Transport protein of Stemphylium solanol A as well as coding gene and application thereof

Abstract

The invention discloses a transport protein of Stemphylium solanol A as well as a coding gene and application thereof, and relates to the technical field of microbial genetic engineering. The transporter is any one of (1), (2) and (3): (1) SvMFS1 protein, the amino acid sequence of which is as shown in SEQ ID NO.3; (2) carrying out substitution, deletion and / or addition of one or more amino acid residues on the amino acid sequence of the SvMFS1 protein to obtain a protein which has more than 90% of identity with the SvMFS1 protein and is related to crosslink solanol A transport; and (3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1) or (2). Research finds that overexpression of the coding gene of the transporter in Stemphylium cystosum can significantly improve the yield of interlinked solanol A, thereby providing a new direction for developing microbial-sourced interlinked solanol A.

Description

Technical Field

[0001] The invention relates to the technical field of microbial genetic engineering, and in particular to a transport protein of Stoloniferum solanol A and a coding gene and application thereof. Background Art

[0002] The Major Facilitator Superfamily is a ubiquitous conserved protein superfamily present in a variety of organisms, including bacteria, archaea, and eukaryotes. The MFS family is responsible for the transport of a variety of small molecules, including monosaccharides, oligosaccharides, drug molecules, inositol, organophosphates, neurotransmitters, cofactors, amino acids, peptides, 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 are also inextricably linked to drug resistance caused by the exocytosis of various toxic compounds. MFS proteins are able to specifically transport a range of toxic exogenous compounds, including secondary metabolites and antibiotics. In filamentous fungi, many MFS transporters have the ability to regulate toxin secretion.

[0003] Toxins are non-enzymatic compounds produced by plant pathogenic fungi during metabolism, which can destroy the normal physiological functions of plants at extremely low concentrations. Stemphylium spp. is a typical necrotrophic pathogen that can produce a variety of toxins. The most prominent of these is Altersolanol A, which is a tetrahydroanthraquinone compound with an orange-yellow color and a molecular formula of C 16 H 16 O8, CAS No. 22268-16-2, structural formula is as follows:

[0004]

[0005] Tetrahydroanthraquinone compounds are an important class of microbial secondary metabolites with biological activities such as anticancer, antibacterial, antiviral, antidiabetic, and antimalarial. These substances can also show anticancer activities such as inhibiting cell proliferation, invasion, metastasis, and angiogenesis by inducing cell apoptosis, blocking the cell cycle, or inhibiting the action of related enzymes. However, Altersolanol A has not yet become a clinical drug, and the research on its activity is not in-depth enough, especially the literature reports on its mechanism of action are few. It is speculated that this 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 microbial sources, and further research and development of Altersolanol A may help create new anticancer, antibacterial, antiviral and other drugs. Summary of the invention

[0006] The purpose of the present invention is to provide a transporter of Altersolanol A from Spatholobus, its encoding gene and application, so as to solve the problems existing in the above-mentioned prior art. Overexpression of the encoding gene of the transporter in Spatholobus can significantly increase the yield of Altersolanol A, thereby providing a new direction for the development of Altersolanol A from microbial sources.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a transporter of solanol A from Stolonifera stolonifera, wherein the transporter is any one of (1), (2) and (3):

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

[0010] (2) Substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of the SvMFS1 protein to obtain a protein that has more than 70% identity with the SvMFS1 protein and is associated with solanol A transport;

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

[0012] The present invention also provides a gene encoding the above transport protein.

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

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

[0015] The present invention also provides a recombinant overexpression vector, comprising the above gene expression cassette.

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

[0017] The present invention also provides the use of the above-mentioned coding gene, gene expression cassette, recombinant overexpression vector or recombinant host cell in promoting the production of solanol A by Stolonifera, and the yield of solanol A is increased by overexpressing the coding gene in the Stolonifera.

[0018] The present invention also provides a method for constructing a solanol A-producing bacterium, comprising the steps of transforming the above-mentioned recombinant overexpression vector into Pseudomonas stoloniferum to construct a recombinant Pseudomonas stoloniferum that overexpresses the encoding gene; the recombinant Pseudomonas stoloniferum is the solanol A-producing bacterium.

[0019] The present invention also provides a solanol A producing bacterium constructed according to the above construction method.

[0020] The present invention also provides a method for producing solanol A, comprising the step of fermenting and culturing the solanol A producing bacteria to prepare the solanol A.

[0021] The present invention discloses the following technical effects:

[0022] The present invention compares transcriptome sequencing of two different Stemphylium lycopersici alternavirus 1 (SlAV1) infected fungal virus to obtain differentially expressed genes, and obtains a new Stemphylium lycopersici major promoter superfamily gene, namely, SvMFS1 gene, through analysis and screening. The gene is a key transport protein for transport and synthesis of Altersolanol A in Stemphylium lycopersici, and can significantly increase the yield of Altersolanol A by overexpression in S. lycopersici. Therefore, the discovery and cloning of the SvMFS1 gene of the present invention provides a basis for artificially modifying microorganisms for biosynthesis of Altersolanol A. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a graph showing the changes in the expression of the SvMFS1 gene before and after the SvHN-02 strain was infected with S1MV1;

[0025] Figure 2 This is a schematic diagram of the protein structure encoded by the transporter SvMFS1 gene;

[0026] Figure 3 This is a schematic diagram of the transmembrane domain of the protein encoded by the transporter SvMFS1 gene;

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

[0028] Figure 5The figure is a statistical graph of the content of Altersolanol A in the fermentation products of SvHN-02SvMFS1 gene knockout / complementation strains; 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 complementation strains;

[0029] Figure 6 The phenotypes of the two SvMFS1 gene overexpression transformants obtained for screening and the wild-type S. stolonifera are shown in Figure 2. Among them, SvHN-02 is a wild-type strain; OESvMFS1-5 and OESvMFS1-8 are overexpression strains;

[0030] Figure 7 The figure is a statistical chart of the content of Altersolanol A in the fermentation product of the strain SvHN-02 that overexpresses the SvMFS1 gene; among them, SvHN-02 is a wild-type strain; OESvMFS1-5 and OESvMFS1-8 are overexpression strains. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The strains involved in the embodiments of the present invention: the vesicular stipedium strain SvHN-02 and the tomato stipedium strain SlHN-10 have been disclosed in Chinese patent CN202011153985.3; the above strains are all preserved by Hunan Agricultural University, and it is promised to be open to the public within 20 years from the date of application of this invention.

[0037] The fungal virus Stemphylium lycopersici alternavirus 1 (SlAV1), formerly named Stemphylium lycopersici mycovirus1 (SlMV1), has been disclosed in Chinese patent CN202011153985.3.

[0038] Example 1 Candidates and verification of key transport proteins for the transport synthesis of Altersolanol A in Spatholobus

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

[0040] Design primers based on transcriptome sequencing results:

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

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

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

[0044] 1. Extraction of fungal DNA and RNA

[0045] The experimental methods and steps were based on the instructions of the Tiangen Biochemical Technology (Beijing) Co., Ltd. Rapid DNA Extraction Detection Kit (KG203) and RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441).

[0046] 2. PCR amplification of SvMFS1 gene

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

[0048] Amplification procedure: 94°C pre-denaturation for 5 min; 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, 34 cycles; 72°C extension for 5 min. DNA and cDNA PCR products were detected by 1% agarose gel electrophoresis and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0049] The results showed that the full length of the DNA of the SvMFS1 gene obtained was 1858 bp, and the nucleotide sequence was shown in SEQ ID NO.1.

[0050] SEQ ID NO.1:

[0051]

[0052] RT-PCR amplification of SvMFS1 gene CDS, the specific operation is as follows:

[0053] The reverse transcription of RNA was performed according to the Quant cDNA First-Strand Synthesis Kit (KR103) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The PCR reaction system was based on the SvMFS1 gene PCR amplification system. The amplified product was electrophoresed on a 1% agarose gel, and the RT-PCR product was cloned into a T vector and sent to a sequencing company for sequencing verification. The results showed that the CDS length of the SvMFS1 gene was 1674 bp (SEQ ID NO.2), and the amino acid sequence length of the protein it encoded was 557 Aa (as shown in SEQ ID NO.3). Protein structure prediction showed that the protein contained the MFS family domain ( Figure 2 ) and contains 14 transmembrane domains ( Figure 3 ), which is a typical feature of MFS family proteins.

[0054] SEQ ID NO.2:

[0055]

[0056] SEQ ID NO.3:

[0057] MNEGIGSNSSGVSTPDILSGEKIYLPHEQDAKEEVETTGDGSTSDIKEEYQTGLQLVLLMISILFTVCLTSLDMTIVGTAIPKITDEFHGLGMVSWYGSAYFMTFGGFQPASGKFYRYFPLKWSFLGALLIFELGSLICGIAQNSTTFVVGRAIAGVGASAVVTGAFTIAALSCEPRQRPAIMGLLGVVYGLSSVVGPLLGGVFSDHASWRWCFYINLPIGGVSAALILFFFKTPPQVVTEQTTWQEKLLQMDPVGIALVMGAIVAYILALESGGQKEPWGSSTVIGLLVGFVVIFLVFVLWEIYNGKHAMLPPRLFRQRSIWQPAGFIFFFSSAYIVLLYYLPIYFQSIDNRSAIMSGVLNLPLVLSLAIGSTVSGIVVSKTGLAAPFMLTGAALATLSTGLMYTFDTDTGMGKWIGYQILYGAGVGLGFQMAINTAQANVSIADMSSATATVFFFQTIGGAFSLSASQSGFANRLLATLAKTAPGVNPQLVIRTGATQIRSSFTPGEVPGILEAYMAGIKVTMAIATGLAGASVLVTLFVSRKRLNVQKLQGGAA。

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

[0059] 3.1 Obtaining of the SvMFS1 knockout double fragments

[0060] The principle of Split-marker homologous recombination 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 S. vesicularis. The flanking sequence of the SvMFS1 gene was 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 fragment was obtained by fusion PCR amplification. The knockout double fragments UH1 (upstream homologous arm combined with HYG1 fragment) and DH2 (downstream homologous arm combined with HYG2 fragment) were obtained by the second round of PCR, in which the HYG1 sequence partially overlapped with the HYG2 sequence. The double fragments were introduced into the protoplast cells of S. vesicularis 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. The specific operations are as follows:

[0061] The upstream and downstream flanking sequences of the SvMFS1 gene in the genome of S. vesiculosus were obtained through the NCBI database. Primers SvMFS1-UF-F / R and SvMFS1-DF-F / R were designed using primer 5.0. The upstream fragment UF and downstream fragment DF of SvMFS1 were amplified using the genomic DNA of S. vesiculosus SvHN-02 as a template. Primers HYG-F / R were designed, and the HYG fragment was amplified 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 with primers SvMFS1-UF-F and SPD-SY-R, and the HYG2+DF fragment was amplified with primers SPD-XY-F and SvMFS1-DF-R. The relevant primers are 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 the double fragment of SvMFS1 in situ complementation

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

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

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

[0074] SvMFS1-CoDF-R:5'-CGAGGTGTTTCCAGGTTGGTTAAAGGAATGTACAGTCTATAT-3'(SEQ IDNO.14),

[0075] SvMFS1-CoDF-F:5′-TGCCAAAGGCAATACCCTGCTTGCCTCTGGAAACCAGCATC-3′(SEQ IDNO.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] 3.3PEG is a smooth muscle

[0082] After the mycelium is crushed with a freezing grinder, it is transferred to 50mL YEPD liquid culture medium and cultured in a constant temperature shaker at 28°C, 220rpm for 48 hours. In a clean bench, the mycelium is filtered with three layers of sterile lens paper and washed with 10mL 0.7M NaCl. After that, 1.5mL of enzymatic solution is added to the filtered mycelium. (Four enzymes 2% pectinase, 2% lysozyme, 2% collapse enzyme, 2% Kitalase cell lysing enzyme are dissolved in 0.7M NaCl), 28°C, 80rpm, and cultured for 1 hour. After enzymatic hydrolysis, the mycelium is filtered with three layers of sterile lens paper, and then rinsed three times with 10mL 0.7M NaCl. The filtrate was centrifuged at 6000 rpm for 6 minutes, and the protoplasts at the bottom of the centrifuge tube were collected and resuspended in STC buffer (100 g sucrose, 50 ml 0.5 M Tris-HCl (pH = 8.0), 3.6755 g CaCl2·2H2O, and deionized water to 500 ml) to adjust the protoplasts to 2 × 10 per ml. 7 Then add 20μg fusion fragment, 400μL PTC (PEG8000200g, add STC solution to 500mL, dissolve and stir in 65℃ water bath, sterilize with bacterial filter) and mix well, ice bath for 10min; then add 600μL PTC and let stand for 5min, transfer to 5mL TB3 liquid culture medium, invert and mix well, let stand at 28℃ for 12 hours, finally pour all mycelium into the culture medium containing antibiotics and then pour the plate for screening.

[0083] 3.4 HPLC analysis of fermentation broth of wild-type strain, knockout strain and complemented strain

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

[0085] Use a puncher with an inner diameter of 7 mm to punch out the bacterial cakes of the wild-type strain SvHN-02, the knockout mutant and the complemented transformant, and inoculate 3 bacterial cakes into PD liquid culture medium respectively, with 3 replicates, and culture at 28°C, 200 r / min for 7 days. After filtering through 3 layers of lens paper and 4 layers of gauze into a 50-mL centrifuge tube to obtain the fermentation liquid, and then use filter paper to absorb water and dry and wrap the obtained mycelium, put it into an oven at 65°C for drying, and then measure the dry weight of the mycelium.

[0086] 3.4.2 Extraction and HPLC determination of Altersolanol A toxin

[0087] (1) Extraction: Use a pipette to draw 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, centrifuge for 5 minutes, take the supernatant, and then add an equal volume of ethyl acetate for 3 extractions. When the solution does not show stratification, add a small amount of powdered CaCl2 to the tube to dissolve and stratify it, then blow dry the resulting organic phase with a liquid nitrogen instrument, add 1 mL of methanol to each sample tube and use an ultrasonic instrument to dissolve it for 1 minute. The obtained sample is filtered through a 0.22 μm Nylon 6 microporous filter membrane into a 2 mL brown injection bottle and stored in a 4°C refrigerator for later use.

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

[0089] (3) Plotting of the standard curve: Prepare the working solutions of 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL and 320 μg / mL from the mother solution of 1 mg / mL Altersolanol A. After the standard solution is prepared, filter it into 2 mL brown injection bottles using a 0.22 μm Nylon 6 microporous filter membrane and mark them.

[0090] The results showed that the synthesis of altersolanol A in the SvMFS1 knockout strain was completely inhibited, while the synthesis in the complemented strain was restored ( Figure 4 and Figure 5 ), indicating that SvMFS1 gene is the key transporter protein for the transport and synthesis of AltersolananolA.

[0091] Example 2 Overexpression of SvMFS1 gene in Sphaerotheca vesiculosus to increase the yield of Altersolanol A

[0092] 1. Extraction of fungal RNA was carried out as described in Example 1.

[0093] 2. Construction of SvMFS1 overexpression vector

[0094] The KSTNP vector preserved with glycerol (kindly donated by Dr. Li Yanlin of Hunan Agricultural University, the present invention promises to distribute to the public for 20 years from the date of application of the present invention) was streaked on an LB plate and cultured overnight at 37°C, and a single colony was picked in LB liquid culture medium, cultured in a shaker at 180rpm and 37°C for 16 hours, and then used for plasmid extraction. The plasmid extraction steps refer to the high-purity plasmid mini-extraction kit (DP104) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The vector was digested with the corresponding EcoR I enzyme to obtain a linear vector recovery, and the purified and recovered SvMFS1 gene CDS full-length and KSTNP linear vector were connected with ExnaseⅢ enzyme, and heat-shocked E. coli DH5α were transformed, and positive clones were selected by PCR, and sent to Shanghai Biotech for sequencing verification, and the correct SvMFS1 gene overexpression vector was constructed.

[0095] 3. Genetic transformation and detection of SvMFS1 gene overexpression vector refer to Example 1

[0096] The SvMFS1 gene overexpression vector was transformed into the S. vesicularis strain SvHN-02 to obtain two SvMFS1 gene overexpression transformants (OESvMFS1-5 and OESvMFS1-8). The culture characteristics showed that the synthesis of altersolanol A was significantly increased ( Figure 6 ). Compared with SvHN-02, the content of Altersolanol A in the fermentation broth of SvMFS1 gene overexpression transformants was significantly increased, indicating that the ability of overexpression transformants to synthesize Altersolanol A was significantly increased ( Figure 7 ).

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A transporter protein of solani alcohol A from Stoloniferum, characterized in that: The Sclerotium transporter protein is any one of (1), (2) and (3): (1) SvMFS1 protein, the amino acid sequence of which is shown in SEQ ID NO.3; (2) Substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of the SvMFS1 protein to obtain a protein that has more than 70% identity with the SvMFS1 protein and is associated with solanol A transport; (3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of (1) or (2).

2. A gene encoding the transporter as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A gene expression cassette, characterized in that Comprising the coding gene described in claim 2 or 3.

5. A recombinant overexpression vector, characterized in that: Comprising the gene expression cassette of claim 4.

6. A recombinant host cell, characterized in that Comprising 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 in promoting the production of solanol A by Pseudomonas stoloniferum, characterized in that: The production of the solanol A is increased by over-expressing the encoding gene in the Pseudomonas aeruginosa.

8. A method for constructing a strain for producing solanol A, characterized in that: The method comprises the steps of transforming the recombinant overexpression vector of claim 5 into Pseudomonas aeruginosa to construct a recombinant Pseudomonas aeruginosa that overexpresses the encoding gene; the recombinant Pseudomonas aeruginosa is the solanol A producing bacterium.

9. A solanol A-producing bacterium constructed according to the construction method of claim 8.

10. A method for producing crosslinked solanol A, characterized in that: The method comprises the step of fermenting and culturing the solanol A producing bacteria according to claim 9 to prepare the solanol A.

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