A transporter of rhizanillic acid a and coding gene and application thereof

Abstract

The application discloses a transmembrane transporter of interlinking solap A and an encoding gene and application thereof, and relates to the technical field of microbial genetic engineering. The transmembrane transporter is any one of (1), (2) and (3): (1) a SvMFS1 protein, wherein the amino acid sequence is shown as SEQ ID NO. 3; (2) a protein having more than 90% identity with the SvMFS1 protein and being related to the transport of interlinking solap A, wherein the amino acid sequence of the SvMFS1 protein is subjected to substitution, deletion and / or addition of one or more amino acid residues; and (3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1) or (2). The application researches and finds that the encoding gene of the transmembrane transporter can significantly improve the yield of interlinking solap A when the encoding gene is overexpressed in sacculispora vulgaris, thereby providing a new direction for the development of microbial source interlinking solap A.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial genetic engineering, in particular to a transmembrane transporter of Altersolanol A, its coding gene and application. BACKGROUND

[0002] The Major Facilitator Superfamily (MFS) is a ubiquitous and conserved superfamily of proteins that exists in a wide range of organisms, including bacteria, archaea and eukaryotes. MFS family is responsible for the transport of a variety of small molecules, including monosaccharides, oligosaccharides, drug molecules, inositol, organophosphates, neurotransmitters, co-factors, amino acids, polypeptides, vitamins, bases, nucleosides, nucleotides, iron chelates, metabolites of the tricarboxylic acid cycle and other solutes. They are not only involved in a wide range of physiological activities, such as material exchange and signal transduction, but also have a close relationship with the drug resistance caused by the exocytosis of various toxic compounds. MFS proteins can specifically transport a series of toxic exogenous compounds, including secondary metabolites and antibiotics. In filamentous fungi, many MFS transporters have the ability to regulate the secretion of toxins.

[0003] Toxins are non-enzymatic compounds produced by plant pathogenic fungi during metabolic processes, which can destroy the normal physiological functions of plants in a very low concentration range. Stemphylium spp. is a typical necrotrophic pathogenic fungus, which can produce a variety of toxins. The most prominent one is Altersolanol A, which is a kind of tetrahydroanthraquinone compound, orange yellow, molecular formula is C 16 H 16 The structure formula is as follows:

[0004]

[0005] Tetrahydroanthraquinone compounds are an important class of microbial secondary metabolites, which have anticancer, antibacterial, antiviral, antidiabetic, antimalarial and other biological activities. This kind of substance can also show anticancer activities such as inhibition of cell proliferation, invasion, metastasis and angiogenesis by inducing apoptosis, blocking cell cycle or inhibiting related enzymes. However, Altersolanol A has not yet become a clinical drug, and the research on its activity is not deep enough, especially the literature reports on the mechanism of action are less. It is speculated that it may be because the amount of Altersolanol A obtained from natural channels is small, which has not attracted people's attention. Therefore, it is of great practical significance to study Altersolanol A from microorganisms. Further research on Altersolanol A may help to create new drugs for anticancer, antibacterial, antiviral and other purposes. SUMMARY

[0006] The present application aims to provide a transporter of Altersolanol A, a coding gene thereof and an application thereof, so as to solve the problems existing in the prior art. The coding gene of the transporter can significantly improve the yield of Altersolanol A when overexpressed in Catenaria anguillarum, thereby providing a new direction for the development of microbially derived Altersolanol A.

[0007] To achieve the above-mentioned object, the present application provides the following solutions.

[0008] The present application provides a transporter of Altersolanol A, which is any one of (1), (2) and (3):

[0009] (1) a SvMFS1 protein, the amino acid sequence of which is shown in SEQ ID NO. 3;

[0010] (2) a protein having more than 70% identity with the SvMFS1 protein and being related to the transport of Altersolanol A, which is obtained by substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of the SvMFS1 protein;

[0011] (3) a fusion protein obtained by connecting a protein tag to the N-terminal or / and C-terminal of (1) or (2).

[0012] The present application also provides a coding gene of the above-mentioned transporter.

[0013] Further, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.

[0014] The present application also provides a gene expression cassette comprising the above-mentioned coding gene.

[0015] The present application also provides a recombinant overexpression vector comprising the above-mentioned gene expression cassette.

[0016] The present application also provides a recombinant host cell comprising the above-mentioned recombinant overexpression vector.

[0017] The present application also provides an application of the above-mentioned coding gene, gene expression cassette, recombinant overexpression vector or recombinant host cell in promoting the production of Altersolanol A by Catenaria anguillarum, which improves the yield of Altersolanol A by overexpressing the coding gene in the Catenaria anguillarum.

[0018] The present application also provides a construction method of an Altersolanol A production strain, which comprises the step of transforming the above-mentioned recombinant overexpression vector into Catenaria anguillarum to construct a recombinant Catenaria anguillarum overexpressing the coding gene; and the recombinant Catenaria anguillarum is the Altersolanol A production strain.

[0019] The application also provides a trans-lycopersiconol A production strain constructed according to the construction method.

[0020] The application also provides a trans-lycopersiconol A production method, which comprises the steps of: fermenting and culturing the trans-lycopersiconol A production strain to prepare the trans-lycopersiconol A.

[0021] The application discloses the following technical effects:

[0022] The application obtains differential expression genes by performing transcriptome sequencing comparison on two different Stemphylium strains infected with a fungal virus Stemphylium lycopersici alternavirus 1 (SlAV1), and obtains a new SvMFS1 gene of a major facilitator superfamily gene through analysis and screening, wherein the gene is a key transport protein for transport synthesis of Altersolanol A in Stemphylium, and the yield of Altersolanol A can be significantly improved by overexpression of the gene in Altersolanol A. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0024] Figure 1 Figure for changes in expression amount of SvMFS1 gene before and after infection of S1MV1 in SvHN-02 strain;

[0025] Figure 2 Schematic diagram of protein structure coded by transport protein SvMFS1 gene;

[0026] Figure 3 Schematic diagram of transmembrane structure of protein coded by transport protein SvMFS1 gene;

[0027] Figure 4 Phenotype diagram of wild type and SvMFS1 gene knockout / back complementation strains, wherein SvHN-02 is a wild type strain, KOSvMFS1-1, KOSvMFS1-2 and KOSvMFS1-10 are gene knockout type strains, and COSvMFS1-1, COSvMFS1-29 and COSvMFS1-39 are gene back complementation strains;

[0028] Figure 5Statistical chart of the content of Altersolanol A in the fermentation of the SvHN-02 SvMFS1 gene knockout / backbone strain; wherein, SvHN-02 is a wild type strain; KOSvMFS1-1, KO SvMFS1-2 and KOSvMFS1-10 are gene knockout strains; COSvMFS1-1, COSvMFS1-29 and COSvMFS1-39 are gene backbones strains;

[0029] Figure 6 The phenotype chart of the two obtained SvMFS1 gene super-expression transformants and the wild type Apophysomyces is screened; wherein, SvHN-02 is a wild type strain; OESvMFS1-5 and OESvMFS1-8 are super-expression strains;

[0030] Figure 7 The statistical chart of the content of Altersolanol A in the fermentation of the SvHN-02 strain in which the SvMFS1 gene is super-expressed; wherein, SvHN-02 is a wild type strain; OESvMFS1-5 and OESvMFS1-8 are super-expression strains. DETAILED DESCRIPTION

[0031] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is merely intended to teach a person skilled in the art further details about the various aspects and features of the present application and is not intended to limit the scope of the application. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and for teaching one skilled in the art to variously employ the present application.

[0032] It should be understood that the terms used in the specification of the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and between any other stated value or stated range in the specification is also encompassed within the scope of the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in the specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in the specification is not intended as an admission that the reference is prior art to the application described and claimed herein.

[0034] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from this specification, which is to be regarded in an illustrative manner. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the subject matter disclosed herein. The specification and examples are illustrative only.

[0035] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used to permit a possibility of additional features, components, steps, etc.

[0036] The strains involved in the embodiments of the present application: Stemphylium vesicarium strain SvHN-02 and Stemphylium lycopersici strain SlHN-10 have been disclosed in Chinese patent CN202011153985.3; the above-mentioned strains are preserved by Hunan Agricultural University and are committed to be open to the public for 20 years from the date of application of the present application.

[0037] The fungal virus Stemphylium lycopersici alternavirus 1 (SlAV1), originally named Stemphylium lycopersici mycovirus 1 (SlMV1) has been disclosed in Chinese patent CN202011153985.3.

[0038] Example 1: Candidate and verification of key transporter protein for Altersolanol A transport synthesis

[0039] SlAV1 infected Stemphylium lycopersici strain SlHN-10 will hinder the synthesis of Altersolanol A, and the virus can horizontally transmit to Stemphylium vesicarium strain SvHN-02 and inhibit the synthesis of Altersolanol A. The transcriptome sequencing of the two groups of strains before and after being infected with SlAV1 was compared. Through the analysis of the differentially expressed genes (DEGs) of SlAV1 infected strains by RNA-seq, a group of candidate genes that may be involved in the synthesis of Altersolanol A in Stemphylium lycopersici and Stemphylium vesicarium were determined. One of them is a gene of the major facilitator superfamily, which is named as SvMFS1. After the wild type strain SlHN-02 is infected with SlAV1, the expression amount of SvMFS1 gene decreases significantly. Figure 1 Therefore, it is speculated that this gene is related to the transport regulation of Altersolanol A synthesis. Then the verification of the function of the SvMFS1 gene was carried out, and the specific operation was as follows:

[0040] According to the transcriptome sequencing results, primers were designed:

[0041] SvMFS1-F: 5'-ATGAATGAAGGGATTGGAAGCA-3' (SEQ ID NO. 4);

[0042] SvMFS1-R: 5'-TCATGCGGCCCCTCCCTGAAG-3' (SEQ ID NO. 5).

[0043] PCR amplification of the SvMFS1 gene, the specific operation process is as follows:

[0044] 1. Extraction of fungal DNA and RNA

[0045] The experimental method and step refer to the instructions of the rapid DNA extraction detection kit (KG203) and the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (DP441) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0046] 2. PCR amplification of the SvMFS1 gene

[0047] The PCR reaction system 50.0 μL: 10×PCR Buffer 5.0 μL, 2.5 mmol / L dNTP 4.0 μL, 2.0 μL of each of the upstream and downstream primers, 2.0 μL of DNA or cDNA template, 1.0 μL of Pfu enzyme, and ddH2O to 50.0 μL.

[0048] Amplification procedure: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, for 34 cycles; 72℃ extension for 5 min. The PCR products of DNA and cDNA were detected by 1% agarose gel electrophoresis and entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing.

[0049] The results show that the DNA full length of the SvMFS1 gene is 1858 bp, and the nucleotide sequence is shown as SEQ ID NO. 1.

[0050] SEQ ID NO. 1:

[0051]

[0052] RT-PCR amplification of the CDS of the SvMFS1 gene was performed as follows:

[0053] Reverse transcription of RNA was performed with reference to the Quant cDNA First-Strand Synthesis Kit (KR103) of Tiangeng Biochemical Technology (Beijing) Co., Ltd. The PCR reaction system was performed with reference to the PCR amplification system of the SvMFS1 gene. The amplification product was subjected to 1% agarose gel electrophoresis, and the RT-PCR product was cloned into a T vector and sent to a sequencing company for sequencing verification. The results show that the length of the CDS of the SvMFS1 gene is 1674 bp (SEQ ID NO. 2), the length of the amino acid sequence of the encoded protein is 557 Aa (as shown in SEQ ID NO. 3), and protein structure prediction shows that the protein contains an MFS family domain ( Figure 2 ) and contains 14 transmembrane domains ( Figure 3 ), which are the characteristics of typical MFS family proteins.

[0054] SEQ ID NO. 2:

[0055]

[0056] SEQ ID NO. 3:

[0057] MNEGIGSNSSGVSTPDILSGEKIYLPHEQDAKEEVETTGDGSTSDIKEEYQTGLQLVLLMISILFTVCLTSLDMTIVGTAIPKITDEFHGLGMVSWYGSAYFMTFGGFQPASGKFYRYFPLKWSFLGALLIFELGSLICGIAQNSTTFVVGRAIAGVGASAVVTGAFTIAALSCEPRQRPAIMGLLGVVYGLSSVVGPLLGGVFSDHASWRWCFYINLPIGGVSAALILFFFKTPPQVVTEQTTWQEKLLQMDPVGIALVMGAIVAYILALESGGQKEPWGSSTVIGLLVGFVVIFLVFVLWEIYNGKHAMLPPRLFRQRSIWQPAGFIFFFSSAYIVLLYYLPIYFQSIDNRSAIMSGVLNLPLVLSLAIGSTVSGIVVSKTGLAAPFMLTGAALATLSTGLMYTFDTDTGMGKWIGYQILYGAGVGLGFQMAINTAQANVSIADMSSATATVFFFQTIGGAFSLSASQSGFANRLLATLAKTAPGVNPQLVIRTGATQIRSSFTPGEVPGILEAYMAGIKVTMAIATGLAGASVLVTLFVSRKRLNVQKLQGGAA.

[0058] 3. Verification of the function of SvMFS1 gene

[0059] 3.1 Obtaining of SvMFS1 knockout double fragments

[0060] Split-marker homologous recombination principle was adopted, and the bacterial phosphotransferase B gene (HYG) with hygromycin resistance was used as a selective marker to knock out the SvMFS1 gene of the Clonostachys rosea. The flanking sequences of the SvMFS1 gene were obtained from the whole genome sequence of SvHN-02, and the flanking fragments included the upstream homologous fragment (UF) and the downstream homologous fragment (DF). The complete HYG fragment was obtained from the vector pCB1300, and the combination product of the upstream homologous arm, the downstream homologous arm and the HYG three fragments was obtained by fusion PCR amplification. The knockout double fragments UH1 (the upstream homologous arm combined with the HYG1 fragment) and DH2 (the downstream homologous arm combined with the HYG2 fragment) were obtained by the second round of PCR, wherein the HYG1 sequence partially overlapped with the HYG2 sequence. The double fragments were introduced into the protoplast cells of the Clonostachys rosea by PEG-mediated protoplast transformation, so as to replace the SvMFS1 gene fragment with the HYG fragment, thereby realizing the knockout of the SvMFS1 gene, and the specific operation was as follows:

[0061] The upstream and downstream flanking sequences of the SvMFS1 gene in the genome of the Clonostachys rosea were obtained through the NCBI database, the primers SvMFS1-UF-F / R and SvMFS1-DF-F / R were designed by using primer 5.0, and the upstream fragment UF and the downstream fragment DF of the SvMFS1 were amplified by using the genomic DNA of the Clonostachys rosea SvHN-02 as a template. The HYG fragment was amplified by using the plasmid vector pBC1300 as a template. The recombination frame was obtained by connecting the upstream fragment UF, the downstream fragment DF and the HYG fragment through Double-joint PCR, the double fragments were obtained by amplifying the UF+HYG1 fragment by using the primers SvMFS1-UF-F and SPD-SY-R, and the HYG2+DF fragment by using the primers SPD-XY-F and SvMFS1-DF-R, and the related primers were as follows:

[0062] SvMFS1-UF-F: 5'-GATACCGTTTCGCTTCTTTCG-3' (SEQ ID NO. 6),

[0063] SvMFS1-UF-R: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCGGCGGAGTATGTTGATG CTTT-3' (SEQ ID NO. 7),

[0064] SvMFS1-DF-F: 5'-CGTCCGCAATGTGTTATTAAGTCGACTTGCCTCTGGAAACCAGCA TC-3' (SEQ ID NO. 8),

[0065] SvMFS1-DF-R: 5'-TCGGGAATAGTCGCCATCTCA-3' (SEQ ID NO. 9),

[0066] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3' (SEQ ID NO. 10),

[0067] HYG-R: 5'-GTCGACTTAATAACACATTGCGGACGT-3' (SEQ ID NO. 11),

[0068] SPD-SY-R: 5'-ACTTCGGGGCAGTCCTCG-3' (SEQ ID NO. 12),

[0069] SPD-XY-F: 5'-GAACTCACCGCGACGTCTGT-3' (SEQ ID NO. 13).

[0070] 3.2 Obtaining of in situ back-supplement double fragment of SvMFS1

[0071] Also using the Split-marker principle, the SvMFS1 gene is back-supplemented by in situ back-supplementation method. First, G418 fragment is obtained from vector KSTNP. Two shortened but partially overlapping G418 fragments (G1 and G2 fragments) are obtained by PCR. In the second round of PCR, the upstream homologous arm plus the SvMfs1 gene fragment is fused with the G1 fragment; the downstream homologous arm is fused with the G2 fragment to obtain the in situ back-supplementation double fragment for transformation, the specific operation is as follows:

[0072] The upstream and downstream flanking sequences of the SvMFS1 gene in the genome of G. capsulata are obtained, and the genomic DNA of G. capsulata SvHN-02 is used as a template to design primers 5.0 to amplify the upstream and downstream fragments of the target gene CoUFJY using primers SvMFS1-UF-F and SvMFS1-CoDF-R. The primers SvMFS1-CoDF-F and SvMFS1-DF-R are used to amplify the downstream fragment CoDF. The primers G418-F / R are designed, and the plasmid vector KSTNP is used as a template to amplify the G418 fragment. The CoUFJY fragment, CoDF fragment and G418 fragment are connected by Double-joint PCR to obtain the recombination frame, and the double fragment is obtained: the CoUFJY+G418-1 fragment is amplified using primers SvMFS1-UF-F and SPD-CoUG1-R, and the G418-2+CoDF fragment is amplified using primers SPD-CoDG2-F and SvMFS1-DF-R. The related primers are as follows:

[0073] SvMFS1-UF-F: 5'-GATACCGTTTCGCTTCTTTCG-3' (SEQ ID NO. 6),

[0074] SvMFS1-CoDF-R: 5'-CGAGGTGTTTCCAGGTTGGTTAAAGGAATGTACAGTCTATAT-3' (SEQ ID NO. 14),

[0075] SvMFS1-CoDF-F: 5'-TGCCAAAGGCAATACCCTGCTTGCCTCTGGAAACCAGCATC-3' (SEQ ID NO. 15),

[0076] SvMFS1-DF-R: 5'-TCGGGAATAGTCGCCATCTCA-3' (SEQ ID NO. 9),

[0077] G418-F: 5'-ACCAACCTGGAAACACCTCG-3' (SEQ ID NO. 16),

[0078] G418-R: 5'-GCAGGGTATTGCCTTTGGCA-3' (SEQ ID NO. 17),

[0079] SPD-CoUG1-R: 5'-ACCGTAAAGCACGAGGAAGC-3' (SEQ ID NO. 18),

[0080] SPD-CoDG2-F: 5'-CTGGGCACAACAGACAATCG-3' (SEQ ID NO. 19).

[0081] 3.3 PEG-mediated protoplast transformation method

[0082] The mycelium was crushed by a freezer mill and transferred to 50 mL YEPD liquid medium, incubated at 28°C, 220 rpm for 48 hours in a constant temperature shaker. The mycelium was filtered with three layers of sterile lens paper and washed with 10 mL 0.7 M NaCl in a clean bench. Then, the filtered mycelium was added to 1.5 mL enzymatic solution (2% pectinase, 2% lywallzyme, 2% cellobers, 2% Kitalase cell lysing enzyme dissolved in 0.7 M NaCl), incubated at 28°C, 80 rpm for 1 hour. After enzymatic hydrolysis, the mycelium was filtered with three layers of sterile lens paper and then washed with 10 mL 0.7 M NaCl for three times. The filtrate was centrifuged at 6000 rpm for 6 minutes, and the protoplasts at the bottom of the centrifuge tube were collected and resuspended with STC buffer (sucrose 100 g, 0.5 M Tris-HCl (pH = 8.0) 50 mL, CaCl2·2H2O 3.6755 g, deionized water to 500 mL). The protoplasts were adjusted to 2 x 10 7 Then, 20 μg of the fusion fragment, 400 μL PTC (PEG8000 200 g, STC solution to 500 mL, dissolved and stirred in a 65°C water bath, sterilized with a bacterial filter) were added and mixed, and the mixture was placed in an ice bath for 10 min. Then, 600 μL PTC was added and allowed to stand for 5 min, and then transferred to 5 mL TB3 liquid medium, inverted and mixed, and incubated at 28°C for 12 hours. Finally, all the mycelium was poured into a culture medium containing antibiotics, and then poured into a plate for screening.

[0083] 3.4 High performance liquid chromatography analysis of fermentation broth of wild type strain, gene knockout and complemented strain

[0084] 3.4.1 Preparation of fermentation broth and measurement of mycelium dry weight

[0085] The puncher with an inner diameter of 7 mm was used to punch the mycelium cake of wild type strain SvHN-02, knockout mutant and complemented transformant, and 3 mycelium cakes were inoculated into PD liquid medium, with 3 repeats, incubated at 28°C, 200 r / min for 7 days, then filtered with 3 layers of lens paper and 4 layers of gauze into a 50 mL centrifuge tube to obtain the fermentation broth. Then, the obtained mycelium was dried by absorbing water with filter paper and wrapped, and placed in a 65°C oven for drying to measure the dry weight of the mycelium.

[0086] 3.4.2 Extraction of altersolanol A toxin and determination by high performance liquid chromatography (HPLC)

[0087] (1) Extraction: Pipette 5 mL of fermentation broth into a 10 mL centrifuge tube, add an equal volume of methanol, mix well, and place at 4°C overnight. Place the overnight solution in a centrifuge at 8000 rpm for 5 minutes to obtain the supernatant, then add an equal volume of ethyl acetate for 3 times of extraction. When the solution does not appear to be layered, a small amount of powdered CaCl2 can be added to the tube to dissolve and separate the layers. Then, the obtained organic phase is blown dry by a liquid nitrogen instrument, 1 mL of methanol is added to each sample tube, and the solution is dissolved with the aid of an ultrasonic instrument for 1 min. The obtained sample is filtered through a 0.22 μm Nylon 6 microporous filter membrane into a 2 mL brown sample bottle, and stored in a 4°C refrigerator for standby.

[0088] (2) Chromatographic conditions: The chromatographic column is Sharpsil-AR C18 (250 x 4.6 mm, 5 μm); the mobile phase is methanol and ultrapure water (25:75); the detection wavelength is 215 nm; the injection volume is 10 μL; the flow rate is 1 mL / min; and the column temperature is 35°C.

[0089] (3) Preparation of standard curve: The working solution is prepared by diluting 1 mg / mL Altersolanol A stock solution: 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL. After the preparation of the standard solution, it is filtered through a 0.22 μm Nylon 6 microporous filter membrane into a 2 mL brown sample bottle, and labeled.

[0090] The results show that the synthesis of Altersolanol A in the SvMFS1 gene knockout strain is completely inhibited, while the synthesis of the complemented strain is restored Figure 4 and Figure 5 , indicating that the SvMFS1 gene is a key transport protein for the synthesis of Altersolanol A.

[0091] Example 2: Overexpression of SvMFS1 gene in A. cunicularis to increase the yield of Altersolanol A

[0092] 1. Extraction of fungal RNA according to Example 1.

[0093] 2. Construction of SvMFS1 overexpression vector

[0094] KSTNP vector (Dr. Li Yanlin of Hunan Agricultural University, the present application promises to the public for 20 years from the date of application, and is given) preserved with glycerol was streaked on LB plate and cultured at 37℃ overnight, and a single colony was picked and cultured in LB liquid medium at 180rpm 37℃ for 16h in a shaker for plasmid extraction. The plasmid extraction step refers to the high-purity plasmid extraction kit (DP104) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The vector was recovered by EcoR I enzyme cutting, and the purified recovered full-length SvMFS1 gene CDS and KSTNP linear vector were connected using Exnase III enzyme, and E. coli DH5α was transformed by heat shock, and positive clones were selected by PCR, and sent to Shanghai Sangon for sequencing verification, and the correct SvMFS1 gene overexpression vector was constructed.

[0095] 3. Genetic transformation and detection of SvMFS1 gene overexpression vector

[0096] The SvMFS1 gene overexpression vector was transformed into the strain of Catenaria anguillarum SvHN-02, and two SvMFS1 gene overexpression transformants (OESvMFS1-5, OESvMFS1-8) were obtained, and from the observation of culture traits, the synthesis of Altersolanol A was significantly increased. Figure 6 Compared with SvHN-02, the content of Altersolanol A in the fermentation broth of the SvMFS1 gene overexpression transformant was significantly increased, indicating that the ability of the overexpression transformant to synthesize Altersolanol A was significantly increased. Figure 7

[0097] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.​

Claims

1. A transporter for solanidol A of Herpomyces pruni, characterized by, The Stemphylium transport protein is any one of (1) and (2): (1) a SvMFS1 protein, the amino acid sequence of which is shown as SEQ ID NO. 3; (2) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1).

2. A coding gene of the transport protein according to claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

2.

4. A gene expression cassette, characterized in that, The coding gene according to claim 2 or 3.

5. A recombinant overexpression vector, characterized by, The gene expression cassette according to claim 4.

6. A recombinant host cell, characterized in that, The recombinant overexpression vector according to claim 5.

7. Use of the coding gene according to claim 2 or 3, the gene expression cassette according to claim 4, the recombinant overexpression vector according to claim 5 or the recombinant host cell according to claim 6 for promoting the production of intermedeol A by Stemphylium, characterized in that, The production of the solanone A is improved by overexpressing the coding gene in the Stemphylium; The Stemphylium is Stemphylium lycopersici or Stemphylium vesicarium.

8. A method for constructing a solanoid A-producing strain, characterized by, The step of transforming the recombinant overexpression vector according to claim 5 into the Stemphylium to obtain a recombinant Stemphylium overexpressing the coding gene; the recombinant Stemphylium is the solanone A production strain. The Stemphylium is Stemphylium lycopersici or Stemphylium vesicarium.

9. A solanone A production strain constructed according to the construction method of claim 8.

10. A method for producing solavatone A, characterized by, The step of fermenting the solanone A production strain according to claim 9 to prepare the solanone A. The Stemphylium is Stemphylium lycopersici or Stemphylium vesicarium.

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