Stemphylium alcohol dehydrogenase as well as coding gene and application thereof

Abstract

The invention discloses a Stemphylium alcohol dehydrogenase as well as a coding gene and application thereof, and relates to the technical field of microbial genetic engineering. The alcohol dehydrogenase is any one of (1), (2) and (3): (1) SvADH1 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 SvADH1 protein to obtain a protein which has more than 70% of identity with the SvADH1 protein and is related to cross-linked solanol A redox; 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 alcohol dehydrogenase in Stemphylium cystosum can significantly improve the yield of the interlinked solanol A, so that a new direction is provided for developing the interlinked solanol A from microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and particularly to a Stemphylium alcohol dehydrogenase, its coding gene and application. Background Art

[0002] Alcohol dehydrogenases (ADH) are redox enzymes that use NAD + or NADP + as coenzymes and are involved in the growth and development of various fungi under aerobic or anaerobic conditions. These processes are related to metabolic pathways associated with the production of alcohols, aldehydes, ketones, and acids. The most studied ADHs are related to ethanol metabolism, either as fermentative enzymes involved in ethanol production or as oxidases required to use ethanol as a carbon source. In addition, ADH proteins can specifically participate in the synthesis of secondary metabolites. In filamentous fungi, many ADHs are closely related to the formation of toxins.

[0003] Mycotoxins are non-enzymatic compounds produced by phytopathogenic fungi during metabolism and can disrupt the normal physiological functions of plants at very low concentrations. Stemphylium spp. are a typical group of necrotrophic pathogens that can produce a variety of toxins. The most prominent among them is Altersolanol A, which is a class of tetrahydroanthraquinone compounds, orange-yellow in color, with the molecular formula C 16 H 16 O8, CAS number 22268-16-2, and the structural formula is as follows:

[0004]

[0005] Tetrahydroanthraquinone compounds are an important class of microbial secondary metabolites with biological activities such as anti-cancer, antibacterial, antiviral, anti-diabetic, and anti-malarial. These substances can also exhibit anti-cancer activities such as inhibiting cell proliferation, invasion, metastasis, and angiogenesis by inducing apoptosis, arresting the cell cycle, or inhibiting the action of related enzymes. However, currently, Altersolanol A has not become a clinical drug, and the research on its activity is not deep enough, especially there are few literature reports on its mechanism of action. It is speculated that this may be because the amount of Altersolanol A obtained through natural channels is small and has not attracted people's attention. Therefore, studying Altersolanol A from microorganisms has important practical significance, and further research and development of Altersolanol A may help to create new anti-cancer, antibacterial, antiviral, etc. drugs. Summary of the Invention

[0006] The object of the present invention is to provide a Stemphylium solani alcohol dehydrogenase, its coding gene and application, so as to solve the problems existing in the above-mentioned prior art. Overexpression of the coding gene of this alcohol dehydrogenase in Stemphylium vesicarium can significantly increase the yield of Altersolanol A, thus 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 an alcohol dehydrogenase for Stemphylium solani Altersolanol A, and the alcohol dehydrogenase is any one of (1), (2) and (3):

[0009] (1) SvADH1 protein, whose amino acid sequence is as shown in SEQ ID NO.3;

[0010] (2) A protein obtained by substituting, deleting and / or adding one or several amino acid residues to the amino acid sequence of the SvADH1 protein, which has more than 70% identity with the SvADH1 protein and is related to the oxidation-reduction of Altersolanol A;

[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 the coding gene of the above alcohol dehydrogenase.

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

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

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

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

[0017] The present invention also provides the application of the above coding gene, gene expression cassette, recombinant overexpression vector or recombinant host cell in promoting the production of Altersolanol A by Stemphylium solani. By overexpressing the coding gene in the Stemphylium solani, the yield of the Altersolanol A is increased.

[0018] The present invention also provides a method for constructing an Altersolanol A-producing bacterium, including the step of transforming the above recombinant overexpression vector into Stemphylium solani to construct a recombinant Stemphylium solani overexpressing the coding gene; the recombinant Stemphylium solani is the Altersolanol A-producing bacterium.

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

[0020] The present invention also provides a method for producing Stemphylium alternansol A, which includes the step of fermenting and culturing the above Stemphylium alternansol A-producing bacterium to prepare the Stemphylium alternansol A.

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

[0022] By performing transcriptome sequencing and comparison on two different Stemphylium strains infected with the fungal virus Stemphylium lycopersici alternavirus 1 (SlAV1), the present invention obtains differentially expressed genes, and through analysis and screening, a new Stemphylium alcohol dehydrogenase family gene, SvADH1 gene, is obtained. This gene is the key alcohol dehydrogenase for the processing and synthesis of Altersolanol A in Stemphylium. By overexpressing it in S. vesicarium, the yield of Altersolanol A can be significantly increased. Therefore, the discovery and cloning of the SvADH1 gene in the present invention provide a basis for artificially modifying microorganisms and then using them for the biosynthesis of Altersolanol A. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a graph showing the change in the expression level of the SvADH1 gene before and after the wild-type strain SvHN-02 is infected with S1MV1; among them, SvHN-02 in the figure represents before being infected with S1MV1; S1MV1 Infected represents after being infected with S1MV1;

[0025] Figure 2 It is a schematic diagram of the protein structure encoded by the alcohol dehydrogenase SvADH1 gene;

[0026] Figure 3 It is a phenotypic diagram of the wild-type and SvADH1 gene knockout / complementation strains; among them, WT is the wild-type strain SvHN-02; ΔSvadh1-8, ΔSvadh1-70, and ΔSvadh1-80 are gene knockout strains; ΔSvadh1-Com7, ΔSvadh1-Com17, and ΔSvadh1-Com30 are gene complementation strains;

[0027] Figure 4Statistical chart of the content of Altersolanol A in the fermentation products of SvHN-02 and SvADH1 gene knockout / complemented strains; among them, WT is the wild-type strain SvHN-02; ΔSvadh1-8, ΔSvadh1-70, and ΔSvadh1-80 are gene knockout strains; ΔSvadh1-Com7, ΔSvadh1-Com17, and ΔSvadh1-Com30 are gene complemented strains;

[0028] Figure 5 Phenotype diagrams of two SvADH1 gene overexpression transformants and wild-type Stachybotrys obtained by screening; among them, WT is the wild-type strain SvHN-02; OE-Svadh1-1 and OE-Svadh1-5 are SvADH1 gene overexpression strains;

[0029] Figure 6 Statistical chart of the content of Altersolanol A in the fermentation products of the strains overexpressing the SvADH1 gene in the SvHN-02 strain; among them, WT is the wild-type strain SvHN-02; OE-Svadh1-1 and OE-Svadh1-5 are overexpression strains. Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0035] The strains involved in the embodiments of the present invention: Stemphylium vesicarium strain SvHN-02 and Stemphylium lycopersici strain SlHN-10 have been disclosed in Chinese Patent CN202011153985.3; the above strains are all preserved by Hunan Agricultural University and are committed to being distributed to the public within 20 years from the application date of the present invention.

[0036] The fungal virus Stemphylium lycopersici alternavirus 1 (SlAV1), formerly named Stemphylium lycopersici mycovirus1 (SlMV1), has been disclosed in Chinese Patent CN202011153985.3.

[0037] Example 1 Candidate and Verification of Key Alcohol Dehydrogenase for Altersolanol A Synthesis in Stemphylium

[0038] The infection of Stemphylium lycopersici strain SlHN-10 by SlAV1 will block the synthesis of Altersolanol A. This virus can be horizontally transmitted to Stemphylium vesicarium strain SvHN-02 and inhibit the synthesis of Altersolanol A. The transcriptome sequencing of two groups of strains before and after SlAV1 infection was compared respectively. By analyzing the differentially expressed genes (DEGs) of RNA-seq in SlAV1-infected strains, 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 alcohol dehydrogenase family, which was named SvADH1. After the wild-type strain SlHN-02 was infected by SlAV1, the expression level of the SvADH1 gene decreased significantly ( Figure 1 ). Therefore, it is speculated that this gene is related to the regulation of Altersolanol A synthesis. Then, the function of the SvADH1 gene was verified, and the specific operation is as follows:

[0039] Design primers according to the results of transcriptome sequencing:

[0040] SvADH1-F: 5'-CAAAACTAAGCCTAGCCTTTTTAAA-3' (SEQ ID NO.4);

[0041] SvADH1-R: 5'-GCGGTATCTATCTGGTCTCGAA-3' (SEQ ID NO.5).

[0042] PCR amplify the SvADH1 gene, and the specific operation process is as follows:

[0043] 1. Extraction of fungal DNA and RNA

[0044] The experimental methods and steps refer to the instructions of the Fast DNA Extraction and Detection Kit (KG203) and RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0045] 2. PCR amplification of the SvADH1 gene

[0046] The PCR reaction system is 50.0 μL: 10×PCR Buffer 5.0 μL, 2.5 mmol / L dNTP 4.0 μL, 2.0 μL of each upstream and downstream primer, 2.0 μL of DNA or cDNA template, 1.0 μL of Pfu enzyme, and ddH2O is added to make up to 50.0 μL.

[0047] Amplification program: Pre-denaturation at 94°C for 5 min; 94°C for 30 s, 57°C for 30 s, 72°C for 1 min, for 34 cycles; extension at 72°C for 5 min. The PCR products of DNA and cDNA are detected by 1% agarose gel electrophoresis and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0048] The results show that the full length of the DNA of the SvADH1 gene is 1684 bp, and the nucleotide sequence is as shown in SEQ ID NO.1.

[0049] SEQ ID NO.1:

[0050]

[0051] RT-PCR amplification of the CDS of the SvADH1 gene was performed as follows:

[0052] For reverse transcription of RNA, the Quant cDNA First Strand Synthesis Kit (KR103) from Tiangen Biochemical Technology (Beijing) Co., Ltd. was referred to. The PCR reaction system was referred to the PCR amplification system of the SvADH1 gene. The amplification products were electrophoresed on 1% agarose gel, and the RT-PCR products were cloned into the T vector and sent to a sequencing company for sequencing verification. The results showed that the length of the CDS of the SvADH1 gene was 1008 bp (SEQ ID NO.2), the amino acid sequence length of the encoded protein was 335 Aa (as shown in SEQ ID NO.3), and protein structure prediction indicated that the protein had an ADH catalytic domain and a Rossmann fold domain that binds nicotinamide adenine dinucleotide (NAD + ), which is a characteristic of typical ADH family proteins. Figure 2 )

[0053] SEQ ID NO.2:

[0054]

[0055] SEQ ID NO.3:

[0056] MATTMRGWQFQASSKPFVESLSIPPSGIPIPSIKDDEVLVESYATGLNAIDYKILELGLITRLVFPSLTPGLDIHGRVAKVGSKVSKFQEGDIVFGVIPPGSKHGALAEYLPVPQDALAKVPEGLKRDDLVAIASVGMTVLAGLQPYAKPGNKVFINGSSGGTGVAAVQIAKILGYDVTASCSTANVGLVKSLGADLVLDYTSAPIVEQLKDTGATFDLILDNVGSPADLYRVSSQFLRPEGKFIQVGLGMNLSAGLQFFRNKLAGLLSWGKREYIFVVGRSETDVYEQLAAWMAGGKLRAVIDSTWEFGDVPKAYERLKTGRAKGKVVVHVKDE。

[0057] 3. Verification of the function of the SvADH1 gene

[0058] 3.1 Obtaining of the SvADH1 knockout double fragments

[0059] Using the principle of Split - marker homologous recombination, with the bacterial phosphotransferase B gene (HYG) with hygromycin resistance as the selection marker, the SvADH1 gene of Stachybotrys saccata was knocked out. The flanking sequences of the SvADH1 gene were obtained from the whole - genome sequence of SvHN - 02. 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 a combined product of the upstream homologous arm, downstream homologous arm, and HYG three - fragment was amplified by fusion PCR. 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 PCR, where the HYG1 sequence and the HYG2 sequence partially overlapped. The double fragments were introduced into the protoplast cells of Stachybotrys saccata by PEG - mediated protoplast transformation to achieve the purpose of replacing the SvADH1 gene fragment with the HYG fragment, thereby realizing the knockout of the SvADH1 gene. The specific operations are as follows:

[0060] The upstream and downstream flanking sequences of the SvADH1 gene in the genome of Stachybotrys chartarum were obtained through the NCBI database. Primers SvADH1-UF-F / R and SvADH1-DF-F / R were designed using primer 5.0. Using the genomic DNA of Stachybotrys chartarum SvHN-02 as a template, the upstream fragment UF and downstream fragment DF of SvADH1 were amplified. Primer HYG-F / R was designed, and using the plasmid vector pBC1300 as a template, the HYG fragment was amplified. The recombinant cassette was obtained by connecting the upstream fragment UF, downstream fragment DF, and HYG fragment through Double-joint PCR. For the acquisition of the double-fragment, primers SvADH1-UF-F and SPD-SY-R were used to amplify the UF+HYG1 fragment, and primers SPD-XY-F and SvADH1-DF-R were used to amplify the HYG2+DF fragment. The relevant primers are as follows:

[0061] SvADH1-UF-F: 5'-GTGGCGTCCAATGGAGTTG-3' (SEQ ID NO.6),

[0062] SvADH1-UF-R: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCCGGGCTTGCTGTATTTAT GTTC-3' (SEQ ID NO.7),

[0063] SvADH1-DF-F: 5'-CGTCCGCAATGTGTTATTAAGTCGACGATAGTAGACGAGGAAGA GGCACA-3' (SEQ ID NO.8),

[0064] SvADH1-DF-R: 5'-CGGGATTAAGGAGGGAGCAA-3' (SEQ ID NO.9),

[0065] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3' (SEQ ID NO.10),

[0066] HYG-R: 5'-GTCGACTTAATAACACATTGCGGACGT-3' (SEQ ID NO.11),

[0067] SPD-SY-R: 5'-ACTTCGGGGCAGTCCTCG-3' (SEQ ID NO.12),

[0068] SPD-XY-F: 5'-GAACTCACCGCGACGTCTGT-3' (SEQ ID NO.13).

[0069] Obtaining of the dual fragments for in situ complementation of SvADH1

[0070] Using the Split-marker principle, the SvADH1 gene was complemented by in situ complementation. First, the G418 fragment needed to be 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 homologous arm plus the SvADH1 gene fragment was fused with the G1 fragment; the downstream homologous arm was fused with the G2 fragment. After obtaining the dual fragments for in situ complementation, the transformation work was carried out as follows:

[0071] The upstream sequence of the SvADH1 gene, the SvADH1 gene sequence, and the downstream flanking sequence in the genome of Stachybotrys chartarum were obtained. Using the genomic DNA of Stachybotrys chartarum SvHN-02 as a template, primers SvADH1-UF-F and SvADH1-CoDF-R were designed with primer 5.0 to amplify the upstream and target gene fragment CoUFJY of SvADH1, and the downstream fragment CoDF with primers SvADH1-CoDF-F and SvADH1-DF-R. Primers G418-F / R were designed, and using the plasmid vector KSTNP (kindly provided by Dr. Li Yanlin of Hunan Agricultural University, and this invention promises to make it publicly available to the public for 20 years from the filing date of this invention application) as a template, the G418 fragment was amplified. The CoUFJY fragment, CoDF fragment, and G418 fragment were ligated by Double-joint PCR to obtain a recombinant cassette. Obtaining of the dual fragments: The CoUFJY + G418-1 fragment was amplified with primers SvADH1-UF-F and SPD-CoUG1-R, and the G418-2 + CoDF fragment was amplified with primers SPD-CoDG2-F and SvADH1-DF-R. The relevant primers are as follows:

[0072] SvADH1-UF-F: 5'-GTGGCGTCCAATGGAGTTG-3' (SEQ ID NO.6),

[0073] SvADH1-CoDF-R: 5'-CGAGGTGTTTCCAGGTTGGTTCCTTCCTCACTCATCCTTC-3' (SEQ ID NO.14),

[0074] SvADH1-CoDF-F: 5'-TGCCAAAGGCAATACCCTGCGATAATAGTCGAGGAAGAGGC-3' (SEQ ID NO.15),

[0075] SvADH1-DF-R: 5'-CGGGATTAAGGAGGGAGCAA-3' (SEQ ID NO.9),

[0076] G418-F: 5'-ACCAACCTGGAAACACCTCG-3' (SEQ ID NO.16),

[0077] G418-R: 5'-GCAGGGTATTGCCTTTGGCA-3' (SEQ ID NO.17),

[0078] SPD-CoUG1-R: 5'-ACCGTAAAGCACGAGGAAGC-3' (SEQ ID NO.18),

[0079] SPD-CoDG2-F: 5'-CTGGGCACAACAGACAATCG-3' (SEQ ID NO.19).

[0080] 3.3 PEG-mediated protoplast transformation method

[0081] The mycelia were crushed with a cryogenic grinder and transferred to 50 mL of YEPD liquid medium. They were cultured in a constant temperature shaker at 28 °C and 200 rpm for 48 hours. The mycelia were filtered with three layers of sterile lens paper in a laminar flow hood and washed with 10 mL of 0.7 M NaCl. Then, the filtered mycelia were added to 1.5 mL of enzymatic hydrolysis solution. (Four enzymes were dissolved in 0.7 M NaCl: 2% pectinase, 2% lysing enzyme, 2% naringinase, and 2% Kitalase cell-lysing enzyme), and cultured at 28 °C and 80 rpm for 1 hour. After enzymatic hydrolysis, the mycelia were filtered with three layers of sterilized lens paper and then rinsed three times with 10 mL of 0.7 M 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 with STC buffer (100 g of sucrose, 50 mL of 0.5 M Tris-HCl (pH = 8.0), 3.6755 g of CaCl2·2H2O, and deionized water was added to 500 mL). The protoplasts were adjusted to 2×10 7 per milliliter, transferred to ice for standby. Then, 20 μg of fusion fragments and 400 μL of PTC (200 g of PEG8000, added with STC solution to 500 mL, dissolved and stirred in a 65 °C water bath, and sterilized with a bacterial filter) were added and mixed evenly, and ice-bathed for 10 min; then 600 μL of PTC was added and allowed to stand for 5 min, transferred to 5 mL of TB3 liquid medium, inverted and mixed evenly, cultured statically at 28 °C for 12 hours, and finally all the mycelia were poured into the medium containing antibiotics and then plated for screening.

[0082] 3.4 High performance liquid chromatography analysis of fermentation broths of wild-type strains, gene knockout and complementation strains

[0083] 3.4.1 Preparation of fermentation broth and measurement of dry mycelial weight

[0084] Use a punch with an inner diameter of 7 mm to punch out mycelial discs of the wild-type strain SvHN-02, knockout mutants, and complementation transformants. Inoculate 3 mycelial discs into PD liquid medium respectively, set 3 replicates, culture at 28 °C and 200 r / min for 7 days, then filter through 3 layers of lens paper and 4 layers of gauze into a 50 mL centrifuge tube to obtain the fermentation broth. Then, absorb the water from the obtained mycelia with filter paper, dry them, wrap them up, and put them into an oven at 65 °C for drying, and then measure the dry weight of the mycelia.

[0085] 3.4.2 Extraction of Altersolanol A toxin and determination by high performance liquid chromatography (HPLC)

[0086] (1) Extraction: Pipette 5 mL of the fermentation broth into a 10 mL centrifuge tube, add an equal volume of methanol, mix well, and place at 4 °C overnight. Centrifuge the overnight solution at 8000 rpm for 5 minutes, take the supernatant, then add an equal volume of ethyl acetate for extraction 3 times. When the solution does not show a layering phenomenon, a small amount of powdered CaCl2 can be added to the tube to dissolve and layer it, and then the obtained organic phase is dried by a liquid nitrogen instrument. Add 1 mL of methanol to each sample tube and use an ultrasonic instrument to assist dissolution for 1 min. The obtained samples are filtered through a 0.22 μm Nylon 6 microporous filter membrane into 2 mL brown injection vials and stored in a 4 °C refrigerator for later use.

[0087] (2) Chromatographic conditions: The chromatographic column is Sharpsil-AR C18 (250×4.6 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; the column temperature is 35 °C.

[0088] (3) Preparation of the standard curve: Prepare working solutions with a mother liquor of 1 mg / mL Altersolanol A: 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL. After the standard solution is prepared, filter it through a 0.22 μm Nylon 6 microporous filter membrane into 2 mL brown injection vials respectively and make marks.

[0089] The results showed that the synthesis of Altersolanol A in the SvADH1 gene knockout strain was completely inhibited, while the synthesis in the complementation strain was restored ( Figure 3 and Figure 4 ), indicating that the SvADH1 gene is the key alcohol dehydrogenase for the processing and synthesis of Altersolanol A.

[0090] Example 2 Overexpression of the SvADH1 gene in Stachybotrys chartarum improves the yield of Altersolanol A

[0091] 1. The extraction of fungal RNA was referred to Example 1.

[0092] 2. Construction of the SvADH1 overexpression vector

[0093] The KSTNP vector preserved with glycerol was streaked on an LB plate and cultured overnight at 37 °C. A single colony was picked and cultured in an LB liquid medium. After culturing for 16 h in a shaker at 180 rpm and 37 °C, it was used for plasmid extraction. The plasmid extraction steps were referred to the High Purity Plasmid Mini Kit (DP104) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The vector was digested with the corresponding EcoR I enzyme to obtain a linear vector for recovery. The full-length CDS of the SvADH1 gene and the KSTNP linear vector after purification and recovery were ligated using ExnaseⅢ enzyme, and heat shock transformed into Escherichia coli DH5α. Positive clones were selected by PCR and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The correct SvADH1 gene overexpression vector was constructed.

[0094] 3. The genetic transformation and detection of the SvADH1 gene overexpression vector were referred to Example 1

[0095] The SvADH1 gene overexpression vector was transformed into the strain of Stachybotrys saccata SvHN-02, and two SvADH1 gene overexpression transformants (OE-Svadh1-1, OE-Svadh1-5) were obtained. From the observation of the culture characteristics, the synthesis of Altersolanol A was significantly increased ( Figure 5 ). Compared with SvHN-02, the content of Altersolanol A in the fermentation broth of the SvADH1 gene overexpression transformants was significantly increased, indicating that the ability of the overexpression transformants to synthesize Altersolanol A was significantly increased ( Figure 6 ).

[0096] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. An alcohol dehydrogenase of Stemphylium botryosum solaniol A, characterized in that, The Stemphylium alcohol dehydrogenase is any one of (1), (2), and (3): (1) SvADH1 protein, the amino acid sequence of which is shown in SEQ ID NO.3; (2) A protein obtained by substituting, deleting, and / or adding one or several amino acid residues to the amino acid sequence of the SvADH1 protein, which has more than 70% identity with the SvADH1 protein and is related to the oxidation-reduction of solamargine A; (3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of (1) or (2).

2. A coding gene for the alcohol dehydrogenase according to claim 1.

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

2.

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

5. A recombinant overexpression vector, characterized in that, It includes the gene expression cassette according to claim 4.

6. A recombinant host cell, characterized in that, It includes 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 alternariol A by Stemphylium, characterized in that, By overexpressing the coding gene in the Stemphylium, the yield of solamargine A is increased.

8. A method for constructing a solavetivone A-producing bacterium, characterized in that, It includes the step of transforming the recombinant overexpression vector according to claim 5 into Stemphylium to construct a recombinant Stemphylium overexpressing the coding gene; the recombinant Stemphylium is the solamargine A-producing bacterium.

9. A solamargine A-producing bacterium constructed by the construction method according to claim 8.

10. A production method of solavetivone A, characterized in that, It includes the step of fermenting and culturing the solamargine A-producing bacterium according to claim 9 to prepare the solamargine A.

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