A creeping phytohexol dehydrogenase, its encoding gene, and its applications

Abstract

This invention discloses a *Stachys pubescens* alcohol dehydrogenase, its encoding gene, and its applications, relating to the field of microbial genetic engineering technology. The alcohol dehydrogenase is any one of (1), (2), and (3): (1) the SvADH1 protein, whose amino acid sequence is shown in SEQ ID NO.3; (2) a protein obtained by substituting, deleting, and / or adding one or more amino acid residues to the amino acid sequence of the SvADH1 protein, resulting in a protein with more than 70% identity to the SvADH1 protein and associated with the redox of cross-linked solanol A; (3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of (1) or (2). This invention has found that overexpression of the encoding gene of this alcohol dehydrogenase in *Stachys pubescens* significantly increases the yield of cross-linked solanol A, thus providing a new direction for the development of microbially derived cross-linked solanol A.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, and in particular to a creeping phytohexol dehydrogenase, its encoding gene, and its applications. Background Technology

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

[0003] Fungal toxins are non-enzymatic compounds produced by plant pathogenic fungi during their metabolism. Even at extremely low concentrations, they can disrupt the normal physiological functions of plants. *Stemphylium spp.* is a typical necrotrophic pathogen that produces a variety of toxins. The most prominent of these is altersolanol A, a tetrahydroanthraquinone compound, orange-yellow in color, with the molecular formula C2. 16 H 16 O8, CAS number 22268-16-2, has the following structure:

[0004]

[0005] Tetrahydroanthraquinones are an important class of microbial secondary metabolites with various biological activities, including anticancer, antibacterial, antiviral, antidiabetic, and antimalarial effects. These substances can also exhibit anticancer activities such as inhibiting cell proliferation, invasion, metastasis, and angiogenesis by inducing apoptosis, arresting the cell cycle, or inhibiting related enzymes. However, Altersolanol A is not yet a clinical drug, and research on its activities is insufficient, especially regarding its mechanism of action. This is likely because the availability of Altersolanol A from natural sources is limited, leading to a lack of attention. Therefore, research on microbial-derived Altersolanol A has significant practical implications, and further development of Altersolanol A may contribute to the creation of new anticancer, antibacterial, and antiviral drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a *Stemona stolonifer* alcohol dehydrogenase, its encoding gene, and its applications, in order to solve the problems existing in the prior art. Overexpression of the encoding gene of this alcohol dehydrogenase in *Stemona stolonifer* can significantly increase the yield of Altersolanol A, thus providing a new direction for the development of microbial-derived Altersolanol A.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides an alcohol dehydrogenase for cross-linked solanol A from *Pseudomonas spp.*, wherein the alcohol dehydrogenase is any one of (1), (2), and (3):

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

[0010] (2) The amino acid sequence of the SvADH1 protein is replaced, deleted and / or added by one or more amino acid residues to obtain a protein that has more than 70% identity with the SvADH1 protein and is related to the redox of cross-linked solanol A.

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

[0012] The present invention also provides the encoding gene of the above-mentioned alcohol dehydrogenase.

[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-described encoding gene.

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

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

[0017] The present invention also provides the application of the above-mentioned coding gene, gene expression cassette, recombinant overexpression vector or recombinant host cell in promoting the production of cross-linked solanol A in Crescentella asiatica, thereby increasing the yield of cross-linked solanol A by overexpressing the coding gene in Crescentella asiatica.

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

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

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

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

[0022] This invention compares the transcriptome sequencing of two different *Stemphylium lycopersici* strains infected with the fungal virus *Stemphylium lycopersici* alternavirus 1 (SlAV1) to obtain differentially expressed genes. Through analysis and screening, a novel *Stemphylium lycopersici* alcohol dehydrogenase family gene—SvADH1—was identified. This gene is a key alcohol dehydrogenase in the synthesis of Altersolanol A in *Stemphylium lycopersici*. Overexpression of this gene in *Stemphylium lycopersici* significantly increases Altersolanol A production. Therefore, the discovery and cloning of the SvADH1 gene in this invention provides a foundation for the artificial modification of microorganisms for the biosynthesis of Altersolanol A. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This figure shows the changes in SvADH1 gene expression levels before and after infection with S1MV1 by wild-type strain SvHN-02; where SvHN-02 represents before S1MV1 infection and S1MV1 Infected represents after S1MV1 infection.

[0025] Figure 2 This is a schematic diagram of the protein structure encoded by the SvADH1 gene, an alcohol dehydrogenase.

[0026] Figure 3 Phenotypic diagrams of wild-type and SvADH1 gene knockout / reinforcement strains; where WT is the wild-type strain SvHN-02; ΔSvadh1-8, ΔSvadh1-70 and ΔSvadh1-80 are gene knockout strains; and ΔSvadh1-Com7, ΔSvadh1-Com17 and ΔSvadh1-Com30 are gene-reinforcement strains.

[0027] Figure 4Statistical graph of Altersolanol A content in fermentation products of SvHN-02 and SvADH1 gene knockout / complement strains; where WT is 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 complement strains;

[0028] Figure 5 Phenotypic diagrams of the two SvADH1 gene overexpression transformants obtained through screening and the wild-type *S. spp.*; where WT is the wild-type strain SvHN-02; and OE-Svadh1-1 and OE-Svadh1-5 are SvADH1 gene overexpression strains.

[0029] Figure 6 The graph shows the content of Altersolanol A in the fermentation product of strains overexpressing the SvADH1 gene in SvHN-02 strain; where WT is the wild-type strain SvHN-02; and OE-Svadh1-1 and OE-Svadh1-5 are overexpressing strains. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

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

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

[0037] Example 1: Candidate and Validation of Key Alcohol Dehydrogenases in Altersolanol A Synthesis from Crescentella asiatica

[0038] SlAV1 infection of the *Stemphylium lycopersici* strain SlHN-10 inhibits Altersolanol A synthesis. This virus can horizontally spread to the *S. vesicarium* strain SvHN-02 and inhibit Altersolanol A synthesis. Transcriptome sequencing was performed on both groups of strains before and after SlAV1 infection. Analysis of differentially expressed genes (DEGs) in the RNA-seq of SlAV1-infected strains identified a group of candidate genes potentially involved in Altersolanol A synthesis in *Stemphylium lycopersici* and *S. vesicarium*. One of these genes, belonging to the alcohol dehydrogenase family, was named SvADH1. In wild-type strain SlHN-02, infection with SlAV1 significantly reduced the expression level of the SvADH1 gene. Figure 1 Therefore, it is speculated that this gene is related to the regulation of Altersolanol A synthesis. Next, the function of the SvADH1 gene was verified, and the specific procedures are as follows:

[0039] Primers were designed based on transcriptome sequencing results:

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

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

[0042] The specific procedure for PCR amplification of the SvADH1 gene is as follows:

[0043] 1. Extraction of fungal DNA and RNA

[0044] The experimental methods and procedures were performed in accordance with the instructions of the Rapid DNA Extraction and Detection Kit (KG203) and the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech (Beijing) Co., Ltd.

[0045] 2. PCR amplification of the SvADH1 gene

[0046] PCR reaction system 50.0 μL: 10×PCR Buffer 5.0 μL, 2.5 mmol / L dNTP 4.0 μL, forward and reverse primers 2.0 μL each, DNA or cDNA template 2.0 μL, Pfu enzyme 1.0 μL, ddH2O to make up to 50.0 μL.

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

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

[0049] SEQ ID NO.1:

[0050]

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

[0052] RNA reverse transcription was performed using the Quant cDNA first-strand synthesis kit (KR103) from Tiangen Biotech (Beijing) Co., Ltd. The PCR reaction system followed the SvADH1 gene PCR amplification system. Amplification products were subjected to 1% agarose gel electrophoresis, and RT-PCR products were cloned into a T vector and sent to a sequencing company for verification. Results showed that the SvADH1 gene CDS length was 1008 bp (SEQ ID NO.2), and the amino acid sequence length of the encoded protein was 335 Aa (as shown in SEQ ID NO.3). Protein structure prediction indicated that the protein possesses the ADH catalytic domain and the ability to bind nicotinamide adenine dinucleotide (NAD). + The Rossman folded structural domain ( Figure 2 This is a typical characteristic of ADH family proteins.

[0053] SEQ ID NO.2:

[0054]

[0055] SEQ ID NO.3:

[0056] MATTMRGWQFQASSKPFVESLSIPPSGIPIPSIKDDEVLVESYATGLNAIDYKILELGLITRLVFPSLTPGLDIHGRVAKVGSKVSKFQEGDIVFGVIPPGSKHGALAEYLPVPQDALAKVPEGLKRDDLVAIASVGMTVLAGLQPYAKPGNKVFINGSSGGTGVAAV QIAKILGYDVTASCSTANVGLVKSLGADLVLDYTSAPIVEQLKDTGATFDLILDNVGSPADLYRVSSQFLRPEGKFIQVGLGMNLSAGLQFFRNKLAGLLSWGKREYIFVVGRSETDVYEQLAAWMAGGKLRAVIDSTWEFGDVPKAYERLKTGRAKGKVVVHVKDE.

[0057] 3. Verification of SvADH1 gene function

[0058] 3.1 Obtaining the SvADH1 knockout double fragment

[0059] Using the principle of split-marker homologous recombination, and with the hygromycin-resistant bacterial phosphotransferase B gene (HYG) as a selective marker, the SvADH1 gene of *Stenophyllaria cystis* was knocked out. Flanking sequences of the SvADH1 gene were obtained from the complete genome sequence of SvHN-02, including an upstream homologous fragment (UF) and a downstream homologous fragment (DF). The complete HYG fragment was obtained from the vector pCB1300, and a combination product of the upstream homologous arm, downstream homologous arm, and HYG fragment was obtained by fusion PCR amplification. A second round of PCR was used to obtain the knockout dual fragments UH1 (upstream homologous arm binds to the HYG1 fragment) and DH2 (downstream homologous arm binds to the HYG2 fragment), where the HYG1 and HYG2 sequences partially overlap. The dual fragments were introduced into *Stenophyllaria cystis* protoplast cells via PEG-mediated protoplast transformation, achieving the goal of replacing the SvADH1 gene fragment with the HYG fragment, thus realizing the knockout of the SvADH1 gene. The specific operation is as follows:

[0060] The upstream and downstream flanking sequences of the SvADH1 gene in the *S. cystis* genome were obtained from the NCBI database. Primers SvADH1-UF-F / R and SvADH1-DF-F / R were designed using Primer 5.0. Using genomic DNA from *S. cystis* SvHN-02 as a template, the upstream fragment UF and downstream fragment DF of SvADH1 were amplified. Primer HYG-F / R was designed, and the HYG fragment was amplified using the plasmid vector pBC1300 as a template. The recombination frame was obtained by ligating the upstream fragment UF, downstream fragment DF, and HYG fragment using double-joint PCR. The UF+HYG1 fragment was amplified using primers SvADH1-UF-F and SPD-SY-R, and the HYG2+DF fragment was amplified using primers SPD-XY-F and SvADH1-DF-R. 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] 3.2 Obtaining the SvADH1 in-situ completion of two fragments

[0070] Similarly utilizing the split-marker principle, the SvADH1 gene was imputed through in situ imputation. First, the G418 fragment was obtained from the KSTNP vector. Two shortened but partially overlapping G418 fragments (G1 and G2) were obtained via PCR. In a second round of PCR, the upstream homologous arm was fused with the SvADH1 gene fragment to the G1 fragment; the downstream homologous arm was fused with the G2 fragment. After obtaining the imputed double fragments, transformation was performed. The specific procedures are as follows:

[0071] The upstream and downstream flanking sequences of the SvADH1 gene in the genome of *S. spp.* were obtained. Using the genomic DNA of *S. spp.* SvHN-02 as a template, primers SvADH1-UF-F and SvADH1-CoDF-R were designed using Primer 5.0 to amplify the upstream and target gene fragment CoUFJY of SvADH1, and the downstream fragment CoDF of primers SvADH1-CoDF-F and SvADH1-DF-R. Primers G418-F / R were designed, and the G418 fragment was amplified using the plasmid vector KSTNP (a generous gift from Dr. Li Yanlin of Hunan Agricultural University, which this invention promises to release to the public for 20 years from the date of this application) as a template. The CoUFJY, CoDF, and G418 fragments were ligated using double-joint PCR to obtain the recombinant cassette. The double fragments were obtained as follows: the CoUFJY+G418-1 fragment was amplified using primers SvADH1-UF-F and SPD-CoUG1-R, and the G418-2+CoDF fragment was amplified using 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 IDNO.14),

[0074] SvADH1-CoDF-F: 5'-TGCCAAAGGCAATACCCTGCGATAATAGTCGAGGAAGAGGC-3' (SEQ IDNO.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

[0081] The mycelia were pulverized using a cryogenic grinder and transferred to 50 mL of LYEPD liquid medium. The medium was incubated in a shaker at 28°C and 200 rpm for 48 hours. The mycelia were then filtered through three layers of sterile lens paper in a laminar flow hood and washed with 10 mL of 0.7 M NaCl. Afterward, the filtered mycelia were added to 1.5 mL of enzymatic digestion solution (containing four enzymes dissolved in 0.7 M NaCl: 2% pectinase, 2% lysozyme, 2% lysin, and 2% Kitalase), and incubated at 28°C and 80 rpm for 1 hour. Following enzymatic digestion, the mycelia were filtered through three layers of sterile lens paper and then washed three times with 10 mL of 0.7 M NaCl. Centrifuge the filtrate at 6000 rpm for 6 minutes, collect the protoplasts at the bottom of the centrifuge tube, and resuspend them 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 a final volume of 500 mL). Adjust the protoplast concentration to 2 × 10⁻⁶ cells / mL. 7 Transfer the mycelium to ice for later use. Then add 20 μg of fusion fragment and 400 μL of PTC (PEG8000 200g, add STC solution to 500 mL, dissolve and stir in a 65℃ water bath, and sterilize with a bacterial filter), mix well, and incubate on ice for 10 min; then add 600 μL of PTC and let stand for 5 min, transfer to 5 mL of TB3 liquid medium, invert and mix well, and incubate at 28℃ for 12 hours. Finally, pour all the mycelia into a medium containing antibiotics and then screen by plate selection.

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

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

[0084] Using a 7mm inner diameter punch, mycelial cakes of wild-type strain SvHN-02, knockout mutant, and complemented transformant were collected. Three mycelial cakes were inoculated into PD liquid medium with three replicates. After incubation at 28℃ and 200r / min for 7 days, the mycelial broth was filtered through three layers of lens paper and four layers of gauze into a 50mL centrifuge tube. The obtained mycelium was then dried by absorbing water with filter paper, wrapped, and placed in a 65℃ oven for drying. The dry weight of the mycelium was then measured.

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

[0086] (1) Extraction: Pipette 5 mL of fermentation broth into a 10 mL centrifuge tube, add an equal volume of methanol, mix well, and incubate overnight at 4°C. Centrifuge the overnight solution at 8000 rpm for 5 minutes, collect the supernatant, and then add an equal volume of ethyl acetate for three extractions. If no layering occurs, add a small amount of powdered CaCl2 to the tube to dissolve and separate the layers. Then, dry the obtained organic phase using a liquid nitrogen apparatus. Add 1 mL of methanol to each sample tube and use an ultrasonic instrument to aid dissolution for 1 min. Filter the obtained samples through a 0.22 μm Nylon 6 microporous membrane into a 2 mL brown sample bottle and store at 4°C for later use.

[0087] (2) Chromatographic conditions: The chromatographic column was Sharpsil-AR C18 (250×4.6mm, 5μm); the mobile phase was methanol and ultrapure water (25:75); the detection wavelength was 215nm; the injection volume was 10μL; the flow rate was 1mL / min; and the column temperature was 35℃.

[0088] (3) Construction of the standard curve: Working solutions were prepared using a stock solution 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 solutions were prepared, they were filtered through a 0.22 μm Nylon 6 microporous membrane into 2 mL brown sample vials and labeled accordingly.

[0089] The results showed that AltersolanolA synthesis was completely inhibited in the SvADH1 gene knockout strain, while synthesis was restored in the complement strain. Figure 3 and Figure 4 This indicates that the SvADH1 gene is a key alcohol dehydrogenase in the processing and synthesis of Altersolanol A.

[0090] Example 2: Overexpression of the SvADH1 gene in *Stylosporium sacchariformis* increases Altersolanol A production.

[0091] 1. Fungal RNA extraction was performed according to Example 1.

[0092] 2. Construction of SvADH1 overexpression vector

[0093] The KSTNP vector, preserved in glycerol, was streaked on LB agar plates and incubated overnight at 37°C. Single colonies were picked and cultured in LB liquid medium at 180 rpm for 16 h in a shaker at 37°C for plasmid extraction. Plasmid extraction was performed according to the high-purity plasmid miniprep kit (DP104) from Tiangen Biotech (Beijing) Co., Ltd. The vector was digested with the appropriate EcoRI enzyme to obtain a linear vector, which was then recovered. The purified and recovered full-length SvADH1 gene CDS was ligated to the KSTNP linear vector using Exnase III enzyme. The ligation was performed by heat shock transformation into *E. coli* DH5α. Positive clones were selected by PCR and sent to Shanghai Sangon Biotech for sequencing verification, confirming the construction of the correct SvADH1 gene overexpression vector.

[0094] 3. Genetic transformation and detection of SvADH1 gene overexpression vector were performed as described in Example 1.

[0095] The SvADH1 gene overexpression vector was transformed into the *S. cynomolgus* strain SvHN-02, resulting in two SvADH1 gene overexpression transformants (OE-Svadh1-1 and OE-Svadh1-5). Observation of culture traits showed a significant increase in Altersolanol A synthesis. Figure 5 Compared to SvHN-02, the content of Altersolanol A in the fermentation broth of SvADH1 gene overexpression transformants was significantly increased, indicating that the ability of overexpression transformants to synthesize Altersolanol A was significantly increased. Figure 6 ).

[0096] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An alcohol dehydrogenase of Stemphylium botryosum solanapyrone A, characterized in that, The Stemphylium stoloniferum alcohol dehydrogenase is the SvADH1 protein, and its amino acid sequence is as shown in SEQ ID NO.

3.

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 as shown in SEQ ID NO.

2.

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

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

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

7. Use of a coding gene as described in claim 2 or 3, a gene expression cassette as described in claim 4, a recombinant overexpression vector as described in claim 5, or a recombinant host cell as described in claim 6 in promoting the production of solavetivone A by Stachybotrys chartarum, characterized in that By overexpressing the coding gene in the Stemphylium vesicarium to increase the yield of solavetivone A.

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

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

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

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