阅读说明：本技术 一类四环二萜类化合物及其制备方法与应用 (Tetracyclic diterpenoid compounds and preparation method and application thereof ) 是由 李盛英 李众 于 2019-11-05 设计创作，主要内容包括：本发明公开了一类四环二萜类化合物,包含化学分子式为C<Sub>20</Sub>H<Sub>32</Sub>的委内瑞拉烯A和化学分子式为C<Sub>20</Sub>H<Sub>30</Sub>O的委内瑞拉烯B；该化合物通过委内瑞拉烯A与B的生物合成基因簇的自体沉默基因簇激活和异源表达该基因簇的方式,实现了委内瑞拉烯A和B生物合成基因簇中的基因在链霉菌及大肠杆菌中的表达,整个操作过程简单,工艺成熟,成本低廉,环境友好。本发明获得的产物委内瑞拉烯A和委内瑞拉烯B有望应用到食品、香料、医药化工等领域。(The invention discloses a tetracyclic diterpenoid compound which contains a chemical molecular formula of C 20 H 32 The venezuelan A and the chemical formula of C 20 H 30 Venezuelan B of O; the compound is prepared by reacting venezuelan A with venezuleneThe mode of activating the self-silencing gene cluster of the biosynthesis gene cluster B and heterogeneously expressing the gene cluster realizes the expression of genes in the venezuelan A and B biosynthesis gene clusters in streptomycete and escherichia coli, and the whole operation process is simple, mature in process, low in cost and environment-friendly. The venezuelan A and venezuelan B products obtained by the method are expected to be applied to the fields of food, spice, pharmaceutical chemicals and the like.)

1. A tetracyclic diterpenoid compound characterized by: the compound is a compound containing a 5-5-6-7 tetracyclic diterpene framework, and the chemical molecular formula of the compound is C₂₀H₃₂The structural formula is shown as formula (1) and is named as: venezuelane a (venezuelaene a);

2. a tetracyclic diterpenoid compound characterized by: the compound is a compound containing a 5-5-6-7 tetracyclic diterpene framework, and the chemical molecular formula of the compound is C₂₀H₃₀O, the structural formula is shown as formula (2), and the name is: venezuelane b (venezuelaene b);

3. a process for the preparation of tetracyclic diterpenoids according to claims 1 or 2, characterized in that: the venezuelae A or the venezuelae B is obtained by expressing genes in a biosynthetic gene cluster of venezuelae in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a method of self-silencing gene activation.

4. A process for the preparation of tetracyclic diterpenoids according to claims 1 or 2, characterized in that: the venezuelae A or the venezuelae B is obtained by expressing genes in a biosynthetic gene cluster of venezuelae in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a method of heterologous expression in Escherichia coli.

5. A Venezuelene biosynthetic gene cluster according to claim 3 or 4, characterized in that: the gene cluster contains 4 genes, namely a gene venA for coding diterpene synthase VenA or a functional equivalent thereof, 2 genes venB and venC for coding cytochrome P450 enzymes VenB and VenC or functional equivalents thereof, and a gene venD for coding geranylgeranyl pyrophosphate synthase VenD or functional equivalents thereof; wherein the nucleotide sequence of the venA is shown as SEQ ID NO: 1, the nucleotide sequence of venB is shown as SEQ ID NO: 2, the nucleotide sequence of venC is shown as SEQ ID NO: 3, the nucleotide sequence of venD is shown as SEQ ID NO: 4 is shown in the specification; or the nucleotide sequence of the gene is a corresponding DNA coding sequence with the amino acid sequence consistency of more than 80 percent with the coding proteins VenA, VenB, VenC and VenD respectively.

6. The method for preparing the tetracyclic diterpenoid compound according to claim 1, comprising the steps of:

(1) using a genome of S.venezuelae ATCC15439 as a template, and respectively carrying out PCR amplification on a gene venA of diterpene synthase, a gene venA of geranylgeranyl pyrophosphate synthase and a gene venC of cytochrome P450 enzyme by using primers venA-F/venA-R, venD-F/venD-R and pDR4venC-F/pDR4venC-R to obtain PCR products of the genes venA, nAD and venC; then, constructing a co-expression vector pET28b-venAD of venA and venD by taking an escherichia coli expression vector pET28b or a functional equivalent thereof as a vector; wherein the nucleotide sequences of the primers are respectively:

VenA-F：CCTGGTGCCGCGCGGCAGCCATATGCAGCAACGCCTCCGCCCG

VenA-R：GTCCACCAGTCATGCTAGCCATATGTCAAACCAGCGGTCGGGTGGG

VenD-F：CCTGGTGCCGCGCGGCAGCCATATGACCCAGGCGACACTGTC

VenD-R：GTCCACCAGTCATGCTAGCCATATGTCATCCGTCGCGGTGGCTCA

pDR4VenC-F：CGTGCAGGACTGGGGGAGTTACTAGTATGACCTGGGCCGCTGCGGG

pDR4VenC-R:ATGATTACGAATTCGAGCTCGGTACCTCATGACGCCACCGCCCGGG

(2) converting the co-expression vector pET28b-venAD into the chemical competence of Escherichia coli engineering bacteria Eco-P (Escherichia coli BL21(DE3)/pACYC-mavEmavS & pTrc-low) which can produce dimethylallyl pyrophosphate and isopentenyl pyrophosphate at high yield to obtain Escherichia coli engineering bacteria Eco-P/pET28b-venAD which can produce Venezuelaene A (venezuelane A);

(3) transferring the engineering bacteria Eco-P/pET28b-venAD seed liquid of escherichia coli into TB culture medium containing kanamycin, chloramphenicol and ampicillin according to the volume ratio of 1-2: 100, culturing at the temperature of 37 +/-2 ℃ and the rpm of 220 +/-20, and culturing when OD is obtained₆₀₀When reaching 1.0-1.5, isopropyl- β -D-thiogalactoside with a final concentration of 0.1-1.0mM is added, protein expression is induced and venezuelan A is biosynthesized under the conditions of 18 +/-2 ℃ and 220 +/-20 rpm.

7. The method for preparing the tetracyclic diterpenoid compound according to claim 2, comprising the steps of:

(1) carrying out PCR amplification on genes venA of diterpene synthase, venD pyrophosphate synthase gene venD and cytochrome P450 enzyme gene venC by respectively using primers venA-F/venA-R, venD-F/venD-R and pDR4venC-F/pDR4venC-R by using an S.venezuelae ATCC15439 genome as a template to obtain PCR products of the genes venA, venD and venC; then, by taking a streptomycete integrated vector pDR4-kasOp or a functional equivalent thereof as a vector, integrating the venC into SpeI and KpnI double-restriction enzyme linearized pDR4-kasOp to obtain an expression vector pDR 4-kasOp-venC; wherein the nucleotide sequences of the primers are respectively:

VenA-F：CCTGGTGCCGCGCGGCAGCCATATGCAGCAACGCCTCCGCCCG

VenA-R：GTCCACCAGTCATGCTAGCCATATGTCAAACCAGCGGTCGGGTGGG

VenD-F：CCTGGTGCCGCGCGGCAGCCATATGACCCAGGCGACACTGTC

VenD-R：GTCCACCAGTCATGCTAGCCATATGTCATCCGTCGCGGTGGCTCA

pDR4VenC-F：CGTGCAGGACTGGGGGAGTTACTAGTATGACCTGGGCCGCTGCGGG

pDR4VenC-R:ATGATTACGAATTCGAGCTCGGTACCTCATGACGCCACCGCCCGGG

(2) the expression vector pDR 4-kasOp-venC was transformed into commercial Escherichia coli Et12567/pUZ8002 to obtain Escherichia coli engineering bacteria Et12567/pUZ8002&pDR 4-kasOp-venC; mixing spore suspension of S.venezuelane ATCC15439 wild type with Escherichia coli engineering bacteria Et12567/pUZ8002&pDR 4-kasOp-venC, and uniformly spread on MgCl-containing glass₂And CaCl₂Culturing on MS plates at 30 +/-2 ℃ for 16 +/-1 h, covering each MS plate with sterile water in which hygromycin and nalidixic acid are dissolved, drying, culturing at 30 +/-2 ℃ again, picking a single colony to an MS culture medium containing nalidixic acid and hygromycin after 3-5 days of colony growth, sporulating for 3 +/-1 days, inoculating to 2 XYT culture solution, culturing at 28 +/-1 ℃ and 220 +/-10 rpm for 2 days, extracting a genome, and verifying that the genotype is correct through PCR (polymerase chain reaction), namely the recombinant strain S.venezuelae ATCC 15439/pDR 4-kasOp-venC;

(3) transferring the recombinant strain S.venezuelae ATCC 15439/pDR4-kasOp x-venC seed solution into an SCM culture medium according to the volume ratio of 1-2: 10, culturing at 30 +/-2 ℃ and 220 +/-20 rpm for 12 hours, feeding venezuelan A at the final concentration of 5mg/L, and continuously culturing for 7-8 days to realize the biotransformation of venezuelan A to venezuelan B, thereby obtaining venezuelan B with the conversion rate of 95% detected by GC.

8. The method for preparing tetracyclic diterpenoids according to claims 6 or 7, characterized in that: adding fermentation liquor containing the venezuelan A or the venezuelan B into equal volume of ethyl acetate for extraction, evaporating to obtain an extract, extracting the extract by using normal hexane to remove components with high polarity, evaporating the extract again, and dissolving the extract in acetonitrile for semi-preparation of an inverse phase C18 chromatographic column to obtain purified venezuelan A or venezuelan B; wherein the chromatographic column is Waters Xbridge^TMC-18column, specification 10X 250mm,5 μm, elution program: eluting with 100% methanol for 30min at a flow rate of 1.5-2.5 mL/min.

9. Use of the tetracyclic diterpenoid compound venezuelane a (venezuelae a) according to claim 1 for the preparation of fragrances.

10. Use of the tetracyclic diterpenoid compound venezuelane b (venezuelae b) according to claim 2 for the preparation of fragrances.

Technical Field

The invention belongs to the fields of natural product chemistry, biochemistry, molecular biology and genetic engineering, and particularly relates to tetracyclic diterpenoid compounds and a preparation method and application thereof.

Background

To date, over 80,000 terpenoids have been found in humans, animals, plants and microorganisms. Terpenoids are composed of multiple isoprene building blocks and represent the largest class of natural products^[1,2]. In general, biosynthesis of parent core structure (terpene) of terpenoids involves the catalytic cyclization of a carbon chain-limited polyprenyl pyrophosphate (C5n, n-2, 3, 4, etc.) by terpene synthase to give a complex hydrocarbon backbone containing multiple rings and chiral centers^[3,4]. For example, monoterpenes are cyclized from geranylgeranyl pyrophosphate (C10), sesquiterpenes are cyclized from farnesyl pyrophosphate (C15), diterpenes are cyclized from geranylgeranyl pyrophosphate (C20), and the like. Further, a compound with a more complex and diverse structural skeleton and physicochemical properties is formed by the catalytic action of post-modification enzymes such as cytochrome P450 enzyme, FAD-dependent oxidase, methyl, acyl and glycosyltransferase^[2]. The terpenoid has rich and diverse physiological activities, such as tumor resistance, malaria resistance, bacteria resistance, virus resistance, inflammation resistance, immunosuppression and the like, has wide application value and prospect in the fields of medical care, food, cosmetics, pesticides, veterinary drugs, chemical industry and the like, and is inseparable from the life production of people^[5,6]. Typical terpenoids are represented by the class of "Nozaki" antimalarial drugs artemisinin^[7](ii) a Paclitaxel as clinical candidate drug with strong antitumor activity^[8](ii) a Steroid hormones essential for human and mammals and widely involved in growth, development and metabolic regulation in vivo^[9](ii) a Active steroid saponin compound widely existing in traditional Chinese medicine^[10]. Moreover, the natural pigment and the flavoring agent which are derived from plants are terpenoids, so the natural pigment and the flavoring agent have wide application in the industries of food, essence and flavor^[11]For example, the commonly used natural pigments β -carotene, tomatoErythrogenin, astaxanthin, and the like; food flavoring agents such as menthol, stevioside, etc.; essence, spice, perfume and perfume fixative ambrox, etc.

However, due to resource and technical limitations, especially the disadvantages of long growth cycle, low yield of terpenoids and high acquisition cost of plants (main sources of terpenoids), it is increasingly difficult for people to obtain terpenoids with novel frameworks directly from nature to serve production and life. Meanwhile, the traditional chemical synthesis method for obtaining new terpenoid has the defects of multiple reaction steps, poor catalytic selectivity, inadequately mild conditions, environmental pollution and the like, and more importantly, the method has limited capability of obtaining a brand new structural framework. In recent years, with the rapid development of genome sequencing technology and bioinformatics, it has been found that microorganisms also have considerable terpenoid production potential. Therefore, through the whole genome analysis aiming at single microorganism, the strategy of combining biochemistry, molecular biology and natural product chemistry is an important means for exploring novel terpenoid for basic and application scientific research^[12]。

Streptomyces venezuelae ATCC15439 is a model strain in the field of natural product biosynthesis research, high-quality whole genome data of the strain is published (GenBank: CP013129), secondary metabolites of the strain are rich, and a genetic operation system is complete. However, the search found that there is no report of expressing a gene in the biosynthetic gene cluster (GenBank: MN508361) of venezuelan in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a silent gene activation method to produce a terpenoid, particularly venezuelan A or venezuelan B, or expressing a gene in the biosynthetic gene cluster of venezuelan in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a heterologous expression method in Escherichia coli to produce a terpenoid, particularly venezuelan A or venezuelan B, and isolating and purifying the terpenoid.

Reference to the literature

[1]MURAI K,LAUTERBACH L,TERAMOTOK,et al.An unusual skeletalrearrangement in the biosynthesis of the sesquiterpene trichobrasilenol fromTrichoderma[J].Angew Chem,2019,131(42):15188-92.

[2]DICKSCHAT J S.Bacterial terpene cyclases[J].Nat Prod Rep,2016,33(1):87-110.

[3]CHRISTIANSON D W.Structural and chemical biology of terpenoidcyclases[J].Chem Rev,2017,117(17):11570-648.

[4]CHRISTIANSON D W.Structural biology and chemistry of the terpenoidcyclases[J].Chem Rev,2006,106(8):3412-42.

[5]HUANG M,LU J-J,HUANG M-Q,et al.Terpenoids:natural products forcancer therapy[J].Expert Opin Inv Drug,2012,21(12):1801-18.

[6]MORIKAWA T,MATSUDA H,YOSHIKAWA M.A review of anti-inflammatoryterpenoids from the incense gum resins frankincense and myrrh[J].J Oleo Sci,2017,ess16149.

[7]KLAYMAN D L.Qinghaosu(artemisinin):an antimalarial drug from China[J].Science,1985,228(4703):1049-55.

[8]ROWINSKY E K,DONEHOWER R C.Paclitaxel(taxol)[J].New Engl J Med,1995,332(15):1004-14.

[9]GOWER D.Modifiers of steroid-hormone metabolism:a review of theirchemistry,biochemistry and clinical applications[J].J Steroid Biochem,1974,5(5):501-23.

[10]MAHATO S B,SARKAR S K,PODDAR G.Triterpenoid saponins[J].Phytochemistry,1988,27(10):3037-67.

[11]BOHLMANN J,KEELING C I.Terpenoid biomaterials[J].The PlantJournal,2008,54(4):656-69.

[12]YAMADA Y,KUZUYAMA T,KOMATSU M,et al.Terpene synthases are widelydistributed in bacteria[J].Proc Natl Acad Sci,2015,112(3):857-62.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a tetracyclic diterpenoid compound and a preparation method and application thereof.

The tetracyclic diterpenoid compounds comprise venezuelane A (venezuelaene A) and venezuelane B (venezuelane B), and specifically comprise the following components in parts by weight:

a tetracyclic diterpenoid compound characterized by: the compound is a compound containing a 5-5-6-7 tetracyclic diterpene framework, and the chemical molecular formula of the compound is C₂₀H₃₂The structural formula is shown as formula (1) and is named as: venezuelayene a (venezuelaenea);

a tetracyclic diterpenoid compound characterized by: the compound is a compound containing a 5-5-6-7 tetracyclic diterpene framework, and the chemical molecular formula of the compound is C₂₀H₃₀O, the structural formula is shown as formula (2), and the name is: venezuelanene b (venezuelaeneb);

the preparation method of the tetracyclic diterpenoid compound is characterized by comprising the following steps: the venezuelae A or the venezuelae B is obtained by expressing genes in a biosynthetic gene cluster of venezuelae in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a method of self-silencing gene activation.

The preparation method of the tetracyclic diterpenoid compound is characterized by comprising the following steps: the venezuelae A or the venezuelae B is obtained by expressing genes in a biosynthetic gene cluster of venezuelae in Streptomyces venezuelae (Streptomyces venezuelae ATCC 15439) by a method of heterologous expression in Escherichia coli.

The venezuelan biosynthetic gene cluster described in the above method is characterized in that: the gene cluster contains 4 genes, namely a gene venA for coding diterpene synthase VenA or a functional equivalent thereof, 2 genes venB and venC for coding cytochrome P450 enzymes VenB and VenC or functional equivalents thereof, and a gene venD for coding geranylgeranyl pyrophosphate synthase VenD or functional equivalents thereof; wherein the nucleotide sequence of the venA is shown as SEQ ID NO: 1, the nucleotide sequence of venB is shown as SEQ ID NO: 2, the nucleotide sequence of venC is shown as SEQ ID NO: 3, the nucleotide sequence of venD is shown as SEQ ID NO: 4 is shown in the specification; or the nucleotide sequence of the gene is a corresponding DNA coding sequence with the amino acid sequence consistency of more than 80 percent with the coding proteins VenA, VenB, VenC and VenD respectively.

Specifically, the preparation method of the tetracyclic diterpenoid compound venezuelan A comprises the following steps:

(1) carrying out PCR amplification on gene venA of diterpene synthase, geranylgeranyl pyrophosphate synthase gene venD and cytochrome P450 enzyme gene venC by using a genome (GenBank: CP013129) of S.venezuelae ATCC15439 as a template and primers venA-F/venA-R, venD-F/venD-R and pDR4venC-F/pDR4venC-R respectively to obtain PCR products of the genes venA, venD and venC; then, constructing a co-expression vector pET28b-venAD of venA and venD by taking an escherichia coli expression vector pET28b or a functional equivalent thereof as a vector; wherein the nucleotide sequences of the primers are respectively:

VenA-F：CCTGGTGCCGCGCGGCAGCCATATGCAGCAACGCCTCCGCCCG

VenA-R：GTCCACCAGTCATGCTAGCCATATGTCAAACCAGCGGTCGGGTGGG

VenD-F：CCTGGTGCCGCGCGGCAGCCATATGACCCAGGCGACACTGTC

VenD-R：GTCCACCAGTCATGCTAGCCATATGTCATCCGTCGCGGTGGCTCA

pDR4VenC-F：CGTGCAGGACTGGGGGAGTTACTAGTATGACCTGGGCCGCTGCGGGpDR4VenC-R:ATGATTACGAATTCGAGCTCGGTACCTCATGACGCCACCGCCCGGG

(2) converting the co-expression vector pET28b-venAD into the chemical competence of Escherichia coli engineering bacteria Eco-P (Escherichia coli BL21(DE3)/pACYC-mavEmavS & pTrc-low) which can produce dimethylallyl pyrophosphate and isopentenyl pyrophosphate at high yield to obtain Escherichia coli engineering bacteria Eco-P/pET28b-venAD which can produce Venezuelaene A (venezuelane A);

(3) transferring the engineering bacteria Eco-P/pET28b-venAD seed liquid of escherichia coli into TB culture medium containing kanamycin, chloramphenicol and ampicillin according to the volume ratio of 1-2: 100, culturing at the temperature of 37 +/-2 ℃ and the rpm of 220 +/-20, and culturing when OD is obtained₆₀₀When the concentration reaches 1.0-1.5, adding isopropyl- β -D-thiogalactose with final concentration of 0.1-1.0mMGlycoside, inducing protein expression and biosynthesizing venezuelan A under the conditions of 18 +/-2 ℃ and 220 +/-20 rpm.

Specifically, the preparation method of the tetracyclic diterpenoid compound venezuelan B comprises the following steps:

(1) carrying out PCR amplification on gene venA of diterpene synthase, gene venD of geranylgeranyl pyrophosphate synthase and gene venC of cytochrome P450 enzyme by taking S.venezuelae ATCC15439 genome (GenBank: CP013129) as a template and primers venA-F/venA-R, venD-F/venD-R and pDR4venC-F/pDR4venC-R respectively to obtain PCR products of the genes venA, venD and venC; then, by taking a streptomycete integrated vector pDR4-kasOp or a functional equivalent thereof as a vector, integrating the venC into SpeI and KpnI double-restriction enzyme linearized pDR4-kasOp to obtain an expression vector pDR 4-kasOp-venC; wherein the nucleotide sequences of the primers are respectively:

VenA-F：CCTGGTGCCGCGCGGCAGCCATATGCAGCAACGCCTCCGCCCG

VenA-R：GTCCACCAGTCATGCTAGCCATATGTCAAACCAGCGGTCGGGTGGG

VenD-F：CCTGGTGCCGCGCGGCAGCCATATGACCCAGGCGACACTGTC

VenD-R：GTCCACCAGTCATGCTAGCCATATGTCATCCGTCGCGGTGGCTCA

pDR4VenC-F：CGTGCAGGACTGGGGGAGTTACTAGTATGACCTGGGCCGCTGCGGG

pDR4VenC-R:ATGATTACGAATTCGAGCTCGGTACCTCATGACGCCACCGCCCGGG

(2) the expression vector pDR 4-kasOp-venC was transformed into commercial E.coli ET12567/pUZ8002 to obtain E.coli engineering bacteria Et12567/pUZ8002&pDR 4-kasOp-venC; mixing spore suspension of S.venezuelane ATCC15439 wild type with Escherichia coli engineering bacteria Et12567/pUZ8002&pDR 4-kasOp-venC, and uniformly spread on MgCl-containing glass₂And CaCl₂Culturing on MS plate at 30 + -2 deg.C for 16 + -1 h, covering each MS plate with sterile water containing hygromycin and nalidixic acid, drying, culturing at 30 + -2 deg.C, culturing for 3-5 days, collecting single colony on MS culture medium containing nalidixic acid and hygromycin, sporulating for 3 + -1 days, inoculating into 2 XYT culture solution, culturing at 28 + -1 deg.C and 220 + -20 rpm for 2 days, and extractingTaking a genome, and verifying that the genotype is correct by PCR to obtain a recombinant strain S.venezuelae ATCC 15439/pDR 4-kasOp-venC;

(3) transferring the recombinant strain S.venezuelae ATCC 15439/pDR4-kasOp x-venC seed solution into an SCM culture medium according to the volume ratio of 1-2: 10, culturing at 30 +/-2 ℃ and 220 +/-20 rpm for 12 hours, feeding venezuelan A at the final concentration of 5mg/L, and continuously culturing for 7-8 days to realize the biotransformation of venezuelan A to venezuelan B, thereby obtaining venezuelan B with the conversion rate of 95% detected by GC.

The preparation method of the tetracyclic diterpenoid compound comprises the following steps: the fermentation liquor containing the venezuelan A or the venezuelan B can be added with equal volume of ethyl acetate for extraction, and then the mixture is evaporated to dryness to obtain an extract, the extract is extracted by normal hexane to remove components with large polarity, and the extract is evaporated to dryness and dissolved in acetonitrile to be used for semi-preparation of an inverse phase C18 chromatographic column to obtain purified venezuelan A or venezuelan B; wherein the chromatographic column is Waters Xbridge^TMC-18column, specification 10X 250mm,5 μm, elution program: eluting with 100% methanol for 30min at a flow rate of 1.5-2.5 mL/min.

The invention relates to application of a tetracyclic diterpenoid compound venezuelaene A (venezuelaene A) in preparing perfumes.

The invention relates to application of a tetracyclic diterpenoid compound venezuelaene B (venezuelaene B) in preparing perfumes.

The invention provides a tetracyclic diterpenoid compound and a preparation method and application thereof. In the development process, the inventor discovers a novel biosynthetic gene cluster of venezuelan A and B from Streptomyces venezuelae ATCC15439 through a genome mining strategy. The expression of genes in the venezuelan A and B biosynthetic gene clusters in streptomycete and escherichia coli is realized by a silent gene activation and heterologous expression mode. Finally, two 5-5-6-7 tetracyclic diterpenoid compounds with brand-new frameworks, namely venezuelan A and venezuelan B with fragrance are obtained by separation and purification. The innovation point is that a genetic engineering method is utilized to construct a self-sufficient engineering bacterium for generating a novel compound venezuelan A. Then, the transformation of the Venezuelae A to the new compound Venezuelene B is completed in one step by using an engineering bacterium S.venezuelae ATCC 15439/pDR 4-kasOp-venC for expressing the P450 gene venC by using a biotransformation method. 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 venezuelan A and venezuelan B products are expected to be applied to the fields of food, spice, pharmaceutical chemicals and the like.

Drawings

Fig. 1 is a three-dimensional structure ellipsoid diagram of venezuelan a provided in an embodiment of the present invention.

Fig. 2 is a chemical structural diagram of venezuelane a (venezuelane a) and venezuelane b (venezuelane b) according to an embodiment of the present invention.

Fig. 3 is an ultraviolet-visible spectrum of venezuelan a according to an embodiment of the present invention.

FIG. 4 is a gas-phase mass spectrum of Venezuelene A provided by the embodiment of the invention.

FIG. 5 shows Venezuelene A provided in an embodiment of the present invention¹H-¹Key relevant signals for H COSY, HMBC and NOESY.

FIG. 6 shows Venezuelene A in CD provided by an embodiment of the present invention₃In Cl¹HNMR map.

FIG. 7 shows Venezuelene A in CD provided by an embodiment of the present invention₃In Cl¹³A CNMR map.

FIG. 8 shows Venezuelene A in CD provided by an embodiment of the present invention₃In Cl¹³C-DEPT 135 ℃ diagram.

FIG. 9 shows Venezuelene A in CD provided by an embodiment of the present invention₃HSQC pattern in Cl.

FIG. 10 shows Venezuelene A in CD provided by an embodiment of the present invention₃In Cl¹H-¹H COSY picture.

FIG. 11 shows Venezuelene A in CD provided by an embodiment of the present invention₃HMBC pattern in Cl.

FIG. 12 shows Venezuelene A in CD provided by an embodiment of the present invention₃NOSEY pattern in Cl.

FIG. 13 is a graph (2) showing the biotransformation of venezuelan A (1) to venezuelan B by GC assay according to the present invention.

Wherein: (i) venezuelae ATCC15439 was fed 5mg/L venezuelae a; (ii) the engineered bacterium S.venezuelae ATCC 15439/pDR4-kasOp x-venC was fed with 5mg/L of venezuelan A.

Fig. 14 is an ultraviolet-visible spectrum of venezuelan B according to an embodiment of the present invention.

FIG. 15 is a gas mass spectrum of Venezuelene B provided in the example of the present invention.

Fig. 16 is a high-resolution mass spectrum of venezuelan B according to an embodiment of the present invention.

FIG. 17 shows Venezuelene B provided in an embodiment of the present invention¹H-¹Key relevant signals for H COSY, HMBC and NOESY.

FIG. 18 shows Venezuelene B in CD provided by an embodiment of the present invention₃In Cl¹HNMR map.

FIG. 19 shows Venezuelene B in CD provided by an embodiment of the invention₃In Cl¹³C-DEPTQ plot.

FIG. 20 shows Venezuelene B in CD provided by an embodiment of the invention₃HSQC pattern in Cl.

FIG. 21 shows Venezuelene B in CD provided by an embodiment of the invention₃In Cl¹H-¹H COSY picture.

FIG. 22 shows Venezuelene B in CD provided by an embodiment of the present invention₃HMBC pattern in Cl.

FIG. 23 shows Venezuelene B in CD provided by embodiments of the present invention₃NOSEY pattern in Cl.

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.

Basic molecular biology experimental techniques such as PCR amplification, plasmid extraction, transformation, etc., which are used in the examples of the present invention, if no specific description is given, are generally performed according to conventional methods, and specifically, refer to molecular cloning instruction (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peyer, et al, 2002, Beijing, science publishers, etc.), or according to the instructions provided by the relevant manufacturers.

The strain S.venezuelae ATCC15439 used in the embodiment of the invention is purchased from American strain collection; escherichia coli BL21(DE3) competent cells were purchased from Beijing Okame. The construction method of the Escherichia coli engineering bacterium Eco-P (Escherichia coli BL21(DE3)/pACYC-mavEmavS & pTrc-low) is shown in Microb CellFact 15,74 (2016); escherichia coli Et12567/pUZ8002 was purchased from Youbao Bio.

The one-step cloning kit used in the examples of the present invention was purchased from Nanjing Novozam; agarose gel DNA recovery kits were purchased from Omega; streptomyces integrative vector pDR4-kasOp was purchased from Addgene; coli expression vector pET28b was purchased from Invitrogen; high fidelity DNA polymerase was purchased from Takara; restriction enzymes were purchased from semer fly; PCR primer synthesis and DNA sequencing were performed by Beijing Okame.

The sporulation culture medium of the S.venezuelae ATCC15439 related by the embodiment of the invention is an MS culture medium, and the formula is as follows: 20g/L of bean flour, 20g/L of mannitol and 20g/L of agar powder; the seed culture medium is 2 XYT culture medium, and the formula is as follows: tryptone 16g/L, yeast extract 10g/L, NaCl 5 g/L; the fermentation medium is SCM medium, and the formula is as follows: 15g/L of soluble starch, 20g/L of soybean peptone and CaCl₂0.1g/L, yeast extract 1.5g/L, 3-morpholine propanesulfonic acid 10.5g/L, pH 7.2.

The seed culture medium of the enterobacter related to the embodiment of the invention is an LB culture medium, and the formula is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl; the fermentation medium is a TB medium, and the formula is as follows: tryptone 12g/L, yeast extract 24g/L, glycerol 40g/L, K₂HPO₄9.4g/L，KH₂PO₄2.2g/L。