Biosynthetic gene cluster and synthetic method of isopentenylated indole alkaloid exopoids

Abstract

The invention relates to the discovery of a biosynthetic gene cluster of an alkaloid, namely, exopoid C, and the biosynthetic gene cluster of the exopoid. A biosynthetic gene cluster exo of an exopoid compound is obtained from a fungus Penicillium sp.HDN14-431 through genome mining, the gene cluster comprises a heme-dependent oxidase gene exoA which is composed of indole dioxygenase and a cytochrome b5 structural domain, a cytochrome P450 enzyme gene exoB and an isopentenyl transferase gene exoC which is composed of indole dioxygenase and a cytochrome b5 structural domain, and the cytochrome P450 enzyme gene exoB and the isopentenyl transferase gene exoC are used as biosynthetic genes of the exopoid compound. All genes in a gene cluster are expressed through an aspergillus nidulans heterologous expression system, two isopentenyl indole alkaloids are obtained in total, and a compound I is a new compound exopoid C. And the compound II, namely the exopoid, is a known compound. The biosynthesis pathway of the compound is reported for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to an isopentenyl indole alkaloid compound exopoid C, an exopoid biosynthetic gene cluster and a synthesis method. Penicillium sp . HDN14-431 obtains a gene cluster of this type of compound, and utilizes the gene cluster to synthesize this type of compound through heterologous expression. Background Art

[0002] Alkaloids are a general term for nitrogen-containing natural products, which are divided into different subclasses according to the nitrogen-containing part, such as indole alkaloids, isoquinoline alkaloids, piperidine alkaloids, etc. Indole alkaloids are one of the largest subclasses, widely distributed in plants, animals and microorganisms. So far, more than 4,000 indole alkaloids have been isolated. Most indole alkaloids show antibacterial, antimalarial, antioxidant and enzyme inhibitory activities. Marine fungi are well known for producing structurally diverse secondary metabolites, including indole alkaloids, pyridone alkaloids, terpenoids, polyketides and non-ribosomal peptides. In recent years, with the development of genome sequencing technology, more and more fungal genomes have been discovered. Bioinformatics analysis shows that the number of gene clusters in fungal genomes far exceeds the number of isolated natural products, which suggests that fungal genomes have huge development potential and are urgently waiting to be explored and utilized.

[0003] At present, gene mining combined with heterologous expression strategy has become an important means to discover new active natural products. By forcibly activating the "silent" genes in the gene cluster, we can further obtain unknown natural products that are difficult to obtain by traditional natural product separation. Therefore, using gene mining technology to discover the enzymes that may be responsible for the synthesis of novel structures in fungi, and obtaining novel structures and good active compounds through heterologous expression, is of great significance for discovering new indole alkaloid compounds from fungi.

[0004] Exopoid was first reported in 2012 and was isolated from fungi in soil. Exophiala pisciphila In its biological activity study, PHF-9 exopoid showed moderate cytotoxicity against A-549, Hela, PANC-28 and BEL-7402 cell lines, IC 50 The values ​​were 76.3, 77.2, 107.5 and 89.5 μmol·L -1 In 2014, Wang Binggui's research group also found a marine fungus Penicillium paneum In 2016, our research group isolated exopoid from a marine fungus Penicilliumsp. HDN14-431, and similar structures were subsequently reported in natural product-related studies, but its biosynthetic pathway has never been reported.

[0005] We have successfully used gene cluster mining technology to Penicillium sp. HDN14-431, we located the compound responsible for the synthesis of exopoid and a new exopoid-like compound, which we named exopoid C, and then isolated it from Aspergillus nidulans. Aspergillus nidulnas The exopoid C and exopoid biosynthetic gene clusters were reconstructed in A1145. Summary of the invention

[0006] The present invention aims to provide a gene cluster for the biosynthesis of exopoids compounds and a method for the biosynthesis of such compounds; The exopoid C and exopoid indole alkaloid compounds have structural formulas as shown in I and II;

[0007] I II

[0008] The gene cluster of the synthetic compound is the isopentenyl indole alkaloid compound exopoid C, the gene cluster exo of exopoid, which is derived from Penicillium Penicillium sp. HDN14-431, the gene cluster contains a heme-dependent oxidase gene exoA, composed of indole dioxygenase and cytochrome b5 domain, a cytochrome P450 enzyme gene exoB and an isopentenyl transferase gene exoC, the corresponding gene sequences are shown in SEQ ID NO.1-3, and the corresponding protein sequences are shown in SEQ ID NO.4-6; the fungus Penicillium Penicillium The strain HDN14-431 was deposited in the Marine Medicinal Biological Resources Center of Ocean University of China with the deposit number MMBC-MIR-00007113.

[0009] The biosynthesis method of the above compound is as follows: 1) Construction of heterologous gene expression vector First, using PCR technology, Penicillium sp. HDN14-431 genomic DNA was used as a template to amplify the target genes exoA, exoB, and exoC; then, the target genes were connected to Aspergillus nidulans Aspergillus nidulnas The expression plasmids pYTU, pYTP and pYTR of A1145 were used to construct the recombinant expression plasmids pYTU-exoA, pYTP-exoB and pYTR-exoC; The primers include pYTU-exoA-F / pYTU-exoA-R, pYTP-exoB-F / pYTP-exoB-R, pYTR-exoC-F / pYTR-exoC-R, and the sequences of the primers are shown in SEQ ID NO.7-12; 2) Construction of Aspergillus nidulans heterologous expression strain The expression vectors pYTU-exoA, pYTP-exoB, and pYTR-exoC were transformed into Aspergillus nidulans using the PEG-mediated method. A . nidulans In the protoplasts of A1145, the plate used was CDS solid medium for regeneration of Aspergillus nidulans protoplasts, and the transformant AN-exoABC was obtained; 3) Fermentation of heterologous expression strains of Aspergillus nidulans and isolation of compounds The transformant was inoculated into the Aspergillus nidulans defective CD solid culture medium for passage, and then inoculated into the Aspergillus nidulans fermentation CD-ST solid culture medium and fermented at 28°C for 4 days. The solid culture medium was then crushed and extracted three times with ethyl acetate, and concentrated under reduced pressure to obtain an extract. The obtained extract was separated and enriched with the target component by ODS column chromatography; then it was prepared and purified by a high performance liquid chromatography column to obtain a monomer compound.

[0010] The effects of the present invention are as follows: The present invention uses Aspergillus nidulans as a dominant expression host and adopts a heterologous expression method to completely reconstruct exopoid C and the biosynthetic gene cluster of exopoid indole alkaloids in the Aspergillus nidulans, and synthesizes two indole alkaloids, which can be used for the production of complex indole alkaloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a schematic diagram of the gene cluster exo.

[0012] Figure 2 Schematic diagram of the structures of plasmids pYTU-exoA, pYTP-exoB and pYTR-exoC used for heterologous expression of Aspergillus nidulans in the present invention.

[0013] Figure 3 This is the LC-MS detection spectrum of the metabolites of AN-exoABC and AN-wild type strains of the present invention.

[0014] Figure 4 is the HRESIMS spectrum of compound I.

[0015] Figures 5 - 9 is the NMR spectrum of compound I. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0017] Example 1 Bioinformatics analysis of exo compound biosynthetic gene clusters Based on Local Blast homology search, the present invention Penicillium sp. HDN14-431 genome was found to have an exopoid indole alkaloid biosynthesis gene cluster exo. The functions of the exo heavy genes were predicted using the NCBI database, which included a heme-dependent oxidase gene exoA, a cytochrome P450 enzyme gene exoB, and an isopentenyl transferase gene exoC, as shown in the attached Figure 1 shown.

[0018] Example 2 Construction of heterologous gene expression vector by Penicillium sp. HDN14-431 genome was used as a template, and primers pYTU-exoA-F / pYTU-exoA-R, pYTP-exoB-F / pYTP-exoB-R, and pYTR-exoC-F / pYTR-exoC-R were used to amplify exoA, exoB, and exoC by PCR. The PCR reaction system was subjected to agarose gel electrophoresis, and the exoA, exoB, and exoC fragments were recovered after purification with a gel recovery kit. Next, the exoA, exoB, and exoC fragments were integrated into the linearized vectors pYTU, pYTP, and pYTR after NotI, BamHI, and BamHI digestion, respectively, using Yisheng One-Step Cloning Enzyme, and then transformed into E. coli In XL-1, positive clones were screened by ampicillin, and the positive clones were selected for liquid fermentation. The plasmids were extracted and sequenced for verification, and finally the correct expression vectors pYTU-exoA, pYTP-exoB, and pYTR-exoC were obtained; The primers are shown in Table 1; Table 1: Primer list Primer Name Sequence (5’ - 3’) Number pYTU-exoA-F GAGCCTGAGCTTCATCCCCAGCATCATTACACCTCAGCAATGCTCAACACCAGGTTGAC SEQ ID NO.7 pYTU-exoA-R GAGGACATACCCGTAATTTTCTGGGCATTTAAATTTATCTTTTGGGGAGGGGGAAAC SEQ ID NO.8 pYTP-exoB-F CTTGACTAACCATTACCCCGCCACATAGACACATCTAAACAATGGCTATCGCGTCGCTG SEQ ID NO.9 pYTP-exoB-R TAAAGGGTATCATCGAAAGGGAGTCATCCAATTTAAATGAAAGCCGGTATGCAGTGATC SEQ ID NO.10 pYTR-exoC-F CTCCCTTCTCTGAACAATAAACCCCACAGAAGGCATTTATGGCTCTTCTCAGCCAAAC SEQ ID NO.11 pYTR-exoC-R GAGACCCAACAACCATGATACCAGGGGATTTAAATGAAACTACACAGTGCGAGTGATG SEQ ID NO.12 Example 3 Construction of Aspergillus nidulans expression strain In the construction of Aspergillus nidulans transfectant strains, the expression plasmids containing different genes were transfected into Aspergillus nidulans by PEG-mediated protoplast transformation. The specific operation method is as follows: Take out the frozen strain from -80℃ Aspergillus nidulnasA1145 was spread on CD (supplemented with U, R, and P elements) solid plates and cultured at 37°C for 2-3 days. The spores were collected in sterile water with a sterile cotton swab and then μ m cell filter to remove mycelium, etc.; take an appropriate amount of spores and add them to 40 mL CD (supplemented with U, R, and P elements) liquid culture medium, and culture at 37 ℃ and 220rpm for 8-9 h until most of the spores have germinated; add 10 mL protoplast hydrolysate (0.02 g Yatalase™ and 0.03 g Lysing Enzymes from Trichoderma harzianum are weighed and dissolved in 10 mL OM buffer, 0.22 μ m sterile filter membrane, which must be prepared and used immediately), resuspend the cells. 28 ℃, 80 rpm enzymatic hydrolysis overnight; when the protoplasts are completely hydrolyzed by microscopic examination, resuspend the protoplasts with an appropriate amount of pre-cooled STC, then add the correctly constructed recombinant plasmid to the protoplasts, and then add 600 μ l PEG solution, after standing at room temperature for 25 minutes, was coated on the CDS regeneration medium of the corresponding defective type and cultured at 37 degrees Celsius for two days; the grown transformants were transferred to the CD medium of the corresponding defective type for expansion culture, and the correct transfection strain AN-exoABC was obtained after 2 days; The transformants activated on CD plates were inoculated on CD-ST solid plates and fermented at 25°C for 4 days. A. nidulans A1145 was fermented under the same conditions as the control group, and the culture medium was extracted with an equal volume of ethyl acetate, the organic phase was spin-dried, and the crude extract was dissolved in methanol for LC-MS analysis; The Aspergillus nidulans defective CD culture medium comprises: 1% anhydrous glucose, 5% 20x NitrateSalts, 0.1% Trace element, and before use, U mother solution (1%), R mother solution (1‰) and P mother solution (1‰) are added as needed; The CDS solid medium for regenerating Aspergillus nidulans protoplasts comprises: 1% anhydrous glucose, 5% 20xNitrate Salts, 0.1% Trace element, 21.86% D -sorbitol, 2% agar, add U stock solution (1%), R stock solution (1‰) and P stock solution (1‰) as needed before use; The U element mother solution consists of: 0.56% Uracil, 24.442% Uridine; The R element mother solution comprises: 0.0125% Riboflavin; The P element mother solution comprises: 0.05% Pyridoxal hydrochloride; The nitrogen source 20x Nitrate Salts consists of: 12% NaNO3, 1.04% KCl, 1.04% MgSO4·7H2O, 3.04% KH2PO4; The trace element composition of the reagent is: 0.22% ZnSO4·7H2O, 0.11% H3BO3, 0.05% MnCl2·4H2O, 0.016% FeSO4·7H2O, 0.016% CoCl2·5H2O, 0.016% CuSO4·5H2O, 0.011% (NH4)6Mo7O 24 4H2O, 0.5%Na4EDTA; Depend on Figure 3 The HPLC-MS results showed that compared with the blank control group (A. nidulans wild type), AN-exoABC produced obvious differential peaks of compounds I and II at 6.16min and 6.34min, and the molecular ion peaks were 244 and 228, respectively; The HPLC-MS analysis method is as follows Analytical instrument: Waters SO Detector; Column: Waters ACOUITY UPLC Shield RP18 1.7 μ m 21x50 mm; Mobile phase: acetonitrile-water (containing 0.1% formic acid) was used as the mobile phase, and gradient elution was performed at a flow rate of 0.5 mL / min; Gradient elution program: 5%-100% acetonitrile (0-20min).

[0019] Example 4 Isolation and structural identification of metabolites of Aspergillus nidulans transfected strain AN-exoABC 10L of Aspergillus nidulans transfected strain was fermented and cultured in CD-ST solid medium for 4 days. The fermentation medium was extracted with ethyl acetate, and the organic phase was spin-dried to obtain 3.4g of crude extract. The crude extract was first separated by ODS (octadecylsilyl) column, and the target compound was found in the 55% methanol / water component. The component containing the target compound was separated by high performance liquid chromatography (HPLC) to obtain a pure compound, and the compound structure was analyzed by NMR, HRESIMS, etc. The CD-ST medium for Aspergillus nidulans fermentation comprises: 2% soluble starch, 2% acid hydrolyzed casein, 5% 20x Nitrate Salts, 0.1% Trace element, and before use, U mother liquor (1%), R mother liquor (1‰) and P mother liquor (1‰) are added as needed; The present invention determines the structures of compounds I and II by NMR; II is a known compound identified as an exopoid, and I is a new compound. The difference between I and II is that the aldehyde group is changed to a carboxyl group based on the structure of exopoid. Therefore, this gene cluster is determined to be the biosynthetic gene cluster of exopoid, and it is the first biosynthetic pathway constructed by heterologous expression. Compound 1: purple powder. HRESIMS m / z 244.0969 [M+H] + , thus determining that the molecular formula of the compound is C 14 H 14 O3N, NMR data see Table 2, Appendix Figures 5 - 9 .

[0020] SEQUENCE LISTING 1. exoA gene sequence 2. exoB gene sequence 3. exoC gene sequence 4.exoA amino acid sequence * 5.exoB amino acid sequence * 6.exoC amino acid sequence MALLSQTTTPAAWSCASNRQFPTHPEYQDIKPIAWVTRTQTPTKPIQTSSKEVSSSSTTTYTSEATRQLTALSKAVGQDESIRQKLLEALEFSFGDWGQAPVPQQLNRCGWLSDVSNDHAPFEYSVALSQRTGACELRFLVESQPEENTMSALQASTSQLTADIATKYGPSKVSLDRFHLISDLFLPPNAQGTLASWHSFATSKTLEKWKIYLD PQANGKQNAFKITQTAMDRLGLAASWNLLEQVMHGGEDYPVYFSLGLSPNEAEAVKVYVAHPGASATAIAQRHARADPSTNIHDIEQFYSTMAGGSLGPYRGKPGLSCFHFKNKDPSRVPARTVLYPMDNYASNDAEAQARIEQYMEIISAPQVYRDRYRSAINAVRRPLKDGRGIHSWVGMKDKANGDRSNTYYLSAELFGSLADSGIAMK*

[0021] Table 2 shows the NMR data of compound I ( 1 H NMR (500 MHz) and 13 C NMR (100 MHz)spectroscopic data of compoundIin methanol- d 4) No. <![CDATA[ δ C , type]]> <![CDATA[ δ H , much ( J in Hz)]]> <![CDATA[ 1 H- 1 H COSY]]> HMBC 1a 134.5, C 2 132.5, CH 7.83, s 1a, 3, 3a, 13 3 109.2, C 3a 122.18, C 4 122.15, CH 7.78, d(8.6) 5 3, 6 5 113.1, CH 6.66, d (8.6) 4 3a, 4, 6, 7 6 150.3, C 7 107.0, C 8 117.9, CH 6.77, d (9.8) 9 1a, 6, 7 9 131.0, CH 5.73, d (9.8) 8 7 10 76.9, C 11 <![CDATA[27.7, CH3]]> 1.43, s 9, 10, 12 12 <![CDATA[27.7, CH3]]> 1.43, s 9, 10, 11 13 169.3, C

Claims

1. A gene cluster for synthesizing isopentenylated indole alkaloid compounds, characterized in that: The gene cluster is exopoid C, a biosynthetic gene cluster exo of exopoid indole alkaloids, and the gene cluster contains a heme-dependent oxidase gene exoA, which is composed of an indole dioxygenase and a cytochrome b5 domain, a cytochrome P450 enzyme gene exoB and an isopentenyl transferase gene exoC, and its corresponding gene sequence is shown in SEQ ID NO.1-3.

2. A method for synthesizing exopoid compounds using the gene cluster according to claim 1, characterized in that: The following steps are involved: 1) Construction of heterologous gene expression vector Using PCR technology, Penicillium The target genes exoA, exoB, and exoC were amplified using the genomic DNA of sp. HDN14-431 as a template; then, the target genes were connected to the Aspergillus nidulans expression plasmids pYTU, pYTP, and pYTR, respectively, to construct the recombinant expression plasmids pYTU-exoA, pYTP-exoB, and pYTR-exoC; 2) Construction of Aspergillus nidulans heterologous expression strain The expression vectors pYTU-exoA, pYTP-exoB, and pYTR-exoC were transformed into Aspergillus nidulans Aspergillus nidulans The transformant AN-exoABC was obtained by regeneration of Aspergillus nidulans protoplasts and culture on CDS solid medium; 3) Cultivation and fermentation of Aspergillus nidulans heterologous expression strains The spores of the transformant AN-exoABC were enriched in the Aspergillus nidulans defective CD solid medium and then inoculated into the Aspergillus nidulans fermentation CD-ST solid medium for 4 days to obtain metabolites.

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