CN115894401A - Tetracyclic sesterterpene compounds and synthetic gene cluster thereof - Google Patents
Tetracyclic sesterterpene compounds and synthetic gene cluster thereof Download PDFInfo
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- CN115894401A CN115894401A CN202210945218.9A CN202210945218A CN115894401A CN 115894401 A CN115894401 A CN 115894401A CN 202210945218 A CN202210945218 A CN 202210945218A CN 115894401 A CN115894401 A CN 115894401A
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Abstract
The invention relates to a tetracyclic sesterterpene compound and a synthetic gene cluster thereof, belonging to the field of gene engineering. The tetracyclic sesquiterpene compound synthetic gene cluster is derived from fusarium oxysporum 14005, and the gene cluster contains 6 genes, namely a gene fomdE for coding a sesterterpene synthase FoMS or a functional equivalent thereof, genes fomdA, fomdC and fomdD for coding cytochrome P450 enzymes fomdA, fomdC and fomdD, a gene fomdB for coding an aldehyde ketone reductase fomdB or a functional equivalent thereof, and a gene fomdF for coding a hydrolase fomdF or a functional equivalent thereof. The Mangicols gene cluster discovered by the invention catalyzes and generates a new tetracyclic sesterterpene compound, and provides valuable lead compound resources for enriching a natural product compound library and discovering new antibiotics.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a tetracyclic sesterterpene compound and a synthetic gene cluster thereof.
Background
Fungal natural products are an important source of drug development, and decryption of large numbers of genomes reveals that fungi contain large numbers of potential novel biosynthetic gene clusters, "dark substances". Terpenoids are the general term for all isoprene polymers and derivatives thereof, and the abundant structural types and wide biological activities determine the importance of the terpenoids in natural drug research. Among them, diterpenes and sesterterpenes are the most abundant and diverse and important secondary metabolites of structural types in fungi, and are widely used in pharmaceuticals, perfumes, resins and other fine chemicals. With the continuous increase of genome databases released in NCBI, researchers find that a large number of terpene biosynthetic gene clusters are contained in fungal genomes, which means that the metabolic pathways are reconstructed in microorganisms by using terpene synthase genome mining as a guide through metabolic engineering and synthetic biology strategies, so that not only can more compounds with new structures and high activity be obtained, but also biosynthetic pathways of different compounds can be found, and a novel approach is provided for the research and development of new drugs.
Mangicols are tetracyclic sesterterpenes with various potential medicinal values, such as Mangicols A and B, show remarkable anti-inflammatory activity and have good cytotoxic activity on various cancer cell lines. Since the first report in 2000, only 13 Mangicols have been discovered so far. Therefore, the Mangicols compounds have better development and research prospects. By carrying out heterologous expression on the biosynthetic gene cluster, the blindness in the traditional natural product research can be effectively avoided, and more Mangicols compounds with biological activity can be efficiently excavated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a tetracyclic sesterterpene compound and a synthetic gene cluster thereof.
The purpose of the invention can be realized by the following technical scheme:
the invention firstly provides a tetracyclic sesterterpene compound, and the structural formulas of Mangicol H to Mangicol P are respectively shown as follows:
the invention further provides a biosynthetic gene cluster fomd of four-ring sesterterpene compounds Mangicol H to Mangicol P, which contains 6 genes, namely a gene fomdE for coding a sesterterpene synthase FoMS or a functional equivalent thereof, genes fomdA, fomdC and fomdD for coding cytochrome P450 enzymes fomdA, fomdC and fomdD or functional equivalents thereof, a gene fomdB for coding aldone reductase fomdB or functional equivalent thereof, and a gene fomdF for coding hydrolase fomdF or functional equivalent thereof, wherein the nucleotide sequence of the fomdA is shown as SEQ ID NO.1, the nucleotide sequence of the fomdB is shown as SEQ ID NO.2, the nucleotide sequence of the fomdC is shown as SEQ ID NO.3, the nucleotide sequence of the fomdD is shown as SEQ ID NO.4, the nucleotide sequence of the mfodF is shown as SEQ ID NO.5, and the nucleotide sequence of the fomdE 6;
alternatively, the nucleotide sequences of the 6 genes are DNA coding sequences corresponding to more than 80% identity with the amino acid sequences encoding the proteins fomdA, fomdB, fomdC, fomdD, fomdF and FoMS, respectively.
The invention further provides application of biosynthetic gene cluster fomd of tetracyclic sesterterpene compounds from Mangicols H to Mangicols P in preparation of terpenoids.
In one embodiment of the invention, the application of the biosynthetic gene cluster fomd of the tetracyclic sesterterpenoids Mangicol H to Mangicol P in the synthesis of the tetracyclic sesterterpenoids Mangicol H to Mangicol P is provided.
The invention further provides a vector for expressing biosynthetic gene cluster fomd of the tetracyclic sesterterpene compounds Mangicol H to Mangicol P, which is an eukaryotic or prokaryotic expression vector containing genes fomdA, fomdB, fomdC, fomdD, fomdF and fomdE.
The invention further provides a preparation method of the tetracyclic sesterterpene compounds, which is characterized in that genes in biosynthetic gene clusters fomd of the tetracyclic sesterterpene compounds from Mangicol H to Mangicol P in Fusarium oxysporum 14005 (Fusarium oxysporum 14005) are expressed by a heterologous expression method in Aspergillus oryzae NSAR1 to obtain the tetracyclic sesterterpene compounds from Mangicol H to Mangicol P.
Wherein the content of the first and second substances, aspergillus oryzae NSAR1 and Fusarium oxysporum 14005 (Fusarium oxysporum 14005) are all biological materials known to those skilled in the art.
In one embodiment of the invention, the preparation method of the tetracyclic sesterterpene compound comprises the following steps:
(1) PCR amplification is carried out by taking the genome of Fusarium oxysporum 14005 as a template and primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F/fomdB-R and fomdF-F/fomdF-R as well as genes fomdE, cytochrome P450 enzyme genes fomdA, fomdC, fomdD, aldehyde ketone reductase genes fomdB and hydrolase genes fomdF respectively for diterpene synthase to obtain PCR products of the genes fomdE, fomdA, fomdC, fomdD and fomdF; then, constructing a co-expression vector pUARA4-fomdACE of fomdE, fomdA and fomdC by taking Aspergillus oryzae NSAR1 expression vector pUARA4 as a vector; an Aspergillus oryzae NSAR1 expression vector pUSA4 is used as a vector to construct a co-expression vector pUSA 4-fomdDF of fomdD, fomdB and fomdF;
(2) Under the mediation of PEG solvent, co-expression vectors pUARA4-fomdACE and pUA 4-fomdBDDF are co-transformed into protoplasts of a high-yield host Aspergillus oryzae A.oryzae NSAR1 (niaD-, sC-, delta argB, adeA-) which is easy to express terpene synthase genes, so as to obtain Aspergillus oryzae AO-fomdABCDEF capable of generating tetracyclic diterpenoid compounds from Mangicol H to Mangicol P;
(3) Mycelia of Aspergillus oryzae transformants AO-fomdABCDEF were inoculated and cultured to produce four-ring sesterterpene compounds, mangicol H through Mangicol P.
In one embodiment of the invention, the nucleotide sequences of the primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F/fomdB-R and fomdF-F/fomdF-R are, respectively:
fomdA-F：
AAGCTCCGAATTCGAGCTCGATGGAGTACAGTGAGCTTAGCCTAG
fomdA-R：
GAGCTACTACAGATCCCCGGTCAACAGTTCAGCGTAGATAAGTCC
fomdB-F：TTCGAATCGATTTGAGCTAGATGTCTCAGAAAGGCCCCCA
fomdB-R：
ATCGGGTACGAGGCCGCTAGCTATGCTTCAAATACTGACCCAAAG
fomdC-F：AGCTCCGGAATTCGAGCTCGATGGCACACTACGACTTCAA
fomdC-R：
AGCTACTACAGATCCCCGGCTAGTTAGAAAATGACTCAAACATGG
fomdD-F：CCCCACAGCAAGCTCCGTTAATGGACTTCACTTATCGCTACTCTT
fomdD-R：GTGCATATGATTTAAATTTACTATTCCACACGCAGCATCTCAAGA
fomdE-F：TTCGAATCGATTTGAGCTAGATGGGCAATTTCAGGTTAGATAATG
fomdE-R：GTCACTAGTGCGGCCGCTAGCTACTTGATGGTGACTCGAA
fomdF-F:CCCCACAGCAAGCTCCGTTAATGAAGTTACTCGCTCTGTC
fomdF-R:GTGCATATGATTTAAATTTATCACGCCTGGCACAGAGCTC。
in one embodiment of the present invention, in step (3), the method of inoculating and culturing the mycelium of Aspergillus oryzae transformant AO-fomdABCDEF is: a mycelium of an Aspergillus oryzae transformant AO-fomdABCDEF was inoculated into an MPY medium containing 0.1% adenine, cultured at 30 ℃ and 220rpm for 2 days as a seed solution, inoculated into a rice solid medium containing 0.1% adenine in a ratio of 80g of rice to 120mL of deionized water to 5mL of the seed solution, and cultured at 30 ℃ for 18 days with standing.
In one embodiment of the invention, in the step (3), after the mycelium of the aspergillus oryzae transformant AO-fomdABCDEF is inoculated and cultured, solid fermentation product of the AO-fomdABCDEF is added with equal volume of ethyl acetate for extraction for 3 times, and the extract is obtained after evaporation to dryness; extracting the extract with petroleum ether to obtain components with low polarity, evaporating the petroleum ether extract to dryness, separating the positive phase, eluting with petroleum ether and ethyl acetate as mobile phase, and enriching target components Fr.5 and Fr.7; fr.5, performing gel column separation, and finally eluting by using a reversed-phase Cholester chromatographic column and using a mobile phase of acetonitrile/0.1% formic acid water with the volume ratio of 80; fr.7 gel column separation to obtain three fractions Fr.7.1-Fr.7.3 Fr.7.2 semi-preparative on ACE C18-PFP chromatography column eluting with acetonitrile/0.1% formic acid water volume ratio of 75; fraction fr.7.2.1 was further purified by chromatography on a chiral column Chiralpak IA, in a n-hexane/ethanol volume ratio of 95:5 for mobile phase yielding Mangicols H and I, fr.7.3 for semi-preparative on Phenomenex chromatography column eluting with acetonitrile/0.1% formic acid water volume ratio 80; fraction fr.7.3.1 was further purified by chromatography on chiral column Chiralpak IA, with a volume ratio n-hexane/ethanol of 94: and 6, obtaining Mangicol J and Mangicol M for the mobile phase.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides a tetracyclic sesterterpene compound and a preparation method and application thereof. During the development process, the inventors found a novel gene cluster for the biosynthesis of Mangicols H-P from Fusarium oxysporum 14005 (Fusarium oxysporum 14005) by a genome mining strategy. The expression of the genes in the Mangicols H-P biosynthesis gene cluster in Aspergillus oryzae is realized by means of heterologous expression. Finally, 9 brand-new tetracyclic sesterterpene compounds, namely the tetracyclic sesterterpene compounds Mangicol H to Mangicol P, are obtained through separation and purification.
The innovation point of the invention is mainly embodied in that a genetic engineering method is utilized to construct self-sufficient engineering bacteria for generating new tetracyclic sesterterpene compounds from Mangicol H to Mangicol P. The whole operation process is simple, the process is mature, the cost is low, and the whole process does not contain harmful impurities, is nontoxic and is very environment-friendly. The obtained product Mangicols H-P provides a new resource for the biosynthesis of sesterterpene compounds, provides a choice for obtaining the types of the compounds, and provides a valuable lead compound resource for enriching natural product compound libraries and discovering new antibiotics.
Drawings
FIG. 1 is a UV absorption spectrum of Mangicols H-P of the present invention.
FIG. 2 is a HR-EI-MS spectrum of Mangicol H, a compound of the present invention.
FIG. 3 is a HR-EI-MS spectrum of Mangicol I compound of the present invention.
FIG. 4 is a HR-EI-MS spectrum of Mangicol J, a compound of the present invention.
FIG. 5 is a HR-EI-MS spectrum of Mangicol K, a compound of the present invention.
FIG. 6 is a HR-EI-MS spectrum of Mangicol L, a compound of the present invention.
FIG. 7 is a HR-EI-MS spectrum of Mangicol M, a compound of the present invention.
FIG. 8 is a HR-EI-MS spectrum of Mangicol N, a compound of the present invention.
FIG. 9 is a HR-EI-MS spectrum of Mangicol O, a compound of the present invention.
FIG. 10 is a HR-EI-MS spectrum of Mangicol P, a compound of the present invention.
FIG. 11 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 12 shows that the compound of the present invention, manicol I, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 13 shows that a compound of the present invention, mangicol J, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 14 shows that Manicol K, a compound of the present invention, is soluble in pyridine-d 5 In 1 H-NMR spectrum.
FIG. 15 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 16 shows that the compound of the present invention, manicol M, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 17 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 18 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 19 shows that the compound of the present invention, manicol P, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum.
FIG. 20 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 21 shows that a compound of the present invention, manicol I, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 22 shows that a compound of the present invention, manicol J, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 23 shows that Manicol K, a compound of the invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 24 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 25 shows that Manicol M, a compound of the present invention, dissolves in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 26 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 27 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 28 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum.
FIG. 29 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 HSQC spectrum in (1).
FIG. 30 is the bookCompounds of the invention Mangicol I are soluble in pyridine-d 5 HSQC spectrum in (1).
FIG. 31 shows that Manicol J, a compound of the invention, is soluble in pyridine-d 5 HSQC spectrum in (1).
FIG. 32 shows that Manicol K, a compound of the invention, dissolves in pyridine-d 5 HSQC spectrum in (1).
FIG. 33 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 HSQC spectrum in (1).
FIG. 34 shows that Manicol M, a compound of the invention, dissolves in pyridine-d 5 HSQC spectrum in (1).
FIG. 35 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 HSQC spectrum in (1).
FIG. 36 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 HSQC spectrum in (1).
FIG. 37 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 HSQC spectrum in (1).
FIG. 38 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 In 1 H- 1 H COSY spectrum.
FIG. 39 shows that Manicol I, a compound of the present invention, dissolves in pyridine-d 5 In 1 H- 1 H COSY spectrum.
FIG. 40 shows that a compound of the present invention, mangicol J, is soluble in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum.
FIG. 41 shows that Manicol K of the present invention dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrogram.
FIG. 42 shows that a compound of the present invention, mangicol L, is soluble in pyridine-d 5 In 1 H- 1 H COSY spectrogram.
FIG. 43 shows that Manicol M, a compound of the invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum.
FIG. 44 shows that Manicol N, a compound of the invention, is soluble in pyridine-d 5 In 1 H- 1 H COSY spectrogram.
FIG. 45 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum.
FIG. 46 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H- 1 H COSY spectrogram.
FIG. 47 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1).
FIG. 48 shows that the compound of the present invention, manicol I, is soluble in pyridine-d 5 HMBC spectrum in (1).
FIG. 49 shows that a compound of the present invention, mangicol J, is soluble in pyridine-d 5 HMBC spectrum in (1).
FIG. 50 shows that Manicol K, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1).
FIG. 51 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 HMBC spectrum in (1).
FIG. 52 shows that Manicol M, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1).
FIG. 53 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 HMBC spectrum in (1).
FIG. 54 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1).
FIG. 55 shows that a compound of the present invention, manicol P, is soluble in pyridine-d 5 HMBC spectrum in (1).
FIG. 56 shows that Manicol H, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum of (1).
FIG. 57 shows that Manicol I, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum in (1).
FIG. 58 shows that Manicol J, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum of (1).
FIG. 59 shows that Manicol K, a compound of the invention, is soluble in pyridine-d 5 NOESY spectrum in (1).
FIG. 60 shows that the compound of the present invention, manicol L, is soluble in pyridine-d 5 NOESY spectrum of (1).
FIG. 61 shows that Manicol M, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum of (1).
FIG. 62 shows that the compound of the present invention, mangicol N, is soluble inpyridine-d 5 NOESY spectrum in (1).
FIG. 63 is a drawing showing that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 NOESY spectrum in (1).
FIG. 64 shows that Manicol P, a compound of the invention, is soluble in pyridine-d 5 NOESY spectrum of (1).
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the present invention in any way, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention fall within the scope of the technical solution of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The basic molecular biology experimental techniques such as PCR amplification, plasmid extraction, transformation, etc., which are used in the examples of the present invention, are generally performed according to conventional methods, if not specifically described, and may be specifically described in molecular cloning instruction (third edition) (Sambrook J, russell DW, janssen K, argentine J. Huang Peyer, et al, 2002, beijing, science publishers), or performed according to the instructions provided by the relevant manufacturers.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The synthetic gene cluster of the tetracyclic sesterterpene compound Mangicols provided by the invention is cloned from Fusarium oxysporum 14005, and the gene cluster contains 6 genes which are respectively a gene fomdE encoding a sesterterpene synthase FoMS or a functional equivalent thereof, a gene fomdA encoding cytochrome P450 enzyme, fomdC, a functional equivalent thereof, a gene fomdB encoding an aldone reductase fomdB or a functional equivalent thereof, and a gene fomdF encoding a hydrolase fomdF or a functional equivalent thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.1, the nucleotide sequence of the fomdB is shown as SEQ ID NO.2, the nucleotide sequence of the gene is shown as SEQ ID NO.3, the nucleotide sequence of the gene is shown as SEQ ID NO.4, the nucleotide sequence of the gene is shown as SEQ ID NO.5, the nucleotide sequence of the gene fomdC is shown as SEQ ID NO.5, the nucleotide sequence of the gene is shown as SEQ ID NO.5, and the nucleotide sequence of the gene is identical to that of the gene protein is shown as the gene.
Example 1
Heterologous expression of synthetic genes of tetracyclic diterpenoids Mangicols and structural identification of sesterterpene skeleton compounds are provided.
By using a heterologous expression method, a gene cluster for synthesizing Mangicols H-P in fusarium oxysporum 14005 is transferred into a host aspergillus oryzae by constructing an expression plasmid, and the production condition of a heterologous expression strain product is detected. The media formulations used in this example are shown in Table 1.
TABLE 1 formulation of the culture media used in the examples
Construction of heterologous expression vector of Mangicols H-P Gene Cluster
(1) PCR amplification was carried out using the genome of F.oxysporum 14005 as a template, and primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F/fomdB-R and fomdF-F/fomdF-R, respectively, for the gene foms of diterpene synthase, cytochrome P450 enzyme gene fomdA, fomdC, fomdD, aldoketone reductase gene fomdB, hydrolase gene fomdF.
(2) After the PCR products of the genes fomdE, fomdA and fomdC are purified by a nucleic acid purification kit, the fomdE is integrated into a KpnI digested linear vector pUARA4 by using an Ezmax recombination kit, a ligation product is transformed into Escherichia coli DH10B, and a positive transformant is screened by ampicillin. Liquid culture of positive transformant, extract plasmid PCR to verify, obtain pUARA4-fomdE plasmid; on the basis, the fomdA is integrated into the PacI digested linear vector pUARA4-fomdE by utilizing an Ezmax recombination kit, a ligation product is transformed into escherichia coli DH10B, and a positive transformant is screened by ampicillin. Liquid culture of positive transformant, extracting plasmid PCR verification, and obtaining pUARA4-fomdAE plasmid; and finally, integrating the fomdC into the linear vector pUARA4-fomdAE subjected to NheI enzyme digestion by using an Ezmax recombinant kit, transforming the ligation product into escherichia coli DH10B, and screening positive transformants by ampicillin. And (3) liquid culturing the positive transformant, extracting plasmid PCR verification, and obtaining an expression vector pUARA4-fomdACE plasmid.
(3) Also, after the PCR products of the genes fomdD, fomdB and fomdF were purified by the nucleic acid purification kit in the above manner, fomdD was integrated into KpnI digested linear vector posa 4 using Ezmax recombination kit, the ligation product was transformed into escherichia coli DH10B, and positive transformants were selected by ampicillin. Liquid culture of positive transformant, extracting plasmid PCR verification, obtaining pUSA4-fomdD plasmid; on the basis, the Ezmax recombination kit is used for integrating fomdB into a PacI digested linear vector pUA 4-fomdD, a ligation product is transformed into Escherichia coli DH10B, and a positive transformant is screened through ampicillin. Liquid culture of positive transformant, extracting plasmid PCR verification, obtaining pUSA4-fomdBD plasmid; and finally, integrating the fomdF into the linear vector pUSA4-fomdBD digested by NheI enzyme digestion by using an Ezmax recombinant kit, transforming the ligation product into escherichia coli DH10B, and screening positive transformants by ampicillin. And (3) liquid culturing the positive transformant, extracting plasmid PCR verification, and obtaining an expression vector pUA 4-fomdBDF plasmid.
Primer sequences used in the examples of Table 2
2. Transformation of protoplasts
(1) Aspergillus oryzae A. Oryzae NSAR1 was spread on PDA plates and incubated at 30 ℃ for 7 days.
(2) Collection of the beads in 10mL 0.1% Tween-80 (generally 1 plate of beads need to be collected), and counting with a hemocytometer. Inoculation of about 10 7 The spores were cultured in 50mL DPY at 30 ℃ and 220rpm for 2-3 days.
(3) 100mg of Yatalase was weighed, dissolved by addition of dissolution 0, 20ml was sterilized by filtration through a 0.22 μm filter and added to a 50ml centrifuge tube.
(4) And collecting the thallus. Pouring 100ml of cultured mycelium into a P250 glass filter, removing the culture medium, washing with sterile water (or 0.8M NaCl) for 3-5 times, squeezing out water with a sterile medicine spoon, and adding the pressed dry mycelium into a Yatalase solution. Culturing at 30 deg.C and 200rpm under shaking for 1-2 hr until the spherical mycelium disappears and the supernatant is clear and dirty.
(5) The digested bacterial liquid was filtered through Miracloth filter cloth, the protoplasts were collected and transferred to a new 50ml centrifuge tube, centrifuged at 4 ℃ at 800g for 5 min.
(6) The supernatant was removed, and the mixture was resuspended and washed with 20ml of 0.8M NaCl and centrifuged at 4 ℃ at 800g for 5min (twice washing). The supernatant was removed, and 10ml of 0.8M NaCl was added. The number of protoplasts was counted under a microscope using a bacterial counter. Protoplast number = Total count/80x 400ml x10 4 x dilution factor.
(7) Adjusting protoplast concentration to 2x10 8 cell/ml. (sol 2/sol 3= 4/1), 0.5ml-2ml of protoplast can be harvested according to the growth condition of the thalli.
(8) Mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, and 10. Mu.g of the expression plasmids pUARA4-fomdACE and pUA 4-fomdBDF were added, respectively, and gently mixed. Standing on ice for 20min. During this period, the sterilized Top agar is incubated in a water bath at 50 ℃.
(9) To the suspension of 10, 1ml of sol 3 was added and gently mixed with a tip. Standing at room temperature for 20min. Add 10ml of sol 2 and mix gently.
(10) Centrifugation was carried out at 4 ℃ and 800g for 10min, the supernatant was removed, 1ml of sol 2 was added, the suspension was gently suspended by a pipette gun, and 200. Mu.l was added to the center of the solid screening medium (x 3 plate). 5ml of top agar which is kept at 50 ℃ is rapidly added around the culture dish and mixed evenly. After the plate surface was sufficiently dried, it was wound with parafilm, and incubated at 30 ℃ for 3 to 7 days with the lid facing downward.
(11) Each plate was picked 2-3 clones, 8 in total. And carrying out PCR verification on the grown transformant, wherein the positive transformant is the heterologous expression strain AO-fomdABCDEF of the Mangicols H-P gene cluster.
3. Separation and purification of heterologous expression strain AO-fomdABCDEF expression product
Inoculating a heterologous expression strain AO-fomdABCDEF mycelium into an MPY culture medium containing 0.1% adenine, culturing at 30 ℃ and 220rpm for 2 days to serve as a seed solution, inoculating the seed solution into a rice solid culture medium added with one thousandth adenine according to the proportion of 80g of rice, 120mL of deionized water and 5mL of the seed solution, and standing and culturing at 30 ℃ for 18 days.
Adding the solid fermentation product of AO-fomdABCDEF into equal volume of ethyl acetate for extraction for 3 times, and evaporating to dryness to obtain an extract. Extracting the extract with petroleum ether to obtain low-polarity components, evaporating the petroleum ether extract to dryness, separating the positive phase, eluting with petroleum ether and ethyl acetate as mobile phase, and enriching target components Fr.5 and Fr.7.Fr.5 gel column separation, finally using reversed phase Cholester chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 80 as mobile phase elution to obtain Mangicol K. Fr.7 gel column separation to obtain three fractions Fr.7.1-Fr.7.3 Fr.7.2 semi-preparative on ACE C18-PFP chromatography column eluting with acetonitrile/0.1% formic acid water volume ratio of 75; fraction fr.7.2.1 was further purified by chromatography on chiral column Chiralpak IA, in a n-hexane/ethanol volume ratio of 95: mangicols H and I were obtained for the mobile phase 5. Fr.7.3 by half-preparation on a Phenomenex column eluting with acetonitrile/0.1% formic acid water in volume ratio 80; fraction fr.7.3.1 was further purified by chromatography on a chiral column Chiralpak IA, in a n-hexane/ethanol volume ratio of 94: and 6 obtaining Mangicols J and M for the mobile phase.
Isolated tetracyclic sesquiterpenoids NMR measurements were carried out using Bruker 600MHz ( 1 H 600MHz； 13 C150 MHz), the solvent is deuterated pyridine.
4. Identifying tetracyclic diterpenes Mangicols H-P.
Identifying the obtained tetracyclic sesterterpene Mangicols H-P:
(1) Appearance: was a pale yellow transparent oil.
(2) Solubility: is easily dissolved in methanol and hardly dissolved in water.
(3) Ultraviolet spectrum: the ultraviolet spectrum of the methanol solution of the compound Mangicols H-P has a maximum absorption peak at 210nm, the ultraviolet spectrum is shown in figure 1, and figure 1 is the ultraviolet spectrum of the compound Mangicols H-P of the invention. The ultraviolet spectrum testing instrument is a Mariner System 5304instrument.
(4) Mass spectrum: FIG. 2 is a HR-ESI-MS spectrum of Mangicol H, a compound of the present invention, showing [ M + H [ + ] thereof] + Peak is m/z 357.31519, suggesting that the most probable molecular formula is C 25 H 40 And O. FIG. 3 is a HR-ESI-MS spectrum of Mangicol I, a compound of the present invention, showing [ M + H] + Peak is m/z 357.31519, suggesting that the most probable molecular formula is C 25 H 40 And O. FIG. 4 is a HR-ESI-MS spectrum of Mangicol J, a compound of the present invention, showing [ M-H ] 2 O+H] + The peak is m/z 371.29501, which indicates that the most probable molecular formula is C 25 H 39 O 2 . FIG. 5 is a HR-ESI-MS spectrum of Mangicol K, a compound of the present invention, showing [ M + H [ ]] + The peak is m/z 387.28937, which indicates that the most probable molecular formula is C 25 H 38 O 3 . FIG. 6 is a HR-ESI-MS spectrum of Mangicol L, a compound of the present invention, showing [ M + H [ + ] thereof] + The peak was m/z 389.30502, suggesting that the most probable molecular formula is C 25 H 40 O 3 . FIG. 7 is a HR-ESI-MS spectrum of Mangicol M, a compound of the present invention, showing [ M-H 2 O+H] + The peak is m/z 371.29501, which indicates that the most probable molecular formula is C 25 H 39 O 2 . FIG. 8 is a HR-ESI-MS spectrum of Mangicol N, a compound of the present invention, showing [ M-H 2 O+H] + The peak was m/z 389.30557, suggesting that the most probable molecular formula is C 25 H 41 O 3 . FIG. 9 is a HR-ESI-MS spectrum of Mangicol O, a compound of the present invention, showing [ M-H ] 2 O+H] + The peak is m/z 431.31494, suggesting that the most probable molecular formula is C 27 H 45 O 5 . FIG. 10 is a HR-ESI-MS spectrum of Mangicol P, a compound of the present invention, showing [ M-H ] 2 O+H] + The peak is m/z 507.34674, suggesting that the most probable molecular formula is C 33 H 49 O 5 . HR-ESI-MS spectrum test adopts a Thermal Fisher Orbitrap Q active mass spectrometer and methanol as a solvent.
(5) Nuclear magnetic resonance spectroscopy: FIG. 11 shows a compound of the present invention, mangicol H solutionIn pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 12 shows that the compound of the present invention, manicol I, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 13 shows that Manicol J, a compound of the invention, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 14 shows that Manicol K, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 15 shows that the compound of the present invention, mangicol L, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 16 shows that Manicol M, a compound of the present invention, dissolves in pyridine-d 5 In 1 H-NMR spectrum. FIG. 17 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 In 1 H-NMR spectrum. FIG. 18 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In 1 H-NMR spectrum. FIG. 19 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H-NMR spectrum. FIG. 20 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 21 shows that a compound of the present invention, manicol I, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 22 shows that a compound of the present invention, manicol J, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 23 shows that Manicol K, a compound of the invention, is soluble in pyridine-d 5 In 13 C-NMR spectrum. FIG. 24 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 25 shows that Manicol M, a compound of the present invention, dissolves in pyridine-d 5 In 13 C-NMR spectrum. FIG. 26 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 27 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In (1) 13 C-NMR spectrum. FIG. 28 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 In 13 C-NMR spectrum. FIG. 29 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 HSQC spectrum in (1). FIG. 30 shows that the compound of the present invention, manicol I, is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 31 shows that Manicol J, a compound of the invention, is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 32 shows that a compound of the present invention, mangicol K, is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 33 is a drawing of the inventionCompound Mangicol L is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 34 shows that Manicol M, a compound of the invention, dissolves in pyridine-d 5 HSQC spectrum in (1). FIG. 35 shows that the compound of the present invention, manicol N, is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 36 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 HSQC spectrum in (1). FIG. 37 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 HSQC spectrum in (1). FIG. 38 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 39 shows that Manicol I, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 40 shows that Mangicol J, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 41 shows that Manicol K of the present invention dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 42 shows that a compound of the present invention, mangicol L, is soluble in pyridine-d 5 In 1 H- 1 H COSY spectrum. FIG. 43 dissolution of Manicol M, a compound of the invention, in pyridine-d 5 In 1 H- 1 H COSY spectrogram. FIG. 44 shows that the compound of the present invention, manicol N, is soluble in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 45 shows that Manicol O, a compound of the present invention, dissolves in pyridine-d 5 In (1) 1 H- 1 H COSY spectrum. FIG. 46 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 In (1) 1 H- 1 H COSY spectrogram. FIG. 47 shows that Manicol H, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1). FIG. 48 shows that the compound of the present invention, manicol I, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 49 shows that Manicol J, a compound of the invention, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 50 shows that Manicol K, a compound of the present invention, dissolves in pyridine-d 5 HMBC spectrum in (1). FIG. 51 shows that Manicol L, a compound of the present invention, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 52 shows that a compound of the present invention, manicol M, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 53 shows that Manicol N, a compound of the present invention, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 54 shows that a compound of the present invention, manicol O, is soluble in pyridine-d 5 HMBC spectrum of (1)Figure (a). FIG. 55 shows that Manicol P, a compound of the present invention, is soluble in pyridine-d 5 HMBC spectrum in (1). FIG. 56 shows that Manicol H, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum of (1). FIG. 57 shows that Manicol I, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum in (1). FIG. 58 shows that Manicol J, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum of (1). FIG. 59 shows that Manicol K, a compound of the invention, is soluble in pyridine-d 5 NOESY spectrum of (1). FIG. 60 shows that the compound of the present invention, manicol L, is soluble in pyridine-d 5 NOESY spectrum of (1). FIG. 61 shows that Manicol M, a compound of the invention, dissolves in pyridine-d 5 NOESY spectrum in (1). FIG. 62 shows that the compound of the present invention, manicol N, is soluble in pyridine-d 5 NOESY spectrum of (1). FIG. 63 is a drawing showing that a compound of the present invention, mangicol O, dissolves in pyridine-d 5 NOESY spectrum of (1). FIG. 64 shows that a compound of the present invention, manicol P, is soluble in pyridine-d 5 NOESY spectrum of (1).
The final formula was determined as follows:
TABLE 3 preparation of compounds Mangicols H-J 1 H and 13 assignment of peaks in C-NMR spectra
TABLE 4 preparation of the Compound Mangicols K-M 1 H and 13 assignment of peaks in C-NMR spectra
TABLE 5 preparation of compound Mangicols N-P 1 H and 13 assignment of peaks in C-NMR spectra
NMR of the Compound Mangicols H-P the test was carried out using Bruker 600MHz ( 1 H 600MHz； 13 C150 MHz). The solvent of the compound Mangicols H-P is pyridine-d 5 。
Antibacterial activity test of Mangicols sesterterpene compounds
(1) Antibacterial assay for Bacillus subtilis, staphylococcus aureus, staphylococcus epidermidis, pseudomonas aeruginosa and Streptococcus mutans
The strains tested included: bacillus subtilis strain HD11, staphylococcus aureus strain ATCC 6538, staphylococcus epidermidis strain CGMCC1.1757, pseudomonas aeruginosa strain PA01 and Streptococcus mutans strain ATCC UA159. The inhibition of the growth of the selected bacteria by the compound is determined by a continuous dilution method, and the minimum inhibitory concentration of the compound against different bacterial strains is obtained. The Minimum Inhibitory Concentration (MIC) is the minimum concentration of drug required to inhibit bacterial growth. Vancomycin was selected as a positive control in this experiment.
The test bacteria were cultured in Mueller-Hinton Broth (MHB) medium to logarithmic phase, and the cultured bacteria were diluted to 10 with the medium 4 cfu/mL concentration was seeded in 96-well cell culture plates containing 78 μ L of bacterial suspension per well, 2 μ L added to 2-fold serial dilutions of each compound. The sample and control were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO not being higher than 0.05%. A series of sample concentrations of 4000 to 31.3. Mu.g/mL were obtained by serial dilution, and incubated aerobically at 37 ℃ for 16 hours. The absorbance before and after the culture is measured by a microplate reader at 600nm, the Minimum Inhibitory Concentration (MIC) value is calculated according to the change of the absorbance, and all the experiments are carried out in parallel for 3 times.
The results of the experiment are shown in table 7:
TABLE 7 in vitro antibacterial Activity test results for the Compound Mangicols H-P
In vitro antibacterial activity studies show that compound I has strong inhibitory activity against streptococcus mutans (MIC =6.25 μ g/mL), and compounds K and L also have certain inhibitory activity against streptococcus mutans (MIC =12.5 μ g/mL).
(2) Antifungal assay
The strains tested included: candida albicans strain SC 5314. The inhibition of the growth of the selected bacteria by the compound is determined by a continuous dilution method, and the minimum inhibitory concentration of the compound against different bacterial strains is obtained. The Minimum Inhibitory Concentration (MIC) is the minimum concentration of drug required to inhibit bacterial growth. In the experiment, amphotericin B is selected as a positive control drug, and only DMSO treatment is set as a negative control.
Single colony of Candida albicans strain SC 5314 was suspended in RPMI 1640 at a concentration of 1X 10 4 cfu/mL were seeded in 96-well cell culture plates containing 78. Mu.L of fungal suspension per well, 2. Mu.L of each compound added in 2-fold serial dilutions. The sample and control were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of no more than 0.05%. A series of sample concentrations of 4000 to 31.3. Mu.g/mL were obtained by serial dilution and the plates were incubated at 35 ℃ for 16 hours. The absorbance before and after the culture is measured by a microplate reader at 600nm, the Minimum Inhibitory Concentration (MIC) value is calculated according to the change of the absorbance, and all the experiments are carried out in parallel for 3 times.
The results of the experiment are shown in table 8:
TABLE 8 in vitro antifungal Activity test results for the Compound Mangicols H-P
| Cpd.NO. | C.albicans(MIC,μg/mL) |
| H | ≥50 |
| I | ≥50 |
| J | ≥50 |
| K | ≥50 |
| L | ≥50 |
| M | ≥50 |
| N | ≥50 |
| O | ≥50 |
| P | ≥50 |
The above compounds have no obvious inhibition effect on Candida albicans (MIC value is more than or equal to 50 mg/L) by taking DMSO and amphotericin B as controls.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
The sequence listing involved in the present invention is as follows
SEQ ID NO.1: nucleotide sequence of fomdA.
atggagtacagtgagcttagcctagtgactgacccgtcaaaatacggcctcatccccattggcgtggtcatcctcaccctggccattggatcatgtttgctagcccaacactccgagccaccagtgcttaacccagtgaggacattcgaagtcatcaatcttccacgactctaccgcttcatgcgatacagcctggatattctaaatcaaggcgcccagagatttccctatcggccatacaggctcttctgcgaatggggcgaattggtcatcctacctcctgaatatattgacgagatcaagaatgacaagaggttcgattttggagtcgcagccagtgatgtaggtctacggcccgaaggaatagggcttctataagttgactaatggactcaggataatcatgcttatatacctgggtttaatgctcttttacatgatcctttcatgtcaaagattatatcgcgacaactgacccaggccctaggttcgttctgctgagattcgaacccgtctcttcatggttaacaatcaggaacaggcaggctcacggctccattatccgatgaagctgccatggtcatgaaagccgtcattacagactcaaacggtatgtgtcttttcctctgcggaggagtgatgatacgtactttggcaaggaagctaattgttttgcacccagaatggcacgtcataaacctgagagaagatgttgcaaatatcgtaactcgaatgtcctccagagtcttcatgggagaggagctttgtcacgacgaaggatggaacaaagcacaagcacactataccatgaagacattccaagccatgatggtcctttgcatggtacctcggtggcttcgaccttacatccattggatcctgcccccatgttggatagttcgcagatcgctggctgccgcccggaaacaactagctccacacattgcacgtcgtcaagagatcaagactgcggcacttgctcggggagaaaagtctccctttgatgacacgattgaatggttctcacagagtggttctcaagtacccccagcagattgccagatatcgctttctctagctgctattcatacgactaccgacttgctctctcaagtcatgattgatattgccgcatacccagtactcttcgcggagatgcgggaggaggtcatccgcgtcctttcagcgtcaggcctcaccaagactgccctttctaatctcaagttaatggatagtgtcgtaaaagagtcacagagactaaagccaatactgctaggtaagaataattgggccatacgtatttggtatttccactgacccatgccagcctggagaagacaggcccttgctgatgccactctatccaacggtatcaaaatcaagcaaggccaaaaggtagctgtcacgtcaacccatatgtggagcaatgaaaactacgacatggcaaaggagttcaacccctatcgcttcatatcagatacggagaagacatcgtctctagtcagcacaagcccgaatcatctggggtttggtcatggcatgcatgcttgcccaggacgcttctttgctgctaatgaggttaagattgctctatgtcacctacttctcaagtatgactggaaatttaaagacgataagaagccagagcctgtggcgtttggcatggcgtatgtcgctaacccattcgcaaaattgatgattagaagaagagaggaggagatggacttatctacgctgaactgttga
SEQ ID NO.2: nucleotide sequence of fomdB.
atgtctcagaaaggcccccaaattatctttggtacagcgtcaatcggctacggctctcttgccgaagaggatggcgtccaaaaatttctccaagtagcttcagagaatggcatcaagcatcttgacacagctgcaagctaccccactgaggaagccactggcattactgagatccttctcggaaaggcaaagtccggcgcctggacgatagacactagagttgctatcagccgtgatctcagaggagcattgactcctcaagccgttgagaagagtctcgatattagtctggagaagctcaatcaagactcggtcaacattctttcagctcatatgccagaccctgctacacctctggaggatcaggctcgtgcttttgacgaccagttcaagaagggcaagttcaaaaaggtatgtaacacgctgaataggtcgccttgtattatgattcgggctgattcattccacagttgggtctatgcaactggcctctcagcatgatagagaagtacatcgagatctgcgatgataagggctatgtcaaaccaactgttgcacagtacatgtacaactatatctggcgaggaacagacaccaaaatattgtcatttctccgtgaccatgacatggtgctccatgcctacagtccacttgcaggaggcttcctgactgggaagccaactgatggcgatacagctggtacacgttacggggagcaaacccatcctctgactatctacaagtttatctacgacaaggagccttttcacaaggctgtgaaggatctttctggccttgtagctagcggagcggttgatggtcctgctgacgtggccattcggtggttatactaccattctaggcttcagccacaggatgccattattcttggaggttcgaccgttgaacactttgagagaaacatggccatcatcaagaggggacccctctcggaggaggtactcgaggctctcaaggggctggaagttgatgaggccggaatgggagatggcaatgtctttgggtcagtatttgaagcatag
SEQ ID NO.3: nucleotide sequence of fomdC.
atggcacactacgacttcaacatggggcttcagagcttcgcctcgaggtttgattcctccaagattgcccctcttgtgctcatcagcgcaggtctcgtaagtctgtcctctggttcgatacaatgactacagggtcgctaactactgtgaaagcttgtggtgtcccttactgcaactgctatctaccgtatttggtttcaccccctgtccatattccccggcccaaaataccttgccattaccaatgtacccgaacgatggatgagcaatatctctggtacatggatttggaaggtctcaggcctgcaccgcacgtacggccctattgtccgcattggccccaaccgtcttgctatggatggcagtatcggctggttccaagtctacgccatgcgaggcaaagatgacgagttccccaagtatcccgagtacatcttccccggcgacggcctgagcatcctgggagccaatcaggtaaaccatcgtcgccaccggcggcaattctggtccgcgttcaatgaccaggcccttgttgagcaagaaattgtcatccaaccgtacacagatatgctcttacagcgcttgagcgagagagccaaggaaggaaagccaatcaacatagtcgactggatcaactttctcctctttgacattgcaggggagcttgtgttttcccagccctttgattgtctcgataagcaggaataccatccatgggtatccaactttttcagggctgtcaaaggaaatgctgtcaaccgattcgtcaagcactaccccatcacgtcgccccttatcaacttcttgttcacgggcaaggagcagatccagagggaagcggaccagaggaacatgacctttcatcatgcaatgcaacgcatgaagctgggagaacagccgacccctgggcgtcgtgattttatgagcttccttatgcggaggaatcgtgatggaggaggtcttacagaccccgagattctcgtcgattgtcctgtcttgattggtgcgtcgagtgagaccaccacaactgccttatctggcttcttcttctatcttggatcttcgccacaagcatatcaaagacttgttgaggaagtccgatcctctttcaaggcggagagcgagatcgacatgaaaaccactaagaggcttgagtacttgaacgccactgttgatgaagcattacgtgtctatcccccagcagccgagtcaccacccagaatcagccctggtgcagaagttgaaggcaaatacattccgaaaggcgcaagtcactttttcaaccccatgtttgagtcattttctaactag
SEQ ID NO.4: nucleotide sequence of fomdD.
atgggcaatttcaggttagataatgtcaatatcagcattcgtcaatggcttccaagtgagatgccacagagcggaaactggaagctttttgcgctggcaagcattgtcttcgttatttttcgcttctccatcatcgttacctaccggcttttcttctctcctctggccaaattccctggtccaaaggtcgctgcctcaactcacctttatgagtcttactatgactattggaagcaaggccaatattacaaagtcattcagcgcatgcatgagaagtatggtatgttcaagttgccccctcacggccgtgattgtttacgagcagctttctaggtcctatcgtacgcgtcacgcctgatgaactgtcaatcaacgaccccgacttttacgacaccgtctacgttaacggtaacgttcgaaaaaccgagtcctttggacattcattcggtggtggacttggcattgaaggttagttctctctcgcataatactaccgctgcttttcattaatgccctcagataccttcttcgcttccaaggaccatgacctccaccgcaaaagaagaaagccgattgaaccttacttctctcgtagcggtgtcttgaagcttgaagaattaattggggagcgggtcgagaaattattccacagattccatgagctctctggcactggtgttgtagttcgtctcgactatgcttttgaggcctttactggagatgtcatgcaacatatttgcattgagaagcctaagtcgcttcttgcaaccgatgactttggtgagaaatggtatgacatatccttcacgtcgctgtctggaggctgaaattgtgattcataggtttgagatgcttcgaaacgtctcattgagcgtaccgttgatgggcatgctgccatggttggttcagtaagtaatttatttatcatgtactgaaataatgctaagaagtatcagtatactccagtttatccctgagcgctgcattatctggtttgccccatcaacagcttatttccagaactttcgaatcgtatgtaccatcaccatccttcccatacagtacaacagcttagtatataatagcaagcgggtcgtcagattgagcaggccaaacaggagaagttcgagaatgatcgcaaaggcgtcaagtctgttggcggcaaaccaaccctctttcgctacctcattcacgaaagtgacctggcgcctcaagatctcagcacagagagactccagaaggaggcaatggtgcttcttggcggcggaaccactactaccgctcggacagctacaatgacctgtttctggatgctcagcatgccagaaaagggccaacgtcttcgtgatgagctcaggcccgttatggagggatacccgcacaagaagccatctcttacggagcttgagcagcttccttacttgggagctgtcatcaaagaaagtctgaggtacagttggtcctgatccctgtaatcgtcagggggtactaacaaatcacgttagaatggcatatgggtcaatgcgtcgactccctcgcacttcgcccgacacggccctacaatttagagattggacaatcccgcctggcgtaagtacaagtccactcctccttggaacccgtgagcgctgaaatatctttataaccagacacctgttggaatgaatgcttactatctgcatactgatccaattgcgtttcctcagccttttgagtataaacctgagcgatggctcgaaaacgtcacgccagaaatgaagcgcagctttgttccgttctctcgaggttcacgacgatgccctggctccaggtatgttattttctgtcacactgcttcgagaagttgaactgattgctctggtagtttggctcttgctgatctccattttgtccttgccgctttatttggcccttctgggcctaagttcgagcttttcaattcagacagatctgatgtggatgcggttcacgattatttaatgcctcttcctcgtcttgactccaagggagttcgagtcaccatcaagtag
SEQ ID No.5: nucleotide sequence of fomdF.
atgaagttactcgctctgtcatttgccatggtggcctttgtggcctccaagacagtccctttgccccccccgacggctgatactctcattagtcaagctttggtcgcgctcggcggcgagaaggcaattgctggcatcgaaggaatcacctatcactctccaaagtaggtccttcacttacgtccgtcagttgaccactctctgaaccatccagtgtctatcattcccggagtcttatgcagagctacaacctggacaaagcagacacagctgtagcaatttccggatcccagaatatatccttcagctatgcttcggatcagttaactcagcgaattgatcgtgttttcaagcctagtggtatgtaatattatctccatctctagtgtgacgaaaggcttacgccgtatgagagtactggtattgggcatctgctagacttgatgagttcgactactctctcgttgttcgcggcggcaaagatggattcgcttgctatgttcgtggaaatagtcagatttggctccctgcaaatcttacatccggatacgttgatggtaagttgagacttcttttcctgctcctagagtcgggtgatgactgagtcactgttctagctgcccttgcagaatttctcgtcctgcaaggaaacgtcttttctccaaagcttctgttggaggtacgtagtagacattctgttggtaatcatcaactgcagagtttgctgacatttccagatgaaaaatcatcatggtaccgaggctattgaggttttgatcaatggaatcaaaacacctgctggtgggttaccagcctcgaacctggtcatctcgatctaacctagatagtttatgatcccattcttgaaatcaccatggtcttcgatgcgtctagccatctgcctcacatcatacgcaccgacgagaatcatatgatctatggaccttcgacaaacgacctttacgtttctcaatacaaagccatcgagggcatcaaatttcctcacacgtttcagaccatctataactccactacgcagaagctagatgcaacactcgaagagttcatcgtcgaagaaatcaccattaatcccaggtttcccaaggactacttcagtggcctgtcggaaagggaaggattcttcccgaaagaggcacccaagaggactgagggtctcagtcatgcgcacattctagaatttagcagcaacatgctttggtctggtcctgggtccggcattagcaacaactctgtcagcagtatcaagcacaagaacatcgtccccggtctgcccaatgctcattggctcatcgtcaatgacgagtttctaggagttaagcaattcgtgatcgagttcgaggaccacgtcatcgtcggtgatgcacctccccagtggaccaagcaagtaatcgaatggattgacaagaaaattggcaagcctattaaatacctttgggtaagcacccaagatttagtatgaatcaagctttggtgatgagaaactaactctcctgcaatagcctacccaccatcaccgtgatcacagtggaggagctgctgagtatgtccaaattggggcgaagttgattatccccgaaattgcaacgagctactggtcaagcattcccggtgccgagctcatcaccttcaagtatgatttttatgcatcaatcaactaaaatataccatctaatcatctactagcgaaacgcacccctacatccatagcgataacaagcaccaggcctggttcatctgggaggagcaagccacacattccatcgactggtcatatgcatttatcactaacaagtgccccaccaacgaatcaggtattgctatcattgaggctgacgcatggcaccctggtatgcctgatgccaacaatgacaggtgggaaatgagagagtggctcggtcagcttgatagggatggtgttcctgaaagcgcctagtaagtacttcccagtacaagatacaaaaacgaaagcaaagaactgacgaggttccagtgttctgccaacccatggtcagatcagtcaagtctcggaactcattgagcatactgactatgtctatgccgcaagaacaattggtgactggaagaatggaggagctctgtgccaggcgtga
SEQ ID No.6: nucleotide sequence of fomdE.
atggacttcacttatcgctactcttttgagcccacagactatgacactgatggtctttgtgatggtgtccctatccgcaagcataagggcttagaccttgacgaagttgccatttacaaagcccagtatgactgggaagaacacgtgggacccaagctgcctttcaggggtggcatgggcccagtgcataactttatctgcctcacactgcctgagtgtctgcccgagagactcgagatcgtctcatatgccaacgaatttgcattccttcatgatgatatcacagatgtggagtcagcagagacggcaagatataccatagttgacttccacgtttgagagctgaccgtgtataggtggcggctgagaacgatgagttccttaatgccctgcagcaaggtatccgagacggggacatcgaaagtcgtaattctggaaaacgacaccttcaagcttttatcttcaagcaaatggcagccatagatcgaccgcgtgcactagcagccatgaatgcgtgggccacctttgtcaatacaggtgctgggtgcgcccacgacacaaacttcaagacacttgatgaataccttgactatagatcgactgatgttggttatatgtgagttccgtccctaacgatatcaggaacctgttaataagggtcaggttctggcacgctctaatcatctttggatgtgccatcacaatccccgatgatgagatcaacctgtgccaccaactggctctcccagcaatcatgtccgtaaccttgaccaacgacatttggtcttacggcaaagaggctgcggcagcggagaaagctgggaagcctggtgactttgtcaatgctcttgttgttcttcaaagggagcataactgttcgcttcaagaggctgagcgtctctgcagagcacgcaataaaattgaagtcgccaactgtcttcgagtcaccaaggagacgcgagaaagaaacgatgtttctcaagatctcaaagactacctctaccacatgctctttggtgtcagtggaaatgctatctggagtactcagtgccgcagatatgacatgaacgctccgtacaacgagcgtcagcaagccaggctgctgcagacaaaggagcagctcgtctcctcttatgatccggttcaggctgccaaagaagccgctatgaagcaggaatttgagttacacagacttcctacgcctgacagccctggcatcgaagagctttctgtcaagccaatcttgaacaagaacgaccagagtggcgacgagaggccttttgaatcgagtggtggttctgagacctctggacctactactagtgacaactcggctgaaaattgtcatgatgattcttccactacgtacagcgctgaggacacaggcctcaaatccgaaggcagtggcattgaatggactgttcctggattgcctcacactgcgcacaagaaaacatggactgatgcttgtggccagaacctcccacctatggaggacgatatcgtcatggagccctatcgatacctgtgctccttgccttccaagggagttcgaaataagacaatcgatgctctcaatttctggctcaaggttcctgctgaccgagtcaacaccatcaaggccattaccgaaagccttcatgcgtcatcactcatgcttgacgacattgaggatcactctcagctgcgacgcggcaagccatcggctcacgctgtctttggtgaagctcagactattaactcggcgacctaccagtatatccagtctgtcaaccaactgaaccagctcaagagccccaaggcattgggaatttttgtagatgagatcagacagctattcatcgggcaggcatacgagcttcagtggacgtccaccatgatctgtccatcgatagaagagtatcttcaaatggttgatggtaagactggtggtttgttccgcctgctgacgcgactcatgacggccgagtctttggtagatgacgaagtcgacttcactcgcttatgtcaactgtttggtcgatatttccagattcgcgacgactatgccaatctcaaactcgctgatgtgagtttctgttcgcagtcctagctatatgaatgctaactaattcaacagtacactcaacagaagggattctgcgaggatctggacgagggtaaattctcactacccctcgtcattatcttcaatgaacccagcaggtcgcccaacgcaacagctcagctgaggaatctgctggttcagcgctgcatcaacaagagcatgacctttgagcagaagatcttggcgttagatcttatcgaggaggctggtggctttgtggagactgaaaaggtactgcactcgctatacaaagagctagaattagaactgcagagtctgtgtggtgttttcggtgttgagaaccatcagcttgggcttattcttgagatgctgcgtgtggaatag
Claims (10)
1. The tetracyclic sesterterpene compound is characterized by being selected from one of Mangicol H to Mangicol P, and the structural formulas of the Mangicol H to the Mangicol P are respectively shown as follows:
2. the biosynthetic gene cluster fomd of the tetracyclic sesterterpene compound of claim 1, comprising 6 genes, namely the gene fomdE encoding sesterterpene synthase FoMS or a functional equivalent thereof, the genes fomdA, fomdC, fomdD or functional equivalents thereof encoding cytochrome P450 enzymes fomdA, fomdB, fomdD or functional equivalents thereof, the gene fomdB or functional equivalents thereof encoding aldone reductase fomdB, the gene fomdF or functional equivalents thereof encoding hydrolase fomdF, wherein the nucleotide sequence of the fomdA is shown in SEQ ID No.1, the nucleotide sequence of the fomdB is shown in SEQ ID No.2, the nucleotide sequence of the fomdC is shown in SEQ ID No.3, the nucleotide sequence of the fomdD is shown in SEQ ID No.4, the nucleotide sequence of the mdf is shown in SEQ ID No.5, and the nucleotide sequence of the mfode is shown in SEQ ID No. 6;
alternatively, the nucleotide sequences of the 6 genes are DNA coding sequences corresponding to more than 80% identity with the amino acid sequences encoding the proteins fomdA, fomdB, fomdC, fomdD, fomdF and FoMS, respectively.
3. The use of the biosynthetic gene cluster fomd of the tetracyclic sesterterpene compound of claim 2 in the preparation of terpenoids.
4. The use of the biosynthetic gene cluster fomd of the tetracyclic sesterterpenoids according to claim 3 for the preparation of terpenoids, characterized in that the biosynthetic gene cluster fomd of the tetracyclic sesterterpenoids Mangicol H to Mangicol P is used for the synthesis of tetracyclic sesterterpenoids Mangicol H to Mangicol P.
5. The vector for expressing the biosynthetic gene cluster fomd of the tetracyclic sesterterpenoids Mangicol H to Mangicol P according to claim 2, which is a eukaryotic or prokaryotic expression vector containing the genes fomdA, fomdB, fomdC, fomdD, fomdF and fomdE.
6. The method for producing tetracyclic sesterterpenoids according to claim 1, wherein the tetracyclic sesterterpenoids Mangicol H to Mangicol P are obtained by expressing genes in the biosynthetic gene cluster fomd of tetracyclic sesterterpenoids according to claim 2 in Fusarium oxysporum 14005 by heterologous expression in Aspergillus oryzae NSAR 1.
7. The method for preparing tetracyclic sesterterpenes according to claim 6, wherein said method for preparing tetracyclic sesterterpenes comprises the steps of:
(1) PCR amplification is carried out by taking the genome of Fusarium oxysporum 14005 as a template and primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F/fomdB-R and fomdF-F/fomdF-R as well as genes fomdE, cytochrome P450 enzyme genes fomdA, fomdC, fomdD, aldehyde ketone reductase genes fomdB and hydrolase genes fomdF respectively for diterpene synthase to obtain PCR products of the genes fomdE, fomdA, fomdC, fomdD and fomdF; then, the Aspergillus oryzae NSAR1 expression vector pUARA4 is used as a vector to construct a co-expression vector pUARA4-fomdACE of fomdE, fomdA and fomdC; constructing a co-expression vector pUSA 4-fomdDF of fomdD, fomdB and fomdF by taking an Aspergillus oryzae NSAR1 expression vector pUSA4 as a vector;
(2) Under the mediation of PEG solvent, co-expression vectors pUARA4-fomdACE and pUA 4-fomdBDDF are co-transformed into protoplasts of a high-yield host Aspergillus oryzae A.oryzae NSAR1 which is easy to express terpene synthase genes, so as to obtain Aspergillus oryzae transformants AO-fomdABCDEF which can generate four-ring sesterterpene compounds Mangicol H to Mangicol P;
(3) Mycelia of the Aspergillus oryzae transformant AO-fomdABCDEF were inoculated and cultured to produce four-ring sesterterpene compounds, mangicol H through Mangicol P.
8. The method for preparing tetracyclic sesterterpenoids according to claim 7,
the nucleotide sequences of the primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F/fomdB-R and fomdF-F/fomdF-R are respectively:
fomdA-F：
AAGCTCCGAATTCGAGCTCGATGGAGTACAGTGAGCTTAGCCTAG
fomdA-R：
GAGCTACTACAGATCCCCGGTCAACAGTTCAGCGTAGATAAGTCC
fomdB-F：TTCGAATCGATTTGAGCTAGATGTCTCAGAAAGGCCCCCA
fomdB-R：
ATCGGGTACGAGGCCGCTAGCTATGCTTCAAATACTGACCCAAAG
fomdC-F：AGCTCCGGAATTCGAGCTCGATGGCACACTACGACTTCAA
fomdC-R：
AGCTACTACAGATCCCCGGCTAGTTAGAAAATGACTCAAACATGG
fomdD-F：CCCCACAGCAAGCTCCGTTAATGGACTTCACTTATCGCTACTCTT
fomdD-R：GTGCATATGATTTAAATTTACTATTCCACACGCAGCATCTCAAGA
fomdE-F：TTCGAATCGATTTGAGCTAGATGGGCAATTTCAGGTTAGATAATG
fomdE-R：GTCACTAGTGCGGCCGCTAGCTACTTGATGGTGACTCGAA
fomdF-F:CCCCACAGCAAGCTCCGTTAATGAAGTTACTCGCTCTGTC
fomdF-R:GTGCATATGATTTAAATTTATCACGCCTGGCACAGAGCTC。
9. the method for preparing tetracyclic sesterterpenoids according to claim 7, wherein the mycelium of Aspergillus oryzae transformant AO-fomdABCDEF is inoculated and cultured in step (3) by: a mycelium of an Aspergillus oryzae transformant AO-fomdABCDEF was inoculated into an MPY medium containing 0.1% adenine, cultured at 30 ℃ and 220rpm for 2 days as a seed solution, inoculated into a rice solid medium containing 0.1% adenine in a ratio of 80g of rice to 120mL of deionized water to 5mL of the seed solution, and cultured at 30 ℃ for 18 days with standing.
10. The method for preparing tetracyclic sesterterpenoids according to claim 7, wherein in step (3), after the mycelium of Aspergillus oryzae transformant AO-fomdABCDEF is inoculated and cultured, the solid fermentation product of AO-fomdABCDEF is added with equal volume of ethyl acetate for extraction for 3 times, and is evaporated to dryness to obtain an extract; extracting the extract with petroleum ether to obtain components with low polarity, evaporating the petroleum ether extract to dryness, separating the positive phase from the negative phase, eluting with petroleum ether and ethyl acetate as mobile phase, and enriching target components Fr.5 and Fr.7; fr.5, performing gel column separation, and finally eluting by using a reversed-phase Cholester chromatographic column and using a mobile phase of acetonitrile/0.1% formic acid water with the volume ratio of 80; fr.7 gel column separation to obtain three fractions Fr.7.1-Fr.7.3 Fr.7.2 semi-preparative on ACE C18-PFP chromatography column eluting with acetonitrile/0.1% formic acid water volume ratio of 75; fraction fr.7.2.1 was further purified by chromatography on chiral column Chiralpak IA, in a n-hexane/ethanol volume ratio of 95:5 for mobile phase yielding Mangicols H and I, fr.7.3 for semi-preparative on Phenomenex chromatography column eluting with acetonitrile/0.1% formic acid water volume ratio 80; fraction fr.7.3.1 was further purified by chromatography on chiral column Chiralpak IA, with a volume ratio n-hexane/ethanol of 94: and 6, obtaining Mangicol J and Mangicol M for the mobile phase.
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