A biosynthetic gene cluster of a marine pyrenophorol compound and application thereof

Abstract

The application discloses a biosynthesis gene cluster of marine fungus pyrenophorol compounds and application thereof. The nucleotide sequence of the biosynthesis gene cluster is shown in the 1-18549th of SEQ ID NO. 1, and the biosynthesis gene cluster comprises six genes, i.e. a gene responsible for synthesis of a pyl skeleton structure, namely pylA and pylB; a gene responsible for hydroxylation of carbon 4 and 4' in the pyl, namely pylC; a gene responsible for ketone-enol tautomerism, namely pylE; a gene responsible for reduction of ketone groups at carbon 4 and 4' into hydroxyl groups, namely pylD; and a gene responsible for compound efflux, namely pylF. All the gene information related to pyl biosynthesis provided by the application can help understand the biosynthesis mechanism of the sixteen-membered ring dilactone family natural products, and provide materials and knowledge for further structural modification of the compounds.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering, specifically relating to a biosynthetic gene cluster of pyrenophorol-like compounds derived from marine fungi, its preparation method, and its application. Background Technology

[0002] Pyrenophorol (Formula II) is a class of polyketide compounds with a unique C2-symmetric 16-membered ring dilactone structural unit. It was first isolated from the fungus *Byssochlamys nivea* Westling by Swiss scientists. These compounds possess various biological activities, such as antibacterial and herbicidal activity (J Agric Food Chem. 2005, 53(15), 5943-7; Arch Toxicol. 2017, 91(4), 2007-2016). Currently, chemical synthesis techniques for these compounds have been reported (JOrg Chem. 2014, 79(17), 8067-76; 2015, 80(1), 204-16; Nat Prod Res. 2019, 33(19), 2738-2743; 2020, 34(15), 2173-2178). To date, no research has been reported, either domestically or internationally, on the gene clusters encoding pyrenophorol-like compounds derived from marine fungi and their biosynthesis. This invention identifies the biosynthetic gene clusters of these compounds in the marine fungus *Setosphaeria sp. SCSIO41009*, and successfully obtains pyrenophorol and its analogues through gene combination expression in the model fungus *Aspergillus nidus*. This achieves structural diversification of these compounds and is of great significance for the future preparation of pyrenophorol and its analogues using green enzymatic methods.

[0003] Summary of the Invention

[0004] The first objective of this invention is to provide a biosynthetic gene cluster for pyrenophorol-like compounds, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] A second object of the present invention is to provide pyrenophorol-like compounds having structures as shown in any of formula (I):

[0006]

[0007] The biosynthetic gene cluster of the pyrenophorol-like compounds of the present invention is derived from the marine fungus Setosphaeria sp. SCSIO41009, characterized in that the nucleotide sequence of this biosynthetic gene cluster is as shown in the base sequence of positions 1-18549 of SEQ ID NO.1, and comprises 6 genes, specifically:

[0008] (1) The genes responsible for the backbone structure of pyrenophorol-like compounds from marine fungi, namely pylA and pylB, are located at the 1st-7280th and 8256th-9205th bases of the gene cluster nucleotide sequence (SEQ ID NO.1, the same below), respectively, with lengths of 7280 base pairs and 950 base pairs, respectively, encoding polyketide synthase and thioesterase, containing 2386 and 297 amino acids, respectively;

[0009] (2) The gene responsible for the hydroxylation of carbon 4 and 4' in marine fungal pyrenophorol compounds, namely pylC, is located at the nucleotide sequence of the gene cluster from the 12610th to the 14271st base, with a length of 1662 base pairs, and encodes cytochrome monooxygenase, containing 523 amino acids.

[0010] (3) The gene responsible for the ketone-enol tautomerism in pyrenophorol compounds from marine fungi, namely pylE, is located at the nucleotide sequence of the gene cluster from the 16746th to the 18549th base, with a length of 1804 base pairs, and encodes a flavin-dependent oxidase containing 581 amino acids.

[0011] (4) The gene responsible for reducing the ketones at the carbon 4 and 4' positions to hydroxyl groups in pyrenophorol compounds from marine fungi, namely pylD, is located at the nucleotide sequence of the gene cluster from the 15014th to the 15871st base, with a length of 858 base pairs, and encodes ketone reductase, which contains 250 amino acids.

[0012] (5) The gene responsible for efflux of the compound, namely pylF, is located at the 9822-11608th base of the nucleotide sequence of the gene cluster. It is 1787 base pairs long and encodes an efflux pump protein containing 579 amino acids.

[0013] A third objective of this invention is to provide the application of the aforementioned biosynthetic gene cluster in the preparation of any of the pyrenophorol compounds 1-5.

[0014] The fourth object of the present invention is to provide a ketoreductase gene pylD, the nucleotide sequence of which is shown in bases 15014-15871 of SEQ ID NO. 1.

[0015] The fifth object of the present invention is to provide a ketoreductase PylD encoded by the above-mentioned ketoreductase gene pylD, the amino acid sequence of which is shown in SEQ ID NO.2.

[0016] A sixth object of the present invention is to provide the application of the above-mentioned ketoreductase PylD in the reduction of pyrenophorol compounds.

[0017] Preferably, the pyrenophorol compound is pyrenophorol compound 3 or 4, and the reduction is the reduction of the ketone groups at the C4 and C4' positions in the pyrenophorol compound to hydroxyl groups.

[0018] The seventh object of the present invention is to provide a method for preparing the above-mentioned pyrenophorol compounds 1-5, which involves gene combination expression of the genes in the above-mentioned biosynthetic gene cluster in a foreign host.

[0019] Preferably, the exogenous host is Escherichia coli, yeast, Aspergillus nidus, or Aspergillus oryzae.

[0020] The complementary sequence of positions 1-18549 of the sequence shown in SEQ ID NO.1 (sequence listing) can be obtained at any time according to the DNA base complementarity principle. Furthermore, the nucleotide sequence or a portion thereof of positions 1-18549 can be obtained by polymerase chain reaction (PCR), digestion of the corresponding DNA with a suitable restriction endonuclease, DNA in vitro synthesis techniques, or other suitable techniques. This invention provides a method for obtaining a recombinant DNA vector containing at least a portion of the DNA sequence from positions 1-18549 of the sequence shown in SEQ ID NO.1.

[0021] Genes or gene clusters containing the nucleotide sequences or at least a portion thereof provided by this invention can be expressed in heterologous hosts to understand their functions in host metabolism. These heterologous hosts include *Escherichia coli*, yeast, *Aspergillus nidus*, *Aspergillus oryzae*, etc. Expression in heterologous hosts using suitable expression systems yields the corresponding enzymes or other substances with higher bioactivity or production rates.

[0022] This invention targets polyketides derived from marine fungi. Starting with screening biosynthetic gene clusters, it studies their biosynthesis using a combination of heterologous expression and in vitro biochemistry. Through the study of their biosynthetic mechanisms, a series of pyrenophorol-like compounds were obtained using combinatorial biology methods, providing some compound entities for drug screening.

[0023] The gene information provided by this invention, which includes all genes related to pyl biosynthesis, can help people understand the biosynthetic mechanism of sixteen-membered ring dilactone natural products of marine fungi, and provide a material and theoretical basis for further structural modification of such compounds.

[0024] The Setosphaeria sp. SCSIO41009 strain of this invention is disclosed in non-patent literature (Spiro-Phthalides and Isocoumarins Isolated from the Marine Sponge-Derived Fungus Setosphaeria sp. SCSIO41009. Xiaoyan Pang, Xiuping Lin, Jie Yang, Xuefeng Zhou, Bin Yang, Junfeng Wang, and Yonghong Liu. J. Nat. Prod. 2018, 81, 1860-1868). The applicant guarantees that it will be made available to the public from the date of this application. This strain is currently deposited at the RNAM Center for Marine Microbiology (CAS), Guangzhou, Guangdong Province, China, with the accession number SCSIO41009. This strain is available for sale and can be purchased by anyone from this depository. Attached Figure Description

[0025] Figure 1 These are the structural formulas of pyrenophorol compounds 1-5 derived from marine fungi.

[0026] Figure 2 It is the biosynthetic gene cluster of marine pyrenophorol-like compounds (pyl) in Setosphaeria sp.SCSIO41009.

[0027] Figure 3This is an LC-MS analysis of the crude extracts from the fermentation of *Aspergillus nidus* A1145 transformants obtained by biocombining genes from the pyl biosynthetic gene cluster in *Setosphaeria sp. SCSIO41009*: i: *Aspergillus nidus* A1145 ΔEM; ii: Recombinant strain of gene pylA in *Aspergillus nidus* A1145; iii: Recombinant strain of gene pylAB in *Aspergillus nidus* A1145; iv: Recombinant strain of gene pylABC in *Aspergillus nidus* A1145; v: Recombinant strain of gene pylABCE in *Aspergillus nidus* A1145; vi: Recombinant strain of gene pylABCED in *Aspergillus nidus* A1145; vii: Recombinant strain of gene pylABCEDF in *Aspergillus nidus* A1145. Arabic numerals in the figure represent pyrenophorol and its analogues.

[0028] Figure 4 These are the results of the in vitro enzymatic reaction of PylD. a shows the SDS-PAGE result of PylD, with a marker of 180 kDa; b shows the results of the in vitro catalytic conversion of purified PylD into compounds 4 and 5.

[0029] Figure 5 This is a hypothesis about the biosynthetic pathway of pyrenophorol-like compounds.

[0030] Figure 6 It is compound 3. 1 HNMR spectrum (500MHz, DMSO-d6).

[0031] Figure 7 It is compound 3. 13 C10 NMR spectrum (125 MHz, DMSO-d6).

[0032] Figure 8 It is compound 3. 1 H- 1 H COSY spectrum.

[0033] Figure 9 This is the HSQC spectrum of compound 3.

[0034] Figure 10 This is the HMBC spectrum of compound 3.

[0035] Figure 11 It is compound 4. 1 HNMR spectrum (500MHz, DMSO-d6).

[0036] Figure 12 It is compound 4. 13 C10 NMR spectrum (125 MHz, DMSO-d6).

[0037] Figure 13 This is the DEPT135 spectrum (125MHz, DMSO-d6) of compound 4.

[0038] Figure 14 It is compound 4. 1 H- 1 H COSY spectrum.

[0039] Figure 15 This is the HSQC spectrum of compound 4.

[0040] Figure 16 This is the HMBC spectrum of compound 4. Detailed Implementation

[0041] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0042] 1. Genome sequence scan of marine fungus Setosphaeria sp. SCSIO41009 and sequence analysis of Pyl biosynthetic gene clusters.

[0043] A whole-genome scan and annotation of the fungus Setosphaeria sp. SCSIO41009 revealed a biosynthetic gene cluster of approximately 18.5 kb of pyrenophorols. Figure 2 It contains 6 openreading frames (ORFs) (Table 1).

[0044] Table 1: Genes and functions of the Pyl biosynthetic gene cluster

[0045]

[0046] a) Number of amino acids; b) Identity / similarity.

[0047] 2. The biosynthetic gene cluster of Pyl is expressed in Aspergillus nidulans.

[0048] To study the function of each gene in the gene cluster, the pylA-F genes were cloned into the PacI-SwaI sites of pYTU, pYTP, and pYTR, respectively. The recombinant plasmids containing the target gene were constructed by yeast homologous recombination, E. coli DH5α plasmid replication, etc., and transformed into the heterologous host Aspergillus nidulans A1145 (ΔEM, this strain was provided by Professor Tang Yi's research group, reference: Zhao Z, Ying Y, Hung YS, Tang Y. Genome Mining Reveals Neurospora crassa Can Produce the Salicylaldehyde Sordarial. J Nat Prod. 2019 Apr26;82(4):1029-1033. doi:10.1021 / acs.jnatprod.8b00983. The applicant also holds these plasmids and guarantees to provide them to the public from the date of this application). Z, YingY, HungYS, TangY. Genome Mining Reveals Neurospora crassa Can Produce the Salicylaldehyde Sordarial. J Nat Prod. 2019 Apr26;82(4):1029-1033. doi:10.1021 / acs.jnatprod.8b00983. The applicant also holds, and the applicant guarantees to provide to the public from the date of application of this invention) heterologous expression (primer sequences are shown in Table 2). LC-MS analysis of its fermentation crude extract revealed that the fermentation product of ΔEM-pylAB combination could detect compound 1, the fermentation product of ΔEM-pylABC combination could detect compound 2 (NMR of compounds 1 and 2 are shown in Table 3), and the fermentation products of ΔEM-pylABCE, ΔEM-pylABCED, and ΔEM-pylABCEDF combinations could detect compounds 3, 4, and 5 ( Figure 3 The NMR spectra of compounds 3, 4, and 5 are shown in Table 4. Further, by expressing the pylD protein in *E. coli* (primer sequences are shown in Table 5), the obtained purified PylD enzyme, under the action of the cofactor NADPH, was able to catalyze the sequential conversion of compound 3 to compounds 4 and 5. Figure 4 Based on the structures of compounds 1-5 ( Figure 1 This study aims to infer the functions of genes within the pyl biosynthetic gene cluster and the biosynthetic pathways of pyrenophorol-like compounds. Figure 5 ).

[0049] Table 2: Primer names and sequences required for constructing expression plasmids for gene combinations in the pyl gene cluster

[0050]

[0051]

[0052] Table 3: NMR spectra of compounds 1-2 1 H NMR 500MHz, 13 C NMR 125MHz, DMSO-d6, TMS, δppm)

[0053]

[0054] Table 4: 3-5 NMR spectra of compounds ( 1 H NMR 500MHz, 13 C NMR 125MHz, TMS, δppm)

[0055]

[0056]

[0057] a This indicates that the solvent is DMSO-d6. b This indicates that the solvent is CDCl3.

[0058] Table 5: Primer names and sequences required for constructing pylD gene purified protein

[0059]

[0060] The following further provides implementation examples, which are intended to help understand the present invention and are used for illustration only without limiting the scope of the invention.

[0061] Example 1: Extraction of genomic DNA from marine fungus Setosphaeria sp. SCSIO41009

[0062] Fresh spores of the fungus *Setosphaeria sp.* SCSIO41009 were inoculated into PDB medium (26 g potato dextrose solution, diluted to 1 L with water, dissolved, and autoclaved at 121°C for 30 min). The medium was incubated at 28°C with shaking at 200 rpm for 8 days, and mycelia were collected by centrifugation at 3900 rpm for 10 min. Quick-DNA assays were used to analyze the mycelium. TM Fungal / Bacterial Microprep kit (purchased from ZymoResearch) for extraction. Bacterial cells were extracted using ZR BashingBeads. TM Add 750 μL of BashingBead to the Lysis tube. TMBuffer. Vortex in a beadbeater for 20 min. Centrifuge at 10000 rpm for 1 min. Transfer 400 μL of the supernatant to a Zymo-Spin III-F filter, centrifuge at 8000 rpm for 1 min, and discard the filter. Add 1200 μL of Genome Lysis Buffer to the filtrate and mix by pipetting. Transfer 800 μL of the mixture to a Zymo-Spin IC Column, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Repeat the above steps. Add 200 μL of DNA pre-wash buffer to a Zymo-Spin IC Column, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Add 500 μL of gDNAwash buffer to a Zymo-Spin IC Column, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Transfer the Zymo-Spin IC column to a new 1.5 mL centrifuge tube, add 50 μL of DNAElution Buffer to the column matrix, incubate at room temperature for 1 min, centrifuge at 10,000 rpm for 1 min, and the solution in the centrifuge tube is the genomic DNA of the fungus Setosphaeria sp. SCSIO41009.

[0063] Example 2: Obtaining Pyl combinatorial gene transformant strains

[0064] A whole-genome scan and annotation of the marine fungus Setosphaeria sp. SCSIO41009 revealed 581.2 clusters, including a cluster of genes involved in the biosynthesis of pyrenophorol-like compounds. Figure 1 The nucleotide sequence of ) is shown in the base sequence from position 1 to 18549 of SEQ ID NO.1, containing six open reading frames (ORFs) (Table 1). Based on the obtained biosynthetic gene cluster sequence of Pyl, primers for the pylA-F genes were designed, and the primer sequences are shown in Table 2. Heterologous expression plasmids were constructed. The specific operation is as follows, following the Frozen-EZYeast Transformation II... TMThe reagent kit (purchased from Zymo Research) was used to add the pylA target gene fragment and the vector L-YTP obtained by double digestion of pYTP with PacI-SwaI to 30 μL of yeast competent cells RCO1 (RCO1, this strain is disclosed in the reference: Resistance-Gene Directed Discovery of a Natural Product Herbicide with a New Mode of Action. Nature. 2018 Jul; 559(7714):415–418. doi:10.1038 / s41586-018-0319-4. The applicant also holds this strain and guarantees to make it available to the public from the date of this application). 300 μL of EZ 3 solution was added, and after incubation at 30°C for 1 h, it was spread onto a medium containing -U (0.5 g Bacto™ Casamino Acids, 2 g glucose, 88 mL water). After sterilization, when the medium temperature dropped to 40°C, 10 mL of Yeast Nitrogen Base stock solution (NBS) and 1 mL of... Tryptophan stock solution (1 mL L Uracil stock solution) was placed on agar plates and incubated at 30°C for 2 days. Following the instructions of Zymoprep... TMYeast plasmids were extracted using the Yeast PlasmidMiniprep I kit (purchased from Zymo Research). The obtained yeast plasmids were then transformed into E. coli DH5α competent cells for plasmid amplification. Plasmid extraction was performed using the Biospin plasmid DNA mini-extraction kit (Hangzhou Borui Technology Co., Ltd.). The obtained plasmids were correctly sequenced (sequencing performed by Beijing Qingke Biotechnology Co., Ltd.), which is the pylA-pYTP plasmid. Plasmids pylB-pYTR, pylC-pYTU, pylD-pYTU, and pylE-pYTR were obtained using a similar method. For the construction of the pylBC-pYTU plasmid, during yeast homologous recombination, fragments pylB and pylC, the promoter fragment gpdA (a fragment amplified using plasmid pYTU as a template, with amplification primers gpdA-R and gpdA-pYTU-recomb-F), and the vector L-YTU obtained by double digestion of pYTU with PacI-SwaI were added. Then, yeast plasmid extraction and amplification in E. coli DH5α were performed using the same method to finally obtain the plasmid pylBC-pYTU. For the pylDE-pYTR plasmid, during yeast homologous recombination, fragments pylD and pylE, the promoter fragment glaA (a fragment amplified using plasmid pYTU as a template, with amplification primers glaA-R and glaA-pYTR-recomb-F), and the vector L-YTR obtained by double digestion of pYTR with PacI-SwaI were added. Subsequent methods were similar to obtain the plasmid pylDE-pYTR. For plasmid pylDEF-pYTR, during yeast homologous recombination, fragments pylD, pylE, pylF, promoter glaA, promoter amyB (fragment amplified using plasmid pYTP as a template, with amplification primers AmyB-reomb-F and AmyB-reomb-R), and vector L-YTR are added. The plasmid pylDEF-pYTR is then obtained using a similar method.

[0065] For obtaining ΔEM-pylA transformants, 2 μL of plasmids pylA-pYTP, pYTR, and pYTU were added to 100 μL of Aspergillus nidulans A1145 (ΔEM) protoplasts, respectively. After incubating on ice for 50 min, 600 μL of 60% PEG (60 mL PEG 4000, 7.35 g CaCl2, 50 mM Tris-HC, pH 7.5, diluted to 100 mL with water) was added and incubated at room temperature for 20 min. The mixture was then plated on CDS (10 g glucose, 218.6 g sorbitol, 50 mL 20× Nitrate salts, 1 mL Trace Elements, 20 g agar, diluted to 1 L with water) plates and incubated at 37°C for 3 days. Single colonies were then picked and transferred to CDS (10 g glucose, 50 mL 20× Nitrate salts, 1 mL Trace Elements, 20 g agar, diluted to 1 L with water) plates. Elements (20g agar, pH 6.5) plates were incubated at 37°C for 3 days to obtain the ΔEM-pylA transformant. For the ΔEM-pylAB transformant, plasmids pylA-pYTP, pylB-pYTR, and pYTU were added simultaneously during protoplast transformation, and the ΔEM-pylAB transformant was obtained using subsequent methods. For the ΔEM-pylABC transformant, plasmids pylA-pYTP, pylB-pYTR, and pylC-pYTU were added simultaneously during protoplast transformation, and the ΔEM-pylABC transformant was obtained using subsequent methods. For the ΔEM-pylABCE transformant, plasmids pylA-pYTP, pylBC-pYTU, and pylE-pYTR were added simultaneously during protoplast transformation, ultimately yielding the ΔEM-pylABCE transformant. For the ΔEM-pylABCDE transformant, plasmids pylA-pYTP, pylBC-pYTU, and pylDE-pYTR were added simultaneously during protoplast transformation to obtain the ΔEM-pylABCDE transformant. For the ΔEM-pylABCDEF transformant, plasmids pylA-pYTP, pylBC-pYTU, and pylDEF-pYTR were added simultaneously during protoplast transformation to obtain the ΔEM-pylABCDEF transformant. To obtain the ΔEM-pylD transformant, plasmids pylD-pYTU, pYTR, and pYTP were added simultaneously during protoplast transformation, and the ΔEM-pylD transformant was obtained following subsequent methods. The spores of the obtained transformants can be stored at -80°C after being dissolved in 40% glycerol solution for later use.

[0066] The preparation method of yeast competent cells RCO1 is as follows: according to Frozen-EZ Yeast Transformation II TMFollowing the instructions in the kit (purchased from Zymo Research), yeast RCO1 was inoculated onto a PDA plate and incubated at 30°C for two days. A single colony was then inoculated into 30 mL of YPD liquid medium and incubated at 30°C and 200 rpm until OD reached [value missing]. 600 Approximately 0.8–1.0. Centrifuge at 500g for 10 min, discarding the supernatant. Resuspend the cells in 30 mL of EZ 1 solution, centrifuge at 500g for 10 min, discarding the supernatant. Resuspend the cells in 3 mL of EZ 2 solution, aliquoting into new 1.5 mL EP tubes at 30 μL each, and slowly freeze at -80°C to obtain yeast competent cells RCO1.

[0067] The stock solution formula used is as follows:

[0068] YeastNitrogen Base Stock Solution (NBS): 6.7g yeast nitrogen base - amino acid-free and ammonium sulfate-free, 100mL distilled water, filtered through a 0.22μm filter membrane, stored at 4℃. Tryptophan Stock Solution (2mg / mL): 40mg tryptophan, 20mL distilled water, filtered through a 0.22μm filter membrane, stored at 4℃. Uracil Stock Solution (2mg / mL): 40mg tryptophan, 15mL distilled water, dissolved completely in HCl, then diluted to 20mL with distilled water, filtered through a 0.22μm filter membrane, stored at 4℃.

[0069] 20× Nitrate salts: 120g sodium nitrate, 10.4g potassium chloride, 10.4g magnesium sulfate, 30.4g potassium dihydrogen phosphate, add water to a final volume of 1L, autoclave at 121℃ for 30min.

[0070] Trace Elements: 2.20g zinc sulfate heptahydrate, 1.10g boric acid, 0.5g manganese dichloride tetrahydrate, 0.16g ferrous sulfate heptahydrate, 0.16g cobalt dichloride hexahydrate, 0.16g copper sulfate pentahydrate, 0.11g ammonium heptamolybdate tetrahydrate, 5.0g ethylenediaminetetraacetic acid (EDTA), 80mL distilled water. Dissolve completely and bring to a final volume of 100mL. Adjust the pH to 6.5 with KOH granules. Autoclave at 121℃ for 30min.

[0071] Example 3: Obtaining heterologous expression of pyrenophorol-like compounds 1-5

[0072] Taking the heterologous expression of the pyrenophorol-producing strain and the preparation of the compound pyrenophorol as an example, the Aspergillus nidulans A1145 transformant co-expressing the pylABC gene is the pyrenophorol-producing strain. Spores of the Aspergillus nidulans A1145 transformant (ΔEM-PylABC) containing the pylABC gene were inoculated into 5L CD-ST medium (20g soluble starch, 20g casein acid hydrodysate vitamin free, 50mL 20× Nitrate salts, 1mTrace Elements, water added to a final volume of 1L, pH 6.5, autoclaved at 121℃ for 15min) and cultured at 28℃ for 3 days. The resulting fermentation broth was separated into mycelium and bacterial culture. The mycelium was soaked in an equal volume of acetone overnight and then extracted three times. The extracts were concentrated and evaporated to dryness to obtain a crude mycelial extract. The bacterial culture was extracted three times with an equal volume of ethyl acetate. The extracts were concentrated and evaporated to dryness to obtain a crude bacterial culture extract. 0.1 g of crude extracts of mycelia and bacterial suspension were dissolved in 100 μL of methanol. After centrifugation at 15000 rpm for 10 min, 10 μL of each extract was analyzed by LC-MS. The detection conditions were: Eclipse XDB C18 (4.6 × 250 mm, 5 μm) column; mobile phase A: pure water containing 0.1% formic acid; mobile phase B: 100% acetonitrile containing 0.1% formic acid; flow rate: 1 mL / min; detection wavelength: 190 nm–450 nm. The HPLC program was as follows: 0–20 min, 5%–80% B phase; 20–21 min, 80%–100% B phase; 21–25 min, 100% B phase; 25–26 min, 100%–5% B phase; 26–30 min, 5% B phase. The remaining crude extract (9.6 g) was subjected to medium-pressure ODS, with eluent ranging from 10% to 100% acetonitrile, and separated into 10 fractions (Fr-B1 to Fr-B10) under 210 nm UV absorption guidance. Fr-B4 (40% acetonitrile) was further purified by high-performance liquid chromatography (YMC-pack ODS-A, 10×250 mm, 5 μm; flow rate 2 mL / min; isocratic elution with 17% acetonitrile-water (containing 0.4‰ formic acid)) to obtain compound 2 (approximately 200 mg, t). R =23min).

[0073] The transformants ΔEM-pylAB, ΔEM-pylABCE, ΔEM-pylABCED, and ΔEM-pylABCEDF were fermented using the same method, and the fermentation products were detected. LC-MS analysis showed that the fermentation product of the ΔEM-pylAB combination (AN-pylAB in the figure) could detect compound 1 (NMR spectra of compounds 1 and 2 are shown in Table 3). The fermentation products of the ΔEM-pylABCE (AN-pylABCE in the figure), ΔEM-pylABCED (AN-pylABCED in the figure), and ΔEM-pylABCEDF (AN-pylABCEDF in the figure) combinations could all detect compounds 3, 4, and 5. Figure 3 The NMR spectra of compounds 3, 4, and 5 are shown in Table 4. The spectrum of compound 3 is shown below. Figure 6-10 As shown, the spectrum of compound 4 is as follows: Figure 11-16 As shown.

[0074] The specific separation processes for compounds 1, 3, 4, and 5 are as follows:

[0075] The ΔEM-pylAB transformant was fermented in 5L at 28℃ for 3 days using the method described above. The resulting fermentation broth was separated into mycelium and bacterial culture. The mycelium was soaked overnight in an equal volume of acetone and extracted three times. The extracts were concentrated and evaporated to dryness to obtain a crude mycelial extract. The bacterial culture was extracted three times in an equal volume of ethyl acetate and concentrated and evaporated to dryness to obtain a crude bacterial culture extract. The crude extracts (5.3g) obtained from the mycelium and bacterial culture extractions were separated by medium-pressure ODS, with the eluent ranging from 5% to 100% acetonitrile. Under 210nm UV absorption guidance, the extracts were divided into 6 segments (Fr-A1~Fr-A6). Fr-A2 (30% acetonitrile) was further separated by high-performance liquid chromatography (YMC-pack ODS-A, 10×250mm, 5μm; flow rate 2mL / min; 60% methanol-water (containing 0.4‰ formic acid) isocratic elution) to prepare compound 1 (3.6mg, t). R =36min).

[0076] The ΔEM-pyl ABCDE transformant was fermented in a 5L volume using the method described above, and cultured at 28°C for 3 days. The resulting fermentation broth was separated into mycelium and bacterial culture. The mycelium was soaked overnight in an equal volume of acetone and then extracted three times. The extracts were concentrated and evaporated to dryness to obtain a crude mycelial extract. The bacterial culture was extracted three times in an equal volume of ethyl acetate and then concentrated and evaporated to dryness to obtain a crude bacterial culture extract. The crude extract (7.7 g) obtained from mycelial and bacterial broth extractions was subjected to medium-pressure ODS with 20% to 100% methanol as the eluent. Guided by 210 nm UV absorption, it was divided into 13 fractions (Fr-E1 to Fr-E13). Fr-E10 (50% methanol) was further processed by high-performance liquid chromatography (YMC-pack ODS-A, 10×250 mm, 5 μm; flow rate 2 mL / min; isocratic elution with 60% methanol-water (containing 0.4‰ formic acid)) to prepare compound 3 (4.7 mg, t). R =19.4min).

[0077] For the separation of compounds 4 and 5, 2.4 g of the crude fermentation extract of ΔEM-pyl ABCDE was directly loaded onto high-performance liquid chromatography (HPLC) and eluted with a gradient from 10% acetonitrile to 100% acetonitrile. Guided by 210 nm UV absorption, it was divided into 5 fractions (Fr-E21 to Fr-E25). Among them, Fr-E24 (40% acetonitrile) was prepared by HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm; flow rate 2 mL / min; 32% acetonitrile-water isocratic elution) to obtain compound 4 (10.2 mg, t). R =14.0min) and 5 (1.2mg, t R =16.0min).

[0078] Example 4: Expression and purification of the pylD gene in E. coli BL21(DE3)

[0079] RNA was extracted from fresh mycelia of the ΔEM-pylD strain (construction method as described in Example 2) using the Coolaber Fungal RNA Rapid Extraction Kit (RE781-50T). Genomic gDNA was removed using the FastKing cDNA First-Strand Synthesis Kit (KR116-03) to obtain cDNA. The pylD gene was amplified using the obtained cDNA as a template, with primers shown in Table 5, and cloned into pET28a. + pET28a was obtained between the BamHI-NdeI sites of the vector. +The / pylD plasmid was correctly sequenced and transformed into E. coli BL21(DE3) for expression. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured overnight. Positive clones were picked and inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, a 1% (v / v) inoculum was added to 200 mL of LB broth containing 50 μg / mL kanamycin (total 1 L) and cultured at 37°C and 200 rpm until OD (out of control). 600 When the protein expression reaches approximately 0.6–0.8 (3–4 h), the culture temperature is lowered to 16 °C, and IPTG (final concentration 0.1 mM) is added to induce protein expression. The culture is then incubated at 200 rpm for 16–20 h. The induced bacterial culture is then centrifuged at 3900 rpm for 20 min at 4 °C to collect the cells. The cells are resuspended in 50 mM Tris-HCl (pH 8.0), maintaining a bacterial concentration below 20% by volume. The cells are then disrupted on ice using a high-pressure homogenizer (1000 bar, three times). After centrifugation at 12000 rpm for 50 min at 4 °C, the supernatant is collected as the crude enzyme solution containing the target protein. This crude enzyme solution is then loaded onto a Nitrogen electrolyte solution equilibrated with 50 mM Tris-HCl (pH 8.0). 2+ -NTA affinity chromatography column, after adsorption, the liquid was dried dropwise, and impurities were washed away with 10 mL of buffer A (50 mM Hepes buffer, 300 mM NaCl, 10 mM imidazole, pH 7.2) and 10 mL of 1 / 5 buffer B (50 mM Hepes buffer, 300 mM NaCl, 250 mM imidazole, pH 7.2), respectively. Then, 2 mL of buffer B was added to extract the target protein from the Ni... 2+ The NTA affinity column was dissolved. The purified protein was desalted using a PD-10 desalting column and washed with 2 mL of stock solution (50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, pH 7.2). The purified PylD protein was obtained and stored at -80°C. Its protein electrophoresis pattern is shown below. Figure 4 As shown.

[0080] Example 5: In vitro enzymatic reaction and detection of PylD

[0081] The activity of PylD ketoreductase was detected using compound 3 as a substrate: In 100 μL of Tris-HCl buffer (50 mM, pH 8.0), 1 mM NADPH, 4 mg / L substrate, and 20 μL PylD protein were added. The reaction was carried out at 30 °C for 5 s, 30 s, 10 min, 30 min, and 2 h. The reaction was terminated with an equal volume of methanol. After centrifugation at 15000 rpm for 15 min, the supernatant was collected for LC-MS detection. The analytical conditions were: Eclipse XDB C18 (4.6 × 250 mm, 5 μm) column; mobile phase A: pure water containing 0.1% formic acid; mobile phase B: 100% acetonitrile containing 0.1% formic acid; flow rate: 1 mL / min; detection wavelength: 190 nm–450 nm. The HPLC program was as follows: 0-20 min, 5%-80% B phase; 20-21 min, 80%-100% B phase; 21-25 min, 100% B phase; 25-26 min, 100%-5% B phase; 26-30 min, 5% B phase. Through analysis and structural identification, the enzyme-catalyzed reaction products compounds 4 and 5 were obtained. Figure 4 ).

[0082] Based on the structures of compounds 1-5 ( Figure 1 This study speculates on the functions of each gene in the pyl biosynthetic gene cluster and the biosynthetic pathways of pyrenophorol-like compounds. Figure 5 pylA and pylB are responsible for the synthesis of the pyl skeleton structure; pylC is responsible for the hydroxylation of carbons 4 and 4' in pyl; pylE is responsible for keto-enol tautomerism; pylD is responsible for the reduction of keto groups at carbons 4 and 4' to hydroxyl groups; and pylF is responsible for the efflux of the compound.

[0083] SEQ ID NO.1(Setosphaeria sp.SCSIO41009)

[0084] ctagacagcgacaatcttggaaatcactttcttactaaggttagcaagagacgtagcgttgagaacatccaaggttgtaacgagtgcgccaagttcagttcggatccagtttctgagttccactgctgcaagtgagtcgattccgtacacggacaacggtcgctccacgtccatggcttcggggaga

[0085] tgcagcatgcgtacgaatgcgccgttgatcgcgtcaaccaccgctttttgccttgctccttcatcggccgaatcgtttttgagtagtaacaaagcca

[0086] attgaacttctacatctttgccatttgctccacccgcatccccgccgcttgacgagcgaccctcaaaaagtgcagcaaagcgagcgtcctttttcaa

[0087] ttgagagtcgaccggctgaggcacaatcaggcctgtaatcaattgcgcactcgcctctgggctggagcgcgggtgagcatgttgctgcagcac

[0088] cgagacgtagaggatcttggctaacaaaccgctgttgattcctttgaacactcgagtgtcgaagacgttttgcagattctcggtttccgcaatcaca

[0089] ccagaatcttcaattacgcccaaatcgacagagcacgcggcttggccgcgagaacggcgaaaagcggcaaaagagtcgaggaagacattag

[0090] cggcagcatagttggcctgaccgcggtttccaattacaccagagatgctagaaaggagggtgaagaagtccaacctaaggccaagcgattctg

[0091] cggcattgtgcaggttccaggttccgcgggtcttgcattggactgcctcatgatactctgccaacgtcatctggtcaaaaggacgatcctaaggat

[0092] gggttagccaagttctaccgagacgatgtgtgattgatcatactctcaaaaccatcgcgccttggataattcccgcaataggagcctttgtttgttta

[0093] aaggcgtgtaagacctccgtgaagttggtaatatcagcagtgagaaggtcaatgtgggctccaaggccattgatctgcttaacaatgccctgcga

[0094] cttatgatccttgtgcccgctacgagatatgacagcaagattcttagctccttgctttgcgaggtaaacagcaagagaaccgcaaagaccacgga

[0095] ggccaccaacaatgagatagcaaccatcatcccggaatgccagctttggagtcgctcggcggatctatatataaacgtcagcaatcgtgtttcta

[0096] gacttgacggagatctaattgaacaaacaaagcttaccggaacctggaccttggcatcaggcccgtctgataaaacgactttgccgatatgtgttc

[0097] ccggtctgagaaatcgcactgccgtagggatttcggtccacgagaagcggtgcactgggttaatgggcttgatataacccccttccaagagctc

[0098] gaacaatttggccagggagcgctcaattagagaatcaggagctcgttcagcagataagtcaacagctcggaaagagatgttgcgatcaaaggg

[0099] cgtcatcgtcagcttgttacgatcgaggatatccttcttgccgatttcgaccatgacacctccatcagccaggatgcggaaagactcttcaagcata

[0100] tctccagttaacgaattgagcacgacgtcgaccccgcgaccgttggtggcagccaaaatctggtttgcaaagtccgtattgcgtgagttgaaaat

[0101] ctgatcagtgcttaaactgaaagtgtcttgtagaaactgcctcttttcgggactgctaacagtagtaaaaacttgagcacccacgtattgcgcgagtt

[0102] ggactgccgcaatacccacacctccagcagcagagtgaatgagaaccgttttgcctgcagaaagattgcccatgtcgaaaagagcgtgaagg

[0103] ctggtgacatagacgactggaagcgttgcagcatcctcgaacgacagagagtcaggaatgcgatgcacgcgggcgggtacagtccggaccc

[0104] gattagcaaagcaacctttgttccaaaccatgacacgatctcccggagcataggtggttacgcttggactgacttgtttgaccacaccggcagctt

[0105] caaagcccagctgtgactcatcaccgggcacataacccatactaacaaccacgtctttgtaattcagaccagccgcgtaaatctccacttccatgt

[0106] ggccatctggcaatggaagaacatcaggagagacctcgccgaaatggaggctatcaaggtttcccagacgttcgcagcggagtctaacaagc

[0107] gagtcgcactcatggaagtcgacagtgtcgacgggctgttcacttatatcgtcgctttggagcgaggtgagcttggcgtcgggaacaatgcggc

[0108] tgatgtatgttacaccgccgcgctcaacaaattcgtagtcttctgctgagtctggttcttcagtggaaagttggtccaggcatgctgaaatggcggc

[0109] ggaggttgcgttcccggtcgcattctcaacatcgagggtaacgaagcgtacctgctcctcactgcggatagtacggaggagaccagcgatggc

[0110] ggctttgttggggtcattgacgttcaaatgggaacccaccgtgacccacagaaggcgacaccgtttctcgagaacatgtttgacaagttcccact

[0111] gctgttcggtaaagccgtccatgagcgagtggaaaagctcgtcaaccactaacacaatatctttgctggacttgatatcctcggtagggcaaacc

[0112] gtcgactttacagcccatccgtctgagctgagctttctgataacctgggccatttcgtgtgactgcccagccgtcgcctcgacaaggctcatgtga

[0113] gtgatcgtctggcgcacagtctcactcggcttgctctcctcaaccttgcagatgcagcttccatttccagcagtgaaagttgcgccgatgcgcctg

[0114] aggagtccttcaggtaggccgtggccgatgacaaaggcacctgtcttggtggagctgacaaggctttgtgccaagacctcgtcgacgacccca

[0115] gagactggccctttgacaatgatcaagtcaaatttggtgtctgggacaatctcttcacccttgatgacatcggccagatgaacttgcgtaccctgca

[0116] tctgctcttgggttgccaacatgaccttgggatcgcgaagggccaagatgtactgtgagcaagcagaacgcatcgcggactttccatccacgctc

[0117] cacatactggaagcatcttcggagttcaaattaagctccagcacagccagtcccggcctcttgtgaacggcgagatcgattagatcgccagtttc

[0118] cttggtcgcaagatgttgctgcgctgcaacttcgggaatcttgaacaatgtatcgacatcggcgttccatacaaaacgggtgaatgtgtggccgg

[0119] gcttctcgttgtcgcccacctcgatttctccttgctcaagacctctcatctcgaagagacaggtggagtctttggggctaaaaacctggacattggt

[0120] ggtgtaattgcgtggatgctctttgtctccaatgcccacccaatttgccgacgctaacgcaataccttcaccgtcgactggcagattagttccaccct

[0121] ggataaccatgctaccaatgattttgggaacgagcaccgcagaaccggaggtggggggctcaccgttccacaatgacggggaagtagcctgc

[0122] aaacaggagtccaagatagctggatgcattggatagctggactgtccgagagggtttgatggtggtggctcaaggctaacagtagatctcgaag

[0123] tccgtctgccaatgaaagactcgacagcgatgtgtttctggaagcagggtccaaacttgtatcccatgtcggctaatgccttgtaccatacacgag

[0124] caggcgtgggaaattccaacggtcgcagatcatttgctggagcggtagtgcttaggcaatctgtctcaatgcaaacgaaacccgtgcaatgttcc

[0125] tggtcaaccggcgcgtcctcttgcaccgagcacacccgaaactcgtaccaggtacgtgtggaacctcctttgacgggcgtcaatgacaacgtga

[0126] tacgagtctctgcgttctcttctagtaccaaggcacgaaggattttgacatccttcaatcggaagcggtatttttctggtggctggtggttccacttga

[0127] cagtcatagtcgtttggtagatagcttcaactgccatggcaatgtatgctgctcctgggaagacaatttcactgccaagcacatggtctctcagaaa

[0128] cggaacgtcagagagctttaagagctttttgaatgttggtgccacccaagcggtagcagggatctttgatccaagaagatcgtggttgacaaactt

[0129] cttgaaacgccactctttgctggctctggtttcatgccaataccggttggtgtgattccacgagtaattaggcaggtccacgatgactttggcgttgt

[0130] gtttctcgattcgattgactcggctgagactgacgctacccccagcaagatacagctgaccagctgatttgtacagtgcgagaatcgacccctgg

[0131] ccacgaatcaagcaagatgtgtacggaacatcgctaatctttcccgcgagaccattctttatttgtgcaatcgggccagagagcgcattggatgg

[0132] gccaagttcaatcaagaagttggcgccaaattcactatcacggatcagctctgaggcagccactgcaaatcgtacaggtgacaccatgttgctctt

[0133] ccagtatgctgcatcaagcttctcatttggctccaagatttttccagtaacggagctaaacattcggacctgagaactggtgaccttggactgctgct

[0134] tgaaaaggtcatctttgagaagcatctcttcgtagacttcgccgatcttggccatgtagtcagagtggtaagccaggtcaacgagcaggaggcgt

[0135] gcgaaatgaccgtctgtctgcaacctgtctcggagcttctccaatgctgctcgtgtacccgacattgtgagactgtccggactattataacaggca

[0136] atctgaatcttgccttcctctggctggaggtactgctcaattttatcggcgccaataccgactgcaagcattccaacgggctccagaggcgctccg

[0137] actttcttggccgcttgaccacggtaaaatgcagtcttgattgcatcagcgtaggtaagtcgccctgcggcggcggcagcagcgatctcaccag

[0138] aggagtgaccaacaaccgcctcagctttgacgccccaatcatgcaaaacttctagaagcgctagctgcagagctgtcactagcggctgcgaga

[0139] attcgggttgacggagagcttcagcacttcgagccgaagtgagttcttccatcagcgtccacgatggcggctcagacaaagatttgagggcctc

[0140] atcgagttctgcaatgatccttcgcgcaactgggaagttattcagaagatctgctcccatctgagaccactgtgcaccttgtccggtaaagacgaa

[0141] cccaacacgaggggttgaaggggcttgctttccagtcaccaaatctccggttgtcagcttgggcttcttcgattgagtgattgtgaaagcccggta

[0142] gtacagcttgctttttctctcgcttagagtataggccaggtggtcaatatcgattgacaccgtaggatcgagtagatgattggacagggttttgacat

[0143] agttcgtgagcgagctcggctcattcgcagaaaacaccaaaagctttagtggtatcttctctgcctcttcgcctgcatcttcttcatcctcgtcatcaa

[0144] agaagttgctggaaatggtttgataggaagatacgtggggggacgacgtagcattctcgagtacagcgtgggcattagcacctccaaaaccaa

[0145] atgaattaacactaactcgtcggataatcgagcctttgggccattgaaccatggtccggctcgctcgtagtttgagacgctcccaatcgatcctgg

[0146] ggtttggcttgatgaaagtcgggttgcctggaatttggccacgctcaacagccatagtggccttgatcaggcctgagattccagcagcggcctca

[0147] gaatgaccgacattgctctttattgacccgatgatcaactcctggccagccgggcgaccagacgcgaacaccagagcaacaccctgggcctcg

[0148] atggggtctccggtaagtgtaccagttccatggcattcaacatactgcgtagctcggaagtcggtaatcccggcattcttgtaagccattctggtta

[0149] cagctgcctgagcttctgagctcgggttagcgagacccgcagtgcgaccggatgcgttggaagctgtgccccggataacggctctgatggggt

[0150] cgttgtcacggattgcgtcttccagcctcttcaaatagacaatattggcagcctcggccttgacgtagccatctgcgctggcatcaaaacttctgca

[0151] ctggccggaggcggactgtgtcttgttcatagtgccgacttcctcgttatgctctggcgccagccatagattggttcctcccacaatcataccgtcg

[0152] gcctgataggtatctaaaaagcggcaagctacgtctagtgagaccaaactgccagaacatgccgtatcaatagatatactcgggccgttaatgtt

[0153] caagaaatgactcagccgattactgaggatcgaaggtgagagaccaatggtgatggaatctgctctgtcttcgggatcgcgattctgaatagctg

[0154] tatagtcggagaaactgtttccaacgatgacaccagttctggtgccgctgatcttctcgagtgggataccgccgttttccaaagcttcatatgctact

[0155] tctagaagctgacgttgttgtgggtccatggcgacacagtccgcccggctgatgctgaaaaactggccgtcaaaaacggcgggatcgacatact

[0156] ccatgaacatgccgcctggagatttcatcgtccttgctctcctcgttccatcgtaatggccatcgagattgaagcgactttccggtggtgtgttctca

[0157] gctatcccacctcgttgaagaaggtcccataaagctgtcggggatttgctatgtccaggtagtcgacaacccatgccgactacggcaatgggcgt

[0158] ctgcacgcgttcttggggtgttgttggagctggaacatcatttccgtcttcgctggagcacaaactcgtggcggtggaatcttccgggctcatgga

[0159] gctcatgggcgtgctgttcgaggtagagtctgccattgttttgtggttgttgggtacgttgctggagtttaacaactcagttcctgctagagtgaagta

[0160] aatcctgagtggaaaatgacaacgaaatcaaatgggcagctgcaaaggttgcaagtatttacgatatgtaatgaagaaaagaataggagtttact

[0161] gtcaatggcttttatatatactttaaattcgtaagttaatgacttccttgacaccgattattttcctccttcaccttgtcgtgatgaacacttgacactattcc

[0162] gctcttggaaggacatgtttgtttctggagttgtggtagtagacaccacatactatgccaagctatgattatgtgccgcaagattacgtacagtatctt

[0163] ttgatagttttttttgttgcagactagagtcagtgctcggatcccttgggcggctgcataaccacacatggcgatacaagccatgttgttagtgagcg

[0164] cataatctgcatgagggcagcagtgtgggttccgagacttttcaggcgtaccgattaaagacccgcttccaccgcggcttcccaacaatgcaggt

[0165] aagctgcttactatgaacccctgtatccctcagatcctccctggtggtgtttctgaacgggaaactttctcggagttgcttgcagtcgatccgactca

[0166] acaaagtcacgcgcgtaagccgcttcgctatcgtgcacaattcctctgctaatgtctttgaagagttcacatagtacagctttactattttgggcctct

[0167] gccgcagcgctaatgtgcgcacggactgtattgtggaaccaggtcgaccaatggcaactctgatagggtattaccaactgtacacatgtgcttga

[0168] cattttacaagtcaggatgattctcatgaggacgtttataggcggcagtctcctattgcgtttcaacattcactctagcagctatcgaacatagaaatt

[0169] cagtgaccaacgaatcagactatcggcaccatgaaaggcagagaagatgtcagcttcccgacgctcgacggtctcactctgcggggatggctc

[0170] tatcctgcggcacagaaaggcccagccattatcatgacgcctggtgtatgtacatcccctttcattctaaaaacctcaccagcagtctgatctcattt

[0171] tagttcaacatgaccaaagaaactatcatcaatgatgtcgctgaatactatttcgcggcaggcttcacagtcctcagctacgatccacggtcaatcg

[0172] gtgcaagcgaaggtctgccaaaaaatgaagccaacccagtcaagaacatggaggactacatggacgccttaacctttttgaagtcgcacgaga

[0173] tggtggaccctactaaaattgcctattggggttattcattcagtggcatggtagcactatgcgcagctgcacttgacaaacgagccaaggccgtca

[0174] tcgcatcggctccattgaccatctgggagttcaaaaaatggaaaccagtgctggctaaatgcatgaaagaccgagagtctcgtttggcgggcaa

[0175] caggccggtaaatcttcccatgttgaccgaaactggcgaacagccagcaggatttggcgagaatttcgaaagcgaaggtgtgctcaacatcatt

[0176] gcgcgcgcggtcgagctacaacccaatttcagacccgagacagcgctgagtagctactatcacatcgctgctttccaaccattttcgcttcttccct

[0177] tcgtaacaccgacaccagcattcgtcgtatacggcgagcaagatcagatttcgccagctcatcttcagaagaccttaatctacgatgttctttcgga

[0178] accaaaacaactacttacagtaccggacaaggcgcatatgaatcttgtgagcgggcaaggttctgaacggatactagacgagcaggtcgagttc

[0179] ctcaagcgtgtgtggacaaagagttgatgagcgatcttttcactttgtagtacagtagattatttaggacttttatgaagcaagatgccctttaaatttta

[0180] catgattatgtatcttccacatgttgaggttgaacttcacgcgcgtcaaagatggtaagttttcagctacatctcgtccttcagcattgttttatcatcat

[0181] gttgccggccccttggggctctggcactttgtgaagagttgcagacaatatttcagctattgcatagacgcactcaagcccattgaaacatattgtt

[0182] gttacaacaactactactcatcaactttgttccgcctgctcagtcccagaggctctatatgttgtgagatagtggagcaaaatggagattatggtact

[0183] gatcaaggtgtctcggctgcctcgttgcgattatgagactgattgctctctaactgctcacagtcggttcggtctcaccaaatagtcgatgatgcga

[0184] atacgctagaagctttttctacacaacgttgcggttatttgcgcctcaccctgtaaaataccaaaatggtgaaccatgtatccccaaaccatgctcag

[0185] catgcattgatcggtgagaaccctgaaagagttaccgacatcttcatccttctaccccacgtgggccgccacacccctgcccaattttctccgatc

[0186] aacgacaaacgccacaaccgataccaccaacgcagctgcaccaagcgccaaggcaaccgcataagcactctttaggcctcccatgtacgcct

[0187] caaccgcactctgcagctcgtccttgcttagcattgccctcatctcaagcggaccagcgtggaaaagagagtgcgcacgcgctgatccaaacat

[0188] tctgactaatgaggataagagctggtttccgaataaagtctgcgccacgctcaaccagacagcaccagcaagcagttgaaaaaagagggccat

[0189] tgctgagacggtcgagaggtcggaggcatcgacaagggcctgacatgcgatcagggcaatctgcacgctgagaccgaggccaataccggc

[0190] gggaatttgatatccaatccactggtgtgtcggagcattaaccccaatggtataggtcagggcactaccaatagtaatgagaacgttgcctgctatt

[0191] gcaacgagctgaaattctcccgtcatagatatcaagatgccagacacaatggcgaagatggagctcgtggccagcaaagggatgattcgaatg

[0192] ccagactgtgttgggttcgtaccagaaaccgcttggaagtagatggggaggtagtagagcagcacaaagaaggtgctacatacgcacacctgt

[0193] tgcatcatgaggagtgcgatgggtctggtagtcagcagccgcatgttcagagtggcgcgctccttgagaaatttctccacgatgatgaaaaccac

[0194] ggcaatcagccctgctgcgactaacgttccgatgataccaccagagctccaggattgggttataccgccccattgcatggcaagaaggaagca

[0195] gatgacggctccaagcagaagaaatgttcctgcaaagtccatctgcagtagcttttccttccatgacacctggaccggcttaccatgtttgggggt

[0196] ggagaagaagaacattacgaaggcgccggcgaggcctccgataggcaggttgatccaaaagcaccttgcgcaaattagctgcgagcaccatt

[0197] ttcaaagaagtccactcaccacctccaggaaacaaaatccgtgaaagcgcccccaaccaatggtccgactacacttgcaatggcaaagctagc

[0198] agagatgacgccctgtaacgccgccatctttctaggcggtgcgctcaaagccacgatcaaataactccctgtgctaactcctgcccctccaacgc

[0199] ctgcaatcgccctaccgacaatcaatgtgcttgatctcggtgctgcagcacagatcacgctgccgacctcgaatatcaaaattgataccagataaa

[0200] cgaattttacgtcgaagaacttgtataacttgccccacatgctttggaacgcggccatggtgacgaaaaaaccactgccataccaagcaatatcat。

[0201] ccaaggaacggaactcatctgtgatgcgcggaatcgctgtagagattatggtattgtcgagagcaacgagaaagacggacagtgcacaggaa

[0202] gtgagaataagcattagtcgagaagttgctgggtaggttggatcggtgtcttcatggctcgacgcatcgtctaatctaggatgttgctcgcctctttg

[0203] tccatcatatggttcggcttggtctgttgctgccgctgttggaccctttgtctcctcgaatggattgttgtacatggtgcttggagaagatttgttgcata

[0204] acagcgtgtcgtcgtattttgcgggtgattgatcttcttgcattgaatgtattttctgagaacgatactatgggtttattgctgtgtctgtttttggtggtga

[0205] tgctttttagtgtttgatatgcctttaaggtaatattcccttacatacgatatgatgttgtatcacgagttagcttatccatacatctggcaaaggtcaaag

[0206] aaagaaaaaggcctcataaaggaaatcagtctgacaaatcaactagcactcagaaagcgatcccctgctgacaaacgttgtggcagctgtatgt

[0207] gggagcaagaacccctggaacactggtaggctctaaatggaggacatgcttagatgagagacttggcagtggagacttgcagtttgctccttac

[0208] cgtatttgatatctgtacatgaccggtgatcctataggaacgctgacttgcatgcacctaaaaggcgcttatctgcataaaacctgacgctaagatc

[0209] acccaagtccgcggaacgggtgagaatcatagattgtccaatacgaagaaggttacagtaagtagcttatacccgagattaactagcgtgggag

[0210] gcgtgggtcaggcgtggggacaatgtccaataccttgcccaaactagtagcggtaaatggcatgaacaagcaccggaaactcaattattataag

[0211] aggccaatcacaacaatgtcactgtattgccaattcatcataacagtttagcaatcttttggaagacatcagaatgtataggaatattgatcttcacttt

[0212] tcagatcaatgacacatgtacagtttgtgctgccattgcacgttatatgctatgtttgcatgtatgtctggaggtgataaagacgtggagttgtgcaca

[0213] tgttgcgaactcttgaaaagaatatggggtgttttgttttgagaaaggtttctcaaagacgggttctattggagacatgttgaatcatgttctggttcatt

[0214] ggatagagaaggttccacaacacaacgactggtttcccgagtcagcaagttgtgcaggggtgtatctacgtggcgcacttaatgacagctcttac

[0215] gcccttgctctcacggcgtggcattggcactaccatgtccagtacaaaatccacgtcttcccttgtcgtctcatacaattccatttcgaatctgcgga

[0216] aaagggtcgctatcgcgacatacagctccatgtacgcaagctccttgcctaaacaccctctcgatcccttgccaaaagaaaaaaggtaatgcga

[0217] aagtggacgccctccaggtccggtcggattgttgagccaccgctcaggctggaagctaagcgcgtttggaaacaccgtctcgtttgtatgaatag

[0218] agtatgcgtccatgcaaacagtgacacctgccggaatgacccattcattgaattgtaaaggatgcaggcgatttattcgaggcaggctctgaactt

[0219] gaccatatgccatgcgtagagctttggaagagtttgtcagtatcacaatgttcgattttgtgaaacacaattgtaagacttacattcttggataacggc

[0220] acctaggtaggggagtttttcgagattgttgtaaggaagaataacgttcgggtcaggcatgacttcggcaagctcagccttgagccgcgcaagg

[0221] acagcggggttgtccagtaagtggaaagtgccaaccgtgaggacccattttgttgtttccattcctgcgccaaccacgcttgatgcttcttgagtca

[0222] agcgattcactgatttctctgagtcaggcaaagaggagctgaggatatcgtggaatatggttgggtggtcgacgtccaaccctttgacctccctac

[0223] cttccacaatagcggcaacctgatttcgaatactctgtcatctccagtcagtacaagactgagaaaaaatcaagaaatacgtacagttgtaaattct

[0224] acaatgggctttacatgtggcatgagcattaccgtaatggaagttgggattgagttcataacatcgtttatgatcttgaagtgcgcattccaatgtcca

[0225] taccatgcgagagcctgcacgcccagcgccaatggtgactcgaagtcggctgctgcggtacagctcgtcgatcggccaaatgccagtgccgt

[0226] gaccgtatccattgtcatactagaccagacatcattgaagttcacaacctttccggtaccggcgtactcggttgcaaagcgaaacgagatacgctc

[0227] gacgatttctgtcagaccagcgtgcaggcctcgaatgcgctgtttggagaagaaggggttcagggcggctcgcctctgctggtgttgctcggcc

[0228] agtggtgtgctaaacactgcatctgggatgccgaagcgatatctgtacttttcggacttgtccaatggctttgactttgtgctgggtccgtacacattg

[0229] gagaggaatgtggggtcgttgatgtgcagttcagaaggggttatgcgcacaataggaccgtattgttgatggagcttttcgatctgccgcgcaaa

[0230] ctggccaccctctccgccgttgatgaggacatcgtagtatccttcataccaccctgtgacagcggccagctttgggcccgggaattttgctagag

[0231] gactgaagaagagcctgtagatggcgatggctaggcaatagaagaaggaggcgatggcgagtgtcgctaagaaaagaggcatgtctaaaga

[0232] gggcatgttgacgtcatctggcgacgtgcgaagggtgtgctgtcaagcccaagtgctctcgaccaatcttcacagagaaatcatcgtgaagcttt

[0233] atgcttgagtagatgcatggatatcatatcacggccctctgtttttaccctccgctgcgagttgctcttcagctctagaggaatggccttttcagacag

[0234] cttgcaaagtaagcaggcacgccaagagtgaattggtcttgtaccacggcgcatcaatgcgtcgacacatacggcacctggcgatgctctagac

[0235] gattcccatgcacgcttacctgcatatattgaaacatgtgggtcaagggtctagcgtccctggcattctctgcttaggtgcatgcgacggcttaccc

[0236] gagccgcatgggctgcgctagccgtgggggtgcggcagccgcagccactgggccgctcacattgcccccactccatcacgctgagccccga

[0237] catagcggctgcgggtagccacgtcaatctcccctgaagagagctaacgacacgccggacaaaaaatacagccaaccgggaagcgccaag

[0238] ctggaaagcttgctttgagctgctgtgcctcttccttgcgtttctaagctgacaaatgtatctttcagcatgtccagccagccgtgaataacaacccc

[0239] cgaacgtctgctgtacggtatctgactcaaccgactcaatcgactcgctcacactactttcgtcttacattccccactccaaccatgtcttcgtccac

[0240] ggttgtcttcatcacaggtgccggaagaggttcgtgctcggcgtatctggaccaaacaaacaatcccgttaacctgaacaggtatcggtagagg

[0241] cctgactcaagcttacctccagcgcccaaaccacactgtggttgggagtgtccgtgacagcaccgctccagtttgcgaggagctcaagaaactg

[0242] ccagtcgctgaaggatctcgtctgatcctcgtcaccatcgattcttccaagctcgatgatcctgcgaacgccgtcaaggcggctgaagaggctgg

[0243] cattgaccacatcgacattgtcatcgccaatgcaggaacctcgccttcacctggcccgcttgaggcagccaacctccacgaggttgttgatgcgc

[0244] tcaacgtaaacaccatgtcccccattgctctttaccaggccgccaagccactgctagacaagtctgccaaacccatctggatctcgatgagcagc

[0245] gcagccggttctattgggaatgtgcctgttcaccaagcccactggcttctgggctatggaatgtccaaggccgctatgaactttttcaccatggcc

[0246] gtacacgggtcccaacctaattacattgcctactccattcaccctggtcttgtacagactgacctcggcaacaccggcgcgaagatgcaaggaat

[0247] ggaaattgcccctatcacacttgaagacagctgcgccaaagtcatggctactgtatgtcccccacattgaatgataatgaattacaccactgacaa

[0248] gataaccaggccgataatgccacacgtgaaaagacttctggcaagtttcttgacattatgactgatggggagtttccgtggtagattgtagattagg

[0249] ggtttctttgtaaagcaaaacaatacatttgtaacaatgtaatggtataagttactatgagtttagatattaccgttctactaagcataccattcaactcat

[0250] gcaaatactgaatagctattcatatgccgtgaaaaccccataaatgatagtaatcctgttgctcaggcagtagattcttttcatgataattcatgtcgg

[0251] actgtcacaacccccacttctacaattttcatctgagattctagactcctagatatgttcgattatccgcgggacacatatggaagagtctaaggtatt

[0252] ctttttggggcgtttgagttaggattggtaactgtcattaagccccaggataagccattaggctgcaccgtcaaccaggtcgatagggctatttttac

[0253] gcctcatgggatggttcatcctgcgtcagctccatttatttcctcttgtatcagtcattgagtcagcgacatccaatccaaacagtcgtatacagaagt

[0254] tcccgcttggtcagagtacggcgtacaactcggcgccacatgtcagccctcgcccccacgtgtatgggctgtttatgtgccctttccaatcctaac

[0255] aatcccgtgggcgcgctaagagccacctcaaagcctaccaagtcttaggcagctgaagtactaggctggaagtagcatggatttatcgtagcag

[0256] tgcacgggtacacctgttccaagtactggagaagccgtgaggccctaaggcatgctctcataagatctataaaatgaagcgcttgacgctctcgc

[0257] agtttgtttcgatacatatcctcacccacatcattagcttttgctgttttctccagactttcgacgtgcatcatcgattatgctgtcttcgttctataccgca

[0258] ttggccgtgcatgcagcactaagctctgcagcaccgcttattactggttcagacaactgcaaggtgatcccaggcgataagaagtggccaagca

[0259] cgcagacctgggcccatttgaaccagactatcgggggcagattaattgcaacaacacccattgccgaagtgtgccatgcacccaatttcaacga

[0260] ggcacaatgtgcgaaattgagcagtgaatggggctttgcatctctcgtgtgggtctcgctctctgtatgaacttcttgttcgaagttgactaattgtttc

[0261] ttcaagtgttcctacaccggcagagtttatgtctacttggttccagaataacacatgcacgccttttagtgactcttcagcaccctgtaaccttggtaa

[0262] ccgcgccagttattccatcaatgtgcagtctgcggaagacgtgcaacaaggactaaagtttgcccaaaaacacaatgtccgccttgtggttaaaa

[0263] actctggccatgatttcttcgggaaatccactggcaagggcgccctctccctgtgggttcacaaccttaaggacaaaaccgtcatcccccaatac

[0264] cgatcatcgtactacaccggtccggctgttcagattggcgctggtgtttcgggaggcgaggctgccagttttgccgccgataatggataccgtgtt

[0265] gtagtcgggtcctgtccaactgtcaaagctgcgggcggttttacacaaggtggaggtcactcttttcttaccggaatatatggattcagtgcagaca

[0266] atgtcctagaatgggaagtcgtcactgcagctggtgaacatcttattgcgacacctaccaacgagtatgctgacctctactgggcgctgagtgga

[0267] ggcggtggaggaacatatgcggtagtcgtctccatgaagatccgtgcctaccccgatggtaacgttgcagttgcctctttgtcgatcaatgtagcc

[0268] aactcgggaagcgtcgatgcgtattgggatgccgttgaggcctatcatgtcgaattgcaacatctggtagaccaaggcttcgttgccgaatacca

[0269] gctctcaaacgattccctctatcttttcggcatgatcgccccagatcacacgtctgaaactcttcgcgcagctatggtccccatggtctcagccctg

[0270] agctccaggacctcgtcatctctcactgcggagtcagtcaaccttcagatcacgcaccgcgacagctatcacggggcttatgctgccacagtcg

[0271] agccgcttatggctgggaattcgtttgcgcccgccgttgcaggacgtttcctcccacgctccgtgatcgaatcaaacagcaccgcatggcacgct

[0272] gcgctaagagaagtgacttcaatcagcgatggccgatacgccttggctataaccgcactcaatgcgcagaacaagaatcgcaactcgcctgtc

[0273] gcagccaatgcagtgcagcctaatttcaaagaagccttcagcagtatcattatcagtccaaagtgggacaacactgagtcatgggacaacgcag

[0274] gaaagttgttagacgagctccagaatactgtcatgcccattctggaaaaggtgacacctggtgctggcgcatacaaaaatgaagccagctgggc

[0275] tcagaagaacttccaagaagccttttattccggaacctattctaaattgcgggaaatcaaatcaaaatacgaccccaagggattgttttatggtctca

[0276] catcggtcggcagtgagaattgggtggcggattctcagggaagattgtgcgcggcatgaSEQ IDNO.2(Ketoreductase PylD amino acid sequence)

[0277] MSSSTVVFITGAGRGIGRGLTQAYLQRPNHTVVGSVRDSTAPVCEELKKLPVAEGSRLILVTIDSSKLDDPANAVKAAEEAGIDHIDIVIANAGTSPSPGPLEAANLHEVVDALNVNTMSPIALYQAAKPLLDKSAKPIWISMSSAAGSIGNVPVHQAHWLLGYGMSKAAMNFFTMAVHGSQPNYIAYSIHPGLVQTDLGNTGAKMQGMEIAPITLEDSCAKVMATADNATREKTSGKFLDIMTDGEFPW*

Claims

1. A biosynthetic gene cluster of pyrenophorol-like compounds, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of the biosynthetic gene cluster of claim 1 in the preparation of pyrenophorol-like compounds, wherein the structure of the pyrenophorol-like compounds is shown in any of formula (I): Formula (I).

3. A ketoreductase gene pylD, characterized in that, The nucleotide sequence is shown as bases 15014-15871 of SEQ ID NO.

1.

4. The ketoreductase PylD encoded by the ketoreductase gene pylD as described in claim 3.

5. The use of the ketoreductase PylD according to claim 4 in the reduction of pyrenophorol-like compound 3 in claim 2, characterized in that, The reduction involves reducing the ketone group at the C4 or C4' position in pyrenophorol-type compound 3 to a hydroxyl group.

6. The use of the ketoreductase PylD according to claim 4 in the reduction of pyrenophorol-like compound 4 in claim 2, characterized in that, The reduction involves reducing the ketone group at the C4' position of pyrenophorol-like compound 4 to a hydroxyl group.

7. A method for preparing the pyrenophorol-like compounds 1-5 as described in claim 2, characterized in that, The genes in the biosynthetic gene cluster of claim 1 are expressed in a foreign host through gene combination, wherein the genes are pylA, pylB, pylC, pylD, pylE, and pylF; the nucleotide sequence of pylA is shown as the base sequence of SEQ ID NO. 1 from position 1 to 7280, encoding polyketide synthase; the nucleotide sequence of pylB is shown as the base sequence of SEQ ID NO. 1 from position 8256 to 9205, encoding thioesterase; the nucleotide sequence of pylC is shown as the base sequence of SEQ ID NO. 1 from position 12610 to 14271, encoding cytochrome monooxygenase; the nucleotide sequence of pylD is shown as the base sequence of SEQ ID NO. 1 from position 15014 to 15871, encoding ketoreductase; the nucleotide sequence of pylE is shown as the base sequence of SEQ ID NO. 1 from position 16746 to 18549, encoding flavin-dependent oxidase; the nucleotide sequence of pylF is shown as the base sequence of SEQ ID NO. 1 from position 1 to 14271, encoding cytochrome monooxygen ... The sequence of IDNO.1 from position 9822 to 11608 encodes an efflux pump protein.

8. The method according to claim 7, characterized in that, The exogenous host is Aspergillus nidus.

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