinary skill in the art are generally understood that, i.e. Comprise exogenous genes of interest and the cell expressed can be allowed to.Such as, host cell can be prokaryotic host cell (such as, escherichia coli, bacillus subtilis, streptomycete, small single-cell bacteria), eukaryotic host cell (such as, yeast), Plant cell etc..

In a particular embodiment, the host cell of the present invention preferably comprises the expression vector of the present invention, or dyeing The oosporein biosynthesis gene of the external source having single copy or multicopy is integrated on body, or single copy or multicopy Oosporein biological synthesis gene cluster.

In a preferred embodiment, the host cell of the present invention includes beauveria bassiana, Pichia sp..

Preferably, the engineering bacteria of the present invention is by beauveria bassiana (Beauveria bassiana ARSEF2860) Genetic engineering modified and obtain.

The sudden change beauveria bassiana of structural gene inactivation in oosporein biological synthesis gene cluster

The present invention also provides for a kind of beauveria bassiana mutant, and in described mutant, the oosporein of the present invention is biological One or more gene inactivations of synthetic gene, thus described mutant does not produce oosporein.

In a particular embodiment, one or more genes of described inactivation selected from coding such as SEQ ID NO.:9, Gene shown in 11-15.More preferably, the described gene that is deactivated is selected from OpS1, OpS3, OpS4, OpS5, OpS6 Or OpS7.

The ball spore that in the oosporein biological synthesis gene cluster that the present invention provides, one or more structural genes are deactivated Muscardine mutant, can as the model of gene function or control strain in checking oosporein gene cluster, and/or Host cell for heterogenous expression oosporein.

In oosporein biological synthesis gene cluster, red ferment is finished in sudden change beauveria bassiana or the sudden change of structural gene process LAN Female

The present invention also provides for a kind of beauveria bassiana mutant, and in described mutant, the oosporein of the present invention is biological One or more gene overexpressions of synthetic gene, thus described mutant improves or recovers to produce oosporein.

In a particular embodiment, one or more genes of described process LAN are selected from encoding such as SEQ ID NO.: 9, gene shown in 11-15.More preferably, described be deactivated gene selected from OpS1, OpS3, OpS4, OpS5, Ops6 or OpS7.

The ball spore of one or more structural gene process LAN in the oosporein biological synthesis gene cluster that the present invention provides Muscardine mutant, can as the model of gene function or control strain in checking oosporein gene cluster, and/or Host cell for heterogenous expression oosporein.

Main advantages of the present invention include:

(1) present invention is that ooecium mycin provides biosynthesis pathway, explores a kind of new biosynthesis machine System.

(2) clone gene of nucleotide sequence provided by the present invention or at least part of nucleotide sequence can lead to Cross suitable expression system to express to obtain corresponding enzyme or other higher biological activitys in foreign host Or yield.These foreign host include streptomycete, beauveria bassiana, pseudomonas, escherichia coli, spore Bacillus, yeast, plant and animal etc..

(3) present invention the most enough discloses the structure of a kind of ooecium mycin biological synthesis gene cluster, and to respectively The function of individual gene is analyzed and researched.

(4) the invention provides a kind of new method finding reticent intermediate product, i.e. by regulation and control base In cause bunch, the transcription factor of each gene expression carries out process LAN in mutant, thus obtains intermediate product.

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to institute of manufacturer The condition of suggestion.Unless otherwise indicated, otherwise percentage ratio and number be by weight.

Universal method:

1. the method for transformation of Agrobacterium tumefaciems AGL-1

Prepared by 1.1 Agrobacterium tumefaciems AGL-1 competence:

(1) with toothpick picking original strain AGL-1, in YEB flat board (containing 50 μ g/ml Carbenicillins) Go up the most streak culture activation.(2) single bacterium colony on picking flat board, is seeded to 4ml YEB fluid medium and (contains 50 μ g/ml Carbenicillins) in, 28 DEG C, 200rpm, overnight incubation.(3) take the above-mentioned bacterium solution of 2ml It is forwarded in 50ml YEB fluid medium (containing 50 μ g/ml Carbenicillins), 28 DEG C, 200rpm, Cultivation to bacterium solution OD600 is 0.5 (the most about 8h).(4) take out bacterium solution, ice bath 30min, 4 DEG C, 8000 Rpm, 5min are centrifugal collects thalline.(5) abandon supernatant, add the 10ml resuspended thalline of 20mM CaCl2 and sink Form sediment.(6) 4 DEG C, 8,000rpm, 5min, recentrifuge collects thalline.(7) supernatant, with 2ml 20mM The most resuspended thalline of CaCl2, adds sterile glycerol to final concentration of 15～20%.Subpackage is managed to 1.5ml EP, 100 μ l/ pipes, are stored in-80 DEG C after liquid nitrogen flash freezer.

1.2 Plastid transformation Agrobacterium tumefaciems AGL-1 competence:

(1) take out-80 DEG C of frozen Agrobacterium competence, be placed in thawed on ice about 10min.(2) to competence In add 0.5～the plasmid of 1 μ g mesh, stand 30min after mixing on ice.Process as follows the most successively: liquid nitrogen 5min, 37 DEG C of water-bath 5min, are immediately placed on 2min on ice after process.(4) add 1ml nonreactive YEB liquid culture Base, 28 DEG C, 150rpm, recovery 3h.(5) 8,000rpm, 2min are centrifugal collects thalline.(6) abandon Clearly, (blue or green containing 50 μ g/ml carboxylic benzyls to be spread evenly across YEB flat board after the 100 μ resuspended thalline of l nonreactive YEB Mycin and 50 μ g/ml kanamycin), it is inverted for 28 DEG C and cultivates 2 days.(7) PCR verifies transformant, picking Positive transformant is with 3ml YEB liquid (containing 50 μ g/ml Carbenicillins and 50 μ g/ml kanamycin) Overnight shake bacterium, 28 DEG C, 220rpm.(8) bacterium solution taking part and is stored in-80 DEG C, final glycerol concentration is 15%.

The genetic transformation of the most Agrobacterium tumefaciens mediated beauveria bassiana:

(1) the preparation of conidia of beauveria bassiana spore suspension: choose and cultivate on solid potato culture medium PDA plate The beauveria bassiana of two weeks, scrapes appropriate mycelia to 1ml aseptic 0.05%Tween-20 aqueous solution, vortex Vibration separates mycelia and conidium；Filter remove mycelia with glass silk flosssilk wadding or three layers of lens paper, collect filtrate； 12,000rpm, 1min, centrifugal bacterium solution collects spore；Abandon supernatant, with the resuspended spore of 1ml sterilized water, Remove remaining nutrition；12,000rpm, 1min, recentrifuge collects spore；Repeatable step 4～5 one Secondary, thoroughly to remove spore surface remnants nutrition；Abandon supernatant, with the resuspended spore of appropriate amounts of sterilized water, hemocytometer After number device statistics spore number, spore suspension concentration is adjusted to 5 × 10⁵Conidia/ml, uses as working solution Operate in subsequent experimental.(2) muscardine converts: the frozen bacterium of AGL-1 of the successful conversion respective carrier of transferring is extremely 3ml liquid YEB (containing 50 μ g/ml Carbenicillins and 50 μ g/ml kanamycin), 28 DEG C, 220 Rpm, overnight incubation；8,000rpm, 2min, centrifugal collection thalline；Abandon supernatant, with appropriate IM liquid Body weight hangs thalline, adjusts to OD₆₅₀It is 0.15；28 DEG C, 150rpm, inducing culture to OD₆₅₀It is 0.5～0.8, Typically need 6h；Take each 100 μ l of muscardine spore suspension of the Agrobacterium bacterium solution induced and fresh preparation, in 1.5ml EP pipe is mixed evenly；Taking above-mentioned mixed liquor 100 μ l and coat IM flat board, 28 DEG C co-culture 48h；M-100 solid medium temperature after subject to sterilization is down to 60 DEG C, add corresponding antibiotic (cephalothin, Phosphine oxamate/benomyl)；Every piece co-cultures IM flat board and covers with 15ml above-mentioned M-100 culture medium, 25 DEG C Cultivate 3-5 days and occur to single resistant clones；With toothpick, above-mentioned resistant clones picking is resisted to new M-100 On property culture medium flat plate, carry out programmed screening；With SDB fluid medium to resisting on postsearch screening flat board Property bacterium colony shake bacterium and cultivate, and carry out labelling in the relevant position at the M-100 flat board back side；Sucking filtration SDB cultivates bacterium Silk, extracts genome, carries out PCR checking.

The most agriculture bacillus mediated fungal transformation solution and culture medium

The basic salts solution of 2.5 × inducing culture

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
		Distilled water	Finally it is settled to 1 liter

M-100 trace element solution

H3BO3	30mg
		MnCl2·4H2O	70mg
ZnCl2	200mg
		Na2MoO4·2H2O	20mg
FeCl3·6H2O	50mg
		CuSO4·5H2O	200mg
Distilled water	Finally it is settled to 500 milliliters

M-100 saline solution

KH2PO4	16g
		Na2SO4·10H2O	9.064g
KCl	8g
		MgSO4·7H2O	2g
CaCl2	1g
		M-100 trace element solution	8ml
Distilled water	Finally it is settled to 1 liter

Inducing culture (liquid)

Add after being cooled to 50 DEG C after sterilizing:

Inducing culture (solid)

Add after being cooled to 50 DEG C after sterilizing:

M-100(For 1L)

M-100 saline solution	62.5ml
		Glucose	10g
KNO3	3g
		Distilled water	Finally it is settled to 1 liter
Agar powder	15g

MES and AS mother liquor:

	Store liquid concentration	PH (adjusts with 5M KOH)
			2-(morpholine) ethyl sulfonic acid MES	1M	5.3
Acetosyringone AS	10mM	8

4. oosporein isolation and purification method:

The extraction of 4.1 oosporeins:

Separate muscardine mycelia and culture medium with suction method, 50 DEG C of vacuum rotations of fermentation liquor of isolated are steamed Concentrate 1/4th for original volume.In the fermentation liquid concentrated, add trifluoroacetic acid and concussion shakes up to fermentation Liquid is become orange (pH ≈ 2) from aubergine.With the long-pending ethyl acetate extractive fermentation liquid of triploid, separation has Machine phase and aqueous phase.Ethyl acetate is evaporated through vacuum rotation steaming, is finally dissolved in proper amount of methanol.

The separation of 4.2 oosporeins:

First macroporous resin D101 is suspended in industrial alcohol, and fills in chromatographic column.With pH ≈'s 2 Filler macroporous resin in water (adjusting pH with trifluoroacetic acid) balance chromatographic column is to the eluent pH ≈ 2 flowed out (i.e. The aobvious acidity of macroporous resin).Gained oosporein crude extract is uniformly dripped in chromatographic column top, makes sample fill Divide and be adsorbed on macroporous resin.With water (pH ≈ 2) as flowing 3 column volumes of phase eluting, then use hydrogen Potassium oxide aqueous solution (pH ≈ 12) elution samples.Collect the purple solution eluted, adjust with trifluoroacetic acid To pH ≈ 2 (solution becomes orange), with after through 1/10th of vacuum concentrated by rotary evaporation to original volume.Use second Acetoacetic ester extracts solution again, and concentration is evaporated, and dissolves with a little methanol, is loaded in EP pipe.By EP After pipe is placed in-20 DEG C of precipitates overnight, 12000rpm is centrifuged 1min, collects precipitation.Precipitation is precipitation Oosporein crystal.

The Specification Curve of Increasing of oosporein under 5.UV 287nm.

With isolated and purified oosporein be each configured to concentration be 10mg/ml, 8mg/ml, 6mg/ml, The dimethyl of 4mg/ml, 2mg/ml, 1mg/ml, 0.8mg/ml, 0.6mg/ml and 0.4mg/ml Sulfoxide (DMSO) solution.HPLC detection, applied sample amount 3 μ l under the conditions of UV 287nm.Calculate The integrated peak areas of oosporein under variable concentrations, is depicted as oosporein standard curve.Lienarized equation is: Y=2E-07x-0.4486 (y is the quality of oosporein, and x is oosporein peak area).

6. the preparation of beauveria bassiana RNA

The extracting of 6.1 muscardine RNA:

(1) liquid nitrogen grinding freezing muscardine mycelia becomes powder, takes 100mg in EP pipe；(2) immediately Add 1ml Trizol, with liquid-transfering gun piping and druming mixing, count 1/5 volume (200 μ l) chloroform, mixing, 2-3min is placed on ice；(3) in 12000rpm, 4 DEG C of centrifugal 5min, draw supernatant 400 μ l in separately In one EP pipe, add 1/2 volume (200 μ l) chloroform: isoamyl alcohol (24:1), mixing；(4) 12000rpm, 4 DEG C of centrifugal 5min, proceed to supernatant (repeatedly Deproteinization) in another EP pipe, add 1/2 volume (200 μ l) isopropanol, mixing, stand 10min on ice；(5) 12000rpm, 4 DEG C of centrifugal 15min, Abandon supernatant, add 800 μ l 75% ethanol and wash 2 times；(6) 12000rpm, 4 DEG C of centrifugal 5min, abandon Clearly, room temperature is dried；(7) with 50 μ l DEPC water dissolution RNA precipitate；(8) RNA concentration is measured.

6.2 removal DNA impurity:

Configure the reaction system of 50 μ l, degradation of dna:

10×buffer	5μl
		Total serum IgE	45μl
RRI	1μl
		DNase I	1μl

1h is reacted in 37 DEG C.

6.3 recovery purifying RNAs:

(1) 50 μ l reaction system adds 50 μ lDEPC water, adds 100 μ l chloroforms: isoamyl Alcohol (24:1), mixing；(2) 12000rpm, 4 DEG C of centrifugal 5min, move into another EP pipe by supernatant In, add 100 μ l chloroforms: isoamyl alcohol (24:1) mixes；(3) 12000rpm, 4 DEG C of centrifugal 5min, Supernatant is moved in another EP pipe, adds 10 μ l 3M sodium acetates and 250 μ l dehydrated alcohol (ice bath), Mixing, places 20min in-80 DEG C；(4) 12000rpm, 4 DEG C of centrifugal 10min, abandon supernatant, with 800 μ l 75% ethanol washes 2 times；(5)12000rpm.4 DEG C of centrifugal 5min, collect precipitation, dry, with 30 μ l DEPC Water dissolution；(6) RNA concentration is surveyed.

7. the preparation (TOYOBO RNA reversal agents box) of beauveria bassiana cDNA

7.1RNA degeneration:

Configure 12 μ l reaction systems

After 65 DEG C of reaction 5min, it is immediately placed on ice.

7.2RNA inverts:

Configuration reversion reaction system 20 μ l

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

Response procedures

30℃	10min
		42℃	20min
85℃	5min

4℃

5min

8. the method for transformation of Pichia pastoris GS115

8.1 Pichia sp. electricity turn competent preparation:

(1) from YPD flat board, choose the mono-bacterium colony of GS115, be inoculated in 5ml routine YPD culture medium, 30 DEG C of shaking table overnight incubation；(2) take seed culture fluid 0.02-0.1ml, be inoculated in 2 bottles of 50ml respectively In YPD culture medium (being loaded on 250ml triangular flask), incubated overnight to OD₆₀₀=1.3-1.5；(3) by two Bottle bacterium solution is sub-packed in 2 50ml centrifuge tubes, in 1500g, 4 DEG C of centrifugal 5min, use 50ml respectively The outstanding thalline of sterilized water punching of ice bath；(4) 1500g, 4 DEG C of centrifugal 5min, respectively with 25ml ice bath The resuspended thalline of sterilized water；(5) recentrifuge (method is ibid), respectively with the ice bath 1M Pyrusussuriensis of 2ml The resuspended thalline of alcohol；(6) centrifugal (ibid), respectively with the resuspended thalline of 1M sorbitol of 0.1ml ice bath, And be sub-packed in the EP pipe of pre-cooling, often pipe 80 μ l, it is placed in the most standby.

8.2 Pichia sp. electricity shifting methods:

(1) take linearizing plasmid 5-10 μ g to be added on GS115 electricity and turn in competence bacterium solution, mixing；(2) By mixing competence bacterium solution move into pre-cooling electric revolving cup (2mm) in, place 5min on ice；(3) Electricity turns (2kv)；(4) in electricity revolving cup, the 1M sorbitol 1ml of ice bath is added rapidly, by mixed liquor Proceed in 1.5ml EP pipe；(5) bacterium solution is placed in 30 DEG C of stationary incubation 1-2h；(6) bacterium solution is mixed Even, take 200 μ l bacterium solution and be applied on the YPDS flat board of bleomycin/Geneticin, cultivate 2-3 days for 30 DEG C.

The screening of 8.3 Pichia sp. mutants:

(1) going to screen the mutant colonies on flat board with rifle choicest, the YPD being inoculated in 3ml respectively cultivates In base (containing bleomycin/Geneticin), cultivate 24h with 30 DEG C of shaking tables；(2) bacterium solution 1ml is taken respectively In corresponding EP pipe；(3) 12000rpm is centrifuged 1min, abandons supernatant；(4) respectively with 200 μ l Lysate thalline is resuspended, be separately added into the 0.5mm porcelain bead of 100mg；(5) shake on agitator Swing 8 times, each 30s；(6) 12000rpm is centrifuged 1min, is moved into by supernatant in another EP pipe； (7) PCR verifies mutant.

The fermentation of 8.4 Pichia sp. and abduction delivering:

(1) take the Pichia sp. bacterium solution 100 μ l in conventional YPD culture medium, be inoculated in 50ml MGY training Supporting base (being loaded in 250ml triangular flask), 30 DEG C, 24h cultivated by 220rpm shaking table；(2)1500g Centrifugal 5min, abandons supernatant, with the 1ml resuspended thalline of routine MMH culture medium, transfers in 100ml MMH Culture medium (is loaded on 500ml triangular flask), is adjusted to OD₆₀₀=1；(3) in MMH culture medium, add 0.5% Methanol (500 μ l), inducible gene expression, 30 DEG C, 220rpm shaking table cultivate 48h.

9. Pichia sp. converts and expresses used medium

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

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

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

Yeast extract	1g
		Peptone	2g
Glucose	2g
		Distilled water	100ml

MGY culture medium (For 100ml)

YNB	1.34g
		Glycerol	1ml
Vitamin B	0.04mg
		Histidine	4mg
Distilled water	Add to 100ml

MMH culture medium (For 500ml)

1.34%YNB	6.7g
		Histidine	20mg

Vitamin B	0.2mg
		Methanol	2.5ml
Distilled water	Add to 500ml

Lysate

The qualification of embodiment 1 oosporein synthetic gene bunch

Beauveria bassiana ARSEF 2860 is checked order, it is thus achieved that the genomic data of order-checking, to these data Compare genome analysis, have found OpS1 and the gene cluster at place thereof in beauveria bassiana, wherein wrap Include: 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 shows, these genes may be separately encoded following albumen, including: OpS1 encodes Polyketide synthase/orsellinic acid synthase, OpS2 coding transport protein, OpS3 encoding transcription factors, OpS4 coding Hydroxylase, OpS5 encoding laccases, OpS6 coding for glutathion S transferring enzyme, OpS7 encode Cupin albumen (table 1).

Embodiment 2 oosporein synthesis related gene and the qualification of function thereof

Respectively to OpS1 (BBA_08179), OpS2 (BBA_08180), OpS3 (BBA_08181), OpS4 (BBA_08182), OpS5 (BBA_08183), OpS6 (BBA_08184) and OpS7 (BBA_08185) Carry out knocking out and process LAN, and by high performance liquid chromatography (HPLC), the fermentation liquid knocking out mutant has been carried out Detection, the impact that oosporein is synthesized by the disappearance of observation specific gene or process LAN.

The impact of each gene pairs oosporein synthesis in 2.1OpS gene cluster

The pDHt-ks of binary vector pCAMBIA1300 conventional in plant genetic, as skeleton, divides wherein Not Yin Ru cremart resistant gene Bar and benomyl resistance gene Ben, construct for fungal transformation is double Unit carrier pDHt-Bar and pDHt-Ben；Based on carrier pDHt-Bar, construct respectively for knocking out The knockout carrier of gene OpS1, OpS2, OpS3, OpS4, OpS5, OpS6 and OpS7.

Use primer OpS1UF/OpS1UR and OpS1DF/OpS1DR (concrete primer sequence is shown in Table 2), point Other PCR amplifies the upstream and downstream homology arm of knockout carrier, and is connected to upstream and downstream homology arm fragment carry The upstream and downstream site (Fig. 3) of body pDHt-Bar.Plastid transformation crown gall agriculture bar is knocked out with the OpS1 built Bacterium AGL-1 obtains the Agrobacterium containing knockout carrier, is used for converting beauveria bassiana ARSEF 2860.Pass through The screening of cremart resistant panel obtains the OpS1 of beauveria bassiana and knocks out mutant (Δ OpS1)

Knock out the most successively, it is thus achieved that knock out mutant Δ OpS1, Δ OpS2, Δ OpS3, Δ OpS4, Δ OpS5, Δ OpS6 and Δ OpS7, collect each mutant and wild type respectively with the Tween solution of 0.05% The spore of bacterial strain WT, making concentration is 10⁸The spore suspension of individual spore/ml, takes 40 μ l spore suspensions and divides Not being inoculated in the triangular flask equipped with 20ml SDB culture medium, in 25 DEG C, 200rpm shaking table is cultivated 3 days, As seed culture medium.

Take 1ml seed culture medium to be inoculated in respectively in the triangular flask equipped with 50ml SDB culture medium, juxtaposition (light application time is 12h/ days), quiescent culture 5 days in 25 DEG C of illumination boxs.Finally, bacterium is filtered off Filament, collects fermentation liquid.

Result shows, as shown in Figure 4, the fermentation liquid of wild-type strain WT takes on a red color, except Δ OpS2 ferments Liquid redness deepen outside, other genes knock out mutant fermentation liquid all without color.

Being extracted with ethyl acetate fermentation liquid, gained crude extract is after vacuum concentrated by rotary evaporation, by methanol-eluted fractions and use High performance liquid chromatography (HPLC) detects.HPLC detection use CNW company C18 post (, 4.6mm × 250 Mm, 5 μm).Elution requirement is: column temperature 40 DEG C, water (containing 0.1% trifluoroacetic acid): acetonitrile=87:13, Eluting 35min.Ultraviolet detection wavelength is UV 287nm.

Testing result as it is shown in figure 5, in figure the peak of oosporein be marked as redness.Result shows, gene The disappearance of OpS1, OpS3, OpS4, OpS5, OpS6 and OpS7 causes oosporein to synthesize, and base Disappearance because of OpS2 adds the yield of oosporein on the contrary.

Table 2

The regulation and control that oosporein is synthesized by 2.2 transcription factor OpS3

Qualitative experiment:

Based on carrier pDHt-Ben, construct the over-express vector of gene OpS3, use primer GpdA-F/gpdA-R (table 2) amplifies beauveria bassiana glyceraldehyde 3-phosphate dehydro-genase gpdA through PCR (BBA_05480) promoter is connected at the restriction enzyme site SpeI in carrier pDHt-Ben downstream, it is thus achieved that Carrier pDHt-Ben-gpdA.Then, obtain gene OpS3 with primer OpS3-F/OpS3-R (table 2) amplification, And be connected at the restriction enzyme site XbaI of carrier pDHt-Ben-gpdA downstream.With checking primer GpdA-YF/OpS3-YR (table 2) verifies closure, gained over-express vector pDHt-ben-gpdA-OpS3 Structure is as shown in Figure 6.

The carrier built is passed through the mediated transformation beauveria bassiana of Agrobacterium tumefaciems AGL-1, through containing benzene The resistant panel screening of bacterium spirit obtains the process LAN mutant WT::OpS3 of beauveria bassiana OpS3 gene.

Collect beauveria bassiana wild-type strain WT, Δ OpS3 mutant and process LAN mutant respectively The spore of WT::OpS3, making concentration is 10⁸The spore suspension of individual spore/ml, ferment each mutant and open country Raw type bacterial strain, filters and separates mycelium and fermentation liquid, as it is shown in fig. 7, the fermentation liquid of wild-type strain WT Taking on a red color, the fermentation liquid of Δ OpS3 bacterial strain is without color, and the fermentation liquid redness of WT::OpS3 is deepened.

The fermentation liquor HPLC detection that will collect, testing result shows, the process LAN of OpS3 can significantly carry The yield of high oosporein, the disappearance of OpS3 then causes muscardine cannot synthesize oosporein (Fig. 8, ovum The peak of p0-357 is marked as redness).

Quantitative experiment:

Drawing oosporein with isolated and purified oosporein standard specimen is UV 287 at HPLC ultraviolet detection wavelength Standard curve under the conditions of nm.With the peak area ratio of oosporein to standard curve, obtain wild-type strain The oosporein yield of WT and each mutant, as shown in Figure 9.

Result shows, the process LAN of transcription factor gene OpS3 significantly improves the yield of oosporein, from About 150 μ g/ml are brought up to less than 50 μ g/ml.

Study on mechanism:

Extract wild type WT, Δ OpS3 mutant and the mycelium RNA of process LAN mutant WT::OpS3 respectively And reverse transcription obtains cDNA.By Semiquatitative RT-PCR assay detection other genes of OpS gene cluster at wild-type bacteria Expression (Figure 10, tubulin gene is internal standard) in strain WT and OpS3 mutant.

Result shows, compared to wild-type strain, the disappearance of gene OpS3 causes other oosporeins to synthesize The silence of related gene expression, the process LAN of OpS3 then drastically increases the expression of these genes.

The Function Identification of 2.3 polyketide synthase OpS1

Beauveria bassiana Δ OpS1 mutant is carried out orsellinic acid (Formulas I) covering feeding trial.By white for ball spore The seed culture medium of stiff bacterium wild-type strain WT and Δ OpS1 mutant is inoculated in respectively equipped with 50ml SDB In the triangular flask of culture medium.Wild type WT inoculates 3 bottles, and Δ OpS1 mutant inoculates 6 bottles, and wherein 3 Bottle adds orsellinic acid (OrA) ethanol solution of 300 μ l (100mg/ml), and other 3 bottles add 300 μ l Ethanol as comparison, ferment 5 days, collect fermentation liquid (Figure 12).

Result shows, the fermentation liquid of wild type WT takes on a red color, and is not added with the Δ OpS1 mutant of orsellinic acid Fermentation liquid is colourless, and the Δ OpS1 mutant fermentation liquid that with the addition of orsellinic acid also takes on a red color.

Meanwhile, fermentation liquor HPLC of different disposal being detected, ultraviolet detection wavelength is UV 254nm. As shown in figure 13, in figure, the peak of orsellinic acid is marked as yellow to result, and the peak of oosporein is marked as Red.As seen from the figure, the interpolation of orsellinic acid makes to recover in mutant Δ OpS1 the ability of synthesis oosporein, This explanation orsellinic acid is strictly the precursor of oosporein synthesis, and is responsible for synthesis by OpS1.

Pichia sp. heterogenous expression:

By gene OpS1 (BBA_08179) and phosphopantetheine acyltransferase (PPTase) gene BBA_06793 expands out (table 2) from beauveria bassiana cDNA, respectively with carrier pPICZB and PPIC3.5K connects, and obtains heterologous expression vector pPICZB-OpS1 and pPIC3.5K-06793, and successively Convert GS115, after bleomycin and Geneticin screen, obtain different in Pichia sp. of gene OpS1 Mutant GS115::OpS1 is expressed in source.

By wild-type strain GS115 and OpS1 heterogenous expression mutant GS115::OpS1 in MMH culture medium In in 30 DEG C, 220rpm shaking table cultivate 48h.4000rpm is centrifugal collects fermentation liquid, by wild type and The fermentation liquid of heterogenous expression mutant and orsellinic acid standard specimen (OrA) detect through HPLC respectively, ultraviolet detection ripple A length of UV 210nm.

As shown in figure 14, orsellinic acid is marked as yellow to result, Pichia sp. wild-type strain GS115's Fermentation liquid does not accumulate peak, and finds in the fermentation liquid of OpS1 heterogenous expression bacterial strain GS115::OpS1 The accumulation peak of orsellinic acid, molecular weight is MW=168.It is demonstrated experimentally that OpS1 (PKS) is responsible for orsellinic acid Synthesis.

The Function Identification of 2.4 other related genes

As it is shown in figure 5, the disappearance of OpS4, OpS5, OpS6 and OpS7 all causes oosporein not synthesize, But in its fermentation liquid, do not find the accumulation of intermediate product.Therefore, respectively knock out in mutant to find The accumulation peak of oosporein synthetic intermediate, transcription factor OpS3 respectively is being knocked out in mutant by inventor Row process LAN.

Over-express vector pDHt-Ben-gpdA-OpS3 is converted respectively knock out mutant Δ OpS4, Δ OpS5, Δ OpS6 and Δ OpS7, obtain transcription factor process LAN mutant Δ OpS4::OpS3, Δ OpS5::OpS3, Δ OpS6::OpS3 and Δ OpS7::OpS3.Fermentation wild-type strain and mutant (Δ OpS1, Δ OpS2, Δ OpS3, Δ OpS4::OpS3, Δ OpS5::OpS3, Δ OpS6::OpS3 and Δ OpS7::OpS3) and Collect fermentation liquid.

Result shows, in addition to wild type WT and Δ OpS2 mutant fermentation liquid take on a red color, the most colourless OpS5, OpS6 and OpS7 knock out mutant, after process LAN transcription factor, present redness and orange respectively Color (Figure 15).

Subsequently, being detected by above-mentioned fermentation liquid HPLC, ultraviolet detection wavelength is UV 254nm.

Result shows (Figure 16):

(1) in the fermentation liquid of mutant Δ OpS4::OpS3, there is the accumulation peak (molecular weight of an orsellinic acid For MW=168), it is marked as yellow.

(2) in the fermentation liquid of mutant Δ OpS5::OpS3, there is the peak of accumulation, be labeled as blueness, chemical combination Thing molecular weight is MW=154, named " compound 1 ".

(3) having a molecular weight in the fermentation liquid of mutant Δ OpS7::OpS3 is the accumulation of MW=140 Peak, is marked as green, named " compound 2 ", is MW=138 in addition with an accumulation peak molecular weight, It is marked as orange, named " compound 3 ".

(4) in mutant Δ OpS5::OpS3, Δ OpS6::OpS3 and Δ OpS7::OpS3, all have one Individual accumulation peak, molecular weight is MW=274, is labeled as a piece redness, named " compound 4 "；Mutant The fermentation liquid of Δ OpS6::OpS3 is found that the peak (molecular weight is MW=306) of oosporein, labeled For redness.

Thus inferring, hydroxylase OpS4, laccase OpS5 and Cupin albumen OpS7 participate in catalysis with orsellinic acid For the synthesis of the oosporein of precursor, and hydroxylase OpS4 is directly catalyzed the reaction with orsellinic acid as substrate. And the most directly participate in the synthesis of oosporein as the OpS6 of S-glutathione transferase, and it is probably To removing the free radical produced in oosporein building-up process, thus cell is protected to avoid the work of free radical injury With.Hydroxylase OpS4 is the highest with the amino acid sequence homology of salicylate hydroxylase, and salicylate hydroxylase Catalysis bigcatkin willow acid decarboxylation hydroxylating, generates catechol.Therefore inventor speculates that hydroxylase OpS4 is also catalyzed class As react, orsellinic acid is converted into trihydroxytoluene.

2.4.1OpS4 the Function Identification of gene

For the function studying hydroxylase OpS4 further and the reaction being catalyzed thereof, inventor is by gene OpS4 Expand out from the cDNA of beauveria bassiana, and be connected on carrier pPICZB, construct gene OpS4 Heterologous expression vector pPICZB-OpS4, and convert Pichia pastoris GS115, obtain through bleomycin screening The heterogenous expression mutant GS115::OpS4 of gene OpS4.

Respectively with MMH culture medium fermentation wild-type strain GS115 and mutant GS115::OpS4, and respectively Each orsellinic acid adding 300 μ l (100mg/ml) in the fermentation liquid of wild type and mutant, in 30 DEG C, 24h cultivated by 220rpm shaking table.4000rpm is centrifugal collects fermentation liquid, detects with HPLC, ultraviolet detection Wavelength is UV 254nm, and as shown in figure 17, wherein the peak of orsellinic acid is marked as yellow to result.

Result shows, as shown in figure 17, compared to the fermentation liquid of wild-type strain, OpS4 heterogenous expression is dashed forward Orsellinic acid in the fermentation liquid of variant GS115::OpS4 is consumed and is converted into " compound 2 " in a large number, point Son amount is MW=140, is marked as green." compound 2 " is unstable, can convert generation " compound 3 " (MW=138) and " compound 4 " (MW=274), it is respectively labeled as orange and piece redness.

Separating " compound 4 ", identify through nuclear magnetic resonance, NMR, its structure is as shown in formula IV.

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 structure of compound 4 is similar to oosporein, compared with oosporein, only lacks two hydroxyls, by Two trihydroxytoluenes are polymerized, named " 5,5 '-dideoxy oosporein ".Due to " 5,5 '- Dideoxy oosporein " it is " compound 2 " product of being oxidized to form, and the molecule of " compound 2 " Amount is MW=140, therefore conclude that " compound 2 " is trihydroxytoluene, and " compound 3 " (MW=138) For ketone form structure, structure is respectively as shown in Formula II, formula III.Therefore conclude that hydroxylase OpS4 is catalyzed glycosides color The decarboxylation hydroxylation reaction of acid, forms a trihydroxytoluene.

2.4.2OpS7, the Function Identification of OpS5 gene

As shown in figure 16, the fermentation liquid of mutant Δ OpS7::OpS3 has the accumulation of " compound 2 ", I.e. " compound 2 " is the substrate of OpS7." changing in isolated and purified mutant Δ OpS5::OpS3 fermentation liquid Compound 1 " (molecular weight is MW=154), it is accredited as a quinones, structure such as formula through nuclear magnetic resonance, NMR Shown in VI.

Inventor infers that OpS7 is a monooxygenase, the hydroxylating of catalysis " compound 2 " methyl ortho position carbon, Generate a tetrahydroxy toluene (Formula V).When it cannot be utilized, just it is converted into more by enol-type structure Stable ketone form structure, i.e. " compound 1 ".Studying discovery, laccase OpS5 can be by Polyphenols simultaneously Compound is oxidized to quinones, and through radical reaction, between two free radicals, polymerization forms carbon carbon list Key, and laccase OpS5 knocks out in mutant the accumulation having " compound 1 ", therefore inventor infers laccase Enol-type structure " tetrahydroxy toluene " (Formula V) oxidation polymerization of OpS5 catalysis " compound 1 " forms ovum Born of the same parents' rhzomorph (Formula VII).

NMR(CD₃OD,400MHz):

Atom	δ
		H3	4.975
H7	1.877

Confirmatory experiment:

" compound 1 " of inventor's isolated feeds mutant Δ OpS7::OpS3, examines with HPLC Surveying its fermentation liquid, result is as shown in figure 18.

From figure it will be seen that in mutant Δ OpS7::OpS3, add " compound 1 " make ovum The synthesis of p0-357 is recovered, and therefore " compound 1 " is produced by OpS7 catalysis.

Block the synthesis of oosporein in conjunction with knocking out of Figure 16, OpS5, and result in " compound 1 " Accumulation, therefore laccase OpS5 is that substrate synthesizes oosporein with " compound 1 ".

The route of synthesis of embodiment 3 oosporein

Inventor infers the route of synthesis of oosporein as shown in figure 19.Specific as follows:

(1) synthesis that polyketide synthase OpS1 is catalytic material orsellinic acid with S-acetyl-coenzyme-A；

(2) orsellinic acid is catalyzed by hydroxylase Ops4, generates " compound 2 " (three through decarboxylation hydroxylation reaction Hydroxy-methylbenzene)；

(3) " compound 2 " is unstable, can be converted into its ketone form structure " compound 3 ", and chemical combination Thing 2 oxidation polymerization can form a small amount of " compound 4 " (5,5 '-dideoxy oosporein)；

(4) " compound 2 " (trihydroxytoluene) generates tetrahydroxy through Cupin albumen OpS7 hydroxylating Toluene, a small amount of tetrahydroxy toluene can be converted into its ketone form structure " compound 1 "；

(5) tetrahydroxy toluene is catalyzed by laccase OpS5, and oxidized and free radical dimerization generates ovum spore Mycin.

Embodiment 4 oosporein has parasite killing and fungistatic effect

4.1 insecticidal test

Inventor carries out muscardine insecticidal test with greater wax moth for experimental subject.

Take beauveria bassiana wild type WT, the mutant Δ OpS1 that oosporein cannot be synthesized and ovum spore mould (concentration is 10 to the spore suspension of element superior strain WT::OpS3⁶Individual spore/ml), inject greater wax moth respectively Larva, every larva injects the spore suspension of 10 μ l, every kind of bacterium 50 larvas of injection；Separately set one group of sky White comparison, the Tween solution of injection 0.05%.By the greater wax moth larva after injection by injected spore not With supporting in different vessels respectively, it is placed in 25 DEG C of raisings.Remember a number every 12h, observe greater wax moth Death condition.Experimental result is as shown in figure 20.

Result shows, the parasite killing poison of beauveria bassiana wild type WT and oosporein superior strain WT::OpS3 Power is significantly stronger than the mutant Δ OpS1 that cannot synthesize oosporein, and the oosporein parasite killing muscardine is described During play an important role.

4.2 suppression Insect immunity system experimentations

Inventor 24h, 36h and 48h after injection spore respectively takes the blood of infected greater wax moth Lymph carries out microscopy, and result is as shown in figure 21.

From the figure, it can be seen that after injection beauveria bassiana spore 24h, wild type WT and oosporein high yield The spore of bacterial strain WT::OpS3 has been broken through the parcel of greater wax moth hemocyte and has been started to sprout, and mutant Δ OpS1 Spore then cannot break through the parcel of hemocyte；36h after injection, when WT and WT::OpS3 spore Start amount reproduction, and when forming hyphal body, the Δ OpS1 spore of only a few the most just starts to break through parcel and sprouts Send out.As can be seen here, oosporein plays an important role at suppression Insect immunity system aspects.

Meanwhile, we have also observed that the corpse of the greater wax moth infected by WT and WT::OpS3 takes on a red color, And the greater wax moth corpse infected by Δ OpS1 does not takes on a red color, and in polypide, mycelial growth is slow, such as Figure 22 institute Show.

To sum up showing, oosporein is as the main external secretion secondary metabolite of beauveria bassiana, not only shadow Ring the parasite killing process of beauveria bassiana, for the subsequent growth of muscardine and infect most important again.

4.3 bacteriostatic experiment

Compared with WT and WT::OpS3, the greater wax moth larva that mutant Δ OpS1 infects is easier to infect carefully Bacterium, makes worm corpse rot smelly.Therefore, inventor infers that oosporein also has bacteriostasis, helps muscardine The competition of other saprophytic microbes of antagonism.To this end, inventor that oosporein has been carried out gram positive bacteria is withered The bacteriostatic experiment of grass bacillus cereus.

Experimental technique:

The oosporein aqueous solution of preparation variable concentrations, the thick filter paper of infiltration is also affixed on and long has bacillus subtilis Culture medium flat plate on, culture medium is placed in 37 DEG C cultivate 12h.

Experimental result is as shown in figure 23:

At the filter paper that oosporein concentration is 1mg/ml and 0.8mg/ml formed around the most antibacterial Circle, shows that oosporein has good bacteriostasis really.

The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally Please appended claims limited range.

Claims (10)

1. the biological synthesis gene cluster of an oosporein, it is characterised in that described gene cluster includes encoding ovum spore 7 oosporein synthesis related gene: OpS1 involved by mycin biosynthesis, OpS2, OpS3, OpS4, OpS5、OpS6、OpS7；

Wherein, OpS1 is positioned at gene cluster nucleotide sequence 24759-32012 position, encodes polyketide synthase/orsellinic acid Synthase, a length of 2211 aminoacid；

OpS2 is positioned at gene cluster nucleotide sequence 32992-34102 position, encodes transport protein, and a length of 350 Individual aminoacid；

OpS3 is positioned at gene cluster nucleotide sequence 36584-38809 position, encoding transcription factors, and a length of 741 Individual aminoacid；

OpS4 is positioned at gene cluster nucleotide sequence 39149-40901 position, encoding hydroxylase, a length of 427 ammonia Base acid；

OpS5 is positioned at gene cluster nucleotide sequence 41885-44041 position, encoding laccases, a length of 590 amino Acid；

OpS6 is positioned at gene cluster nucleotide sequence 44430-45147 position, coding for glutathion S transferring enzyme, length It is 218 aminoacid；

OpS7 is positioned at gene cluster nucleotide sequence 45713-46768 position, encodes Cupin albumen, and a length of 305 Individual aminoacid.

2. the biosynthesis associated protein of an oosporein, it is characterised in that the aminoacid sequence of described albumen It is selected from the aminoacid sequence as shown in SEQ ID NO.:2-8.

3. the biosynthesis related genes of an oosporein, it is characterised in that described gene code claim The biosynthesis associated protein of oosporein described in 2.

4. an expression vector, it is characterised in that the ovum spore that described expression vector contains described in claim 1 is mould The biological synthesis gene cluster of element or its fragment, or the biosynthesis related genes of the oosporein described in claim 3.

5. the host cell of a restructuring, it is characterised in that described host cell contains described in claim 4 The biosynthesis gene of the oosporein described in claim 1 having external source is integrated on expression vector, or its chromosome Bunch or claim 3 described in the biosynthesis related genes of oosporein.

6. a beauveria bassiana (Beauveria bassiana), it is characterised in that at described ball spore In muscardine, in the biological synthesis gene cluster of oosporein, the one or more genes selected from lower group are deactivated: OpS1, OpS3, OpS4, OpS5, OpS6, OpS7, thus do not produce oosporein.

7. a beauveria bassiana (Beauveria bassiana), in described beauveria bassiana, ovum spore In the biological synthesis gene cluster of mycin, one or more genes are over-expressed, thus improve or recover oosporein Yield,

Wherein, the described gene that is over-expressed is selected from lower group: OpS1, OpS3, OpS4, OpS5, OpS6 And OpS7.

8. an oosporein biosynthesis gene or the purposes of its albumen, it is characterised in that be used for synthesizing The precursor of oosporein or intermediate, wherein said oosporein biosynthesis gene or its albumen are selected from down Group: OpS1, OpS4, OpS5, OpS7.

9. the method preparing ooecium mycin, including step:

I (), in the condition of applicable cultivation and in the case of adding starting compound, is cultivated and is produced ooecium mycin Host cell, thus produce ooecium mycin, wherein said starting compound is selected from lower group: orsellinic acid (Formulas I), Compound 2 (Formula II), tetrahydroxy toluene (Formula V) or a combination thereof；With

(ii) isolated or purified goes out described ooecium mycin.

10. the method that host cell is transformed, it is characterised in that include step:

(a) measure each related gene belonging to ooecium mycin synthetic gene bunch in described host cell expression and/or Activity；

B (), according to measurement result, imports the related gene of ooecium mycin synthetic gene bunch in described host cell, Thus improve/recover described host cell and produce the ability of ooecium mycin,

Wherein, the related gene of described gene cluster include one or more following gene: OpS1, OpS3, OpS4, OpS5, OpS6 or OpS7.