(-)-vinigrol synthetic pathway gene and synthetic method thereof

Abstract

The invention discloses (-)-vinigrol synthesis pathway genes and a synthesis method thereof, the synthesis genes comprise a vniA gene, a vniB gene and a vniC gene, by heterologous expression of the vniA gene, the vniB gene and the vniC gene in aspergillus nidulans or saccharomyces cerevisiae, the synthesis pathway of the compound (-)-vinigrol is found, and more raw material supply is provided for development of (-)-vinigrol.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to a (-)-vinigrol synthesis pathway gene, and also to a synthesis method of the compound (-)-vinigrol. Background Art

[0002] (-)-Vinigrol has been reported to have good pharmacological activity and has a specific inhibitory effect on rabbit and human platelet aggregation induced by adrenaline or platelet activating factor (PAF). 50 The (-)-vinigrol also has good antihypertensive activity. In spontaneously hypertensive rats, oral tests have found that at a dose of 2 mg / kg, it can reduce the mean arterial blood pressure of rats by 15% and last for at least 6 hours. In addition, (-)-vinigrol can also drive the non-apoptotic death of breast cancer cells. In the IC of triple-negative breast cancer cells, 50 The activity of (-)-vinigrol to doxorubicin-resistant breast cancer cells was 41±2.56μM, and it was even better, reaching 8.46±1.2μM. In addition, (-)-vinigrol was also shown to improve rheumatoid arthritis in mice. Therefore, (-)-vinigrol has good drug potential.

[0003] The source of (-)-Vinigrol currently relies mainly on total synthesis. Due to its structural characteristics, it contains 1,5-decahydrobutylnaphthalene, eight consecutive chiral centers and a highly tense eight-membered ring, which makes its chemical synthesis difficult. In the past four decades, more than 20 research groups have conducted total synthesis research on it. However, only five research groups have achieved its total synthesis, among which the Baran, Barriault and Njardarson research groups synthesized its enantiomers. Luo Tuoping and Li Chuangchuang's research groups completed the total synthesis of (-)-vinigrol twice in 2019 and 2024 respectively. It is worth mentioning that Li Chuangchuang's research group (2024) shortened the steps of total synthesis to 12 steps. However, it is extremely difficult to synthesize this molecule, and the scale cannot currently reach the gram level. At the same time, chemical synthesis is relatively unfriendly to the biological synthesis environment.

[0004] In addition, (-)-vinigrol is also directly fermented and isolated from other strains. However, its sources are very limited. Currently, it has only been isolated and identified in strains Virgaria nigra F-5408, Myrothecium sp. ZLW0801-19 and Bloxamia. However, due to its low yield in the original host and the harsh culture conditions of the original strain, it has not been well applied. The present invention achieves efficient synthesis of (-)-vinigrol in a model microorganism, and simultaneously obtains two (-)-vinigrol compounds in the process of producing (-)-vinigrol. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a (-)-vinigrol synthesis pathway gene; another object of the present invention is to provide a method for biosynthesizing (-)-vinigrol compounds.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. (-)-vinigrol synthesis pathway genes, including vniA gene, vniB gene and vniC gene;

[0008] The nucleotide sequence of the vniA gene is shown in SEQ ID NO.1 or a sequence homologous to the vniA gene and having the same function;

[0009] The nucleotide sequence of the vniB gene is shown as SEQ ID NO.2 or a sequence homologous to the vniB gene and having the same function; the nucleotide sequence of the vniC gene is shown as SEQ ID NO.3 or a sequence homologous to the vniC gene and having the same function.

[0010] Preferably, the engineered bacteria are Aspergillus nidulans and Saccharomyces cerevisiae that heterologously express the vniA gene, the vniB gene and the vniC gene.

[0011] Preferably, the (-)-vinigrol synthesis pathway genes are expressed in Aspergillus nidulans or Saccharomyces cerevisiae, wherein the genes include vniA gene, vniB gene and vniC gene;

[0012] The nucleotide sequence of the vniA gene is shown in SEQ ID NO.1 or a sequence homologous to the vniA gene and having the same function;

[0013] The nucleotide sequence of the vniB gene is shown as SEQ ID NO.2 or a sequence homologous to the vniB gene and having the same function; the nucleotide sequence of the vniC gene is shown as SEQ ID NO.3 or a sequence homologous to the vniC gene and having the same function.

[0014] Preferably, the method for expressing (-)-vinigrol synthesis pathway genes in Aspergillus nidulans or Saccharomyces cerevisiae is to construct a recombinant expression vector expressing vniA gene, vniB gene and vniC gene, and transform Aspergillus nidulans or Saccharomyces cerevisiae to obtain the genes.

[0015] Preferably, the recombinant vector expressing the vniA gene is connected to the NotI site of the pANU vector; the recombinant vector expressing the vniC gene is connected to the NotI site of the pANU vector; and the recombinant vector expressing the vniB gene is connected to the BamHI site of the pANR vector.

[0016] Preferably, the vniB gene is expressed in Aspergillus nidulans or Saccharomyces cerevisiae, and compound II is fed during the fermentation process, so that the fermentation product contains a (-)-vinigrol compound; the structural formula of the compound II is as follows:

[0017]

[0018] Preferably, the compound II is heterologously expressed by the gene vniA in Aspergillus nidulans or Saccharomyces cerevisiae, and the fermentation product is repeatedly extracted three times with acetone:ethyl acetate in a volume ratio of 1:3, concentrated to dryness under reduced pressure, separated by a forward silica gel column, eluted with a solution of n-hexane:ethyl acetate in a gradient ratio of 100:0, 50:1, 20:1, and 15:1, and the components containing compound II are collected and concentrated.

[0019] The beneficial effects of the present invention are as follows: the present invention finds the synthesis pathway of the compound (-)-vinigrol by studying the metabolic pathway of Virgaria nigra NBRC9238, and the compound (-)-vinigrol can be separated from the product by placing the vniA gene, the vniB gene and the vniC gene in Aspergillus nidulans or Saccharomyces cerevisiae, thereby providing more raw material supply for the development of (-)-vinigrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0021] Figure 1 for the production of the compound (-)-vinigrol in Aspergillus nidulans;

[0022] Figure 2 The compound (-)-vinigrol is produced in Saccharomyces cerevisiae;

[0023] Figure 3 The homologous gene was expressed in Aspergillus nidulans and compound II was produced. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0025] The nucleotide sequence of the vniA gene in the embodiment of the present invention is shown in SEQ ID NO.1, the nucleotide sequence of the vniB gene is shown in SEQ ID NO.2, and the nucleotide sequence of the vniC gene is shown in SEQ ID NO.3.

[0026] Example 1

[0027] Virgaria nigra NBRC9238 large-scale fermentation and sample pretreatment method: Virgaria nigra NBRC9238 fungal strain was cultured on malt extract agar (MA, malt extract 30g / L) at 25°C for ten days, and after activation by MA, it was inoculated into 8 conical flasks containing CSG medium (1% corn starch, 1% soluble starch, 1% glucose, 1% corn syrup dry powder, 0.5% yeast extract, 0.5% cottonseed powder, adjusted to pH 6.0, and then added with 0.2% CaCO3, 121°C, sterilized for 20 minutes) to prepare seed liquid, each conical flask contained 50mL CSG medium, 220rpm, 25 degrees shaking culture for 3 days. Fermentation was carried out in 40 conical flasks (1000mL), each conical flask contained 250mL CSG medium, and after sterilization, it was cooled to room temperature, and 10mL seed liquid was inoculated in each conical flask. 220rpm, 25°C shaking culture for 7 days. After 7 days, 10 L of the fermentation culture was filtered to separate the bacterial cells and the supernatant. The supernatant and the bacterial cells were extracted three times with ethyl acetate and acetone, respectively, and the extract was concentrated to dryness under reduced pressure to obtain a crude extract.

[0028] Example 2

[0029] Preparation and structural analysis of (-)-vinigrol: The crude extract was subjected to medium pressure reverse phase chromatography (80 g reverse phase silica gel (C18) column, X2 system, BUCHI, Inc), MeOH-H2O system, separation at 25 mL / min. Elution with 30% MeOH for 10 minutes, then elution with 30-100% linear gradient MeOH for 40 minutes, and finally elution with 100% MeOH for 10 minutes to obtain a fraction containing compound IV (213.5 mg) (80% / 20% MeOH / H2O). Semi-preparative HPLC (column: COSMOSIL 5C18-MS-II, 10×250mm, 5μm, Shimadzu LC-20AR) was used for purification with 85% / 15% MeOH / H2O isopycnic program to obtain compound IV (17.8 mg, t R =17.4min).

[0030] Compound (-)-vinigrol Identification: Colorless oil; Optically active (C=1.0, CHCl3), indicating that compound IV is levorotatory, HR TOFMS m / z345.2401[M+Na] in positive ion mode + ,C 20 H 34 The calculated value of NaO3 is 345.2400, indicating that the actual molecular formula of the compound is C 20 H 34 O3, combined with the NMR data in Table 1, the structure of the compound is determined as follows:

[0031]

[0032] Table 1. NMR data assignment of compound (-)-vinigrol

[0033]

[0034] (400MHz for 1 H NMR, 100MHz for 13 C NMR)

[0035] Example 3

[0036] Construction and production of (-)-Vinigrol engineered strains

[0037] Preparation of compound (-)-vinigrol: Genes vniA, vniB and vniC (terpene cyclase, cytochrome P450, GGPPS) from Virgaria nigra NBRC9238 were heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant vector expressed in Aspergillus nidulans was constructed by constructing vniA and vniC at the NotI site of the pANU vector, and vniB at the BamHI site of the pANR vector. The recombinant vector heterologously expressed in Saccharomyces cerevisiae was constructed by constructing vniA and vniC at the BglII site of the pYEU vector, and vniB at the BglII site of the pYEL vector. The heterologous expression strain was fermented in large quantities, and after the fermentation was completed, it was repeatedly extracted three times with acetone:ethyl acetate (1:3), and concentrated to dryness under reduced pressure. The separation method was the same as described in Examples 1 and 2 above. The results are as shown in FIG. Figure 1 and Figure 2 The results showed that the compound (-)-vinigrol was synthesized after VniA, VniB and VniC were expressed in Aspergillus nidulans; the compound (-)-vinigrol was also synthesized after VniA, VniB and VniC were expressed in yeast.

[0038] Example 4

[0039] Preparation of compound II:

[0040] The gene vniA (terpene cyclase) from Virgaria nigra NBRC9238 was heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae, and the heterologous expression strain was fermented in large quantities. After the fermentation, it was repeatedly extracted three times with acetone: ethyl acetate (1:3) and concentrated under reduced pressure. A forward silica gel column was used for separation, and the gradient of n-hexane: ethyl acetate (100 / 0, 50 / 1, 20 / 1, 15 / 1) was used for elution. The components containing compound II were collected and concentrated, and then separated again by a forward silica gel column gradient, and the mobile phase was n-hexane-ethyl acetate 50 / 1, 40 / 1 and 30 / 1 elution) to obtain 38 components, labeled Fr2.1-Fr2.38. Compound II (21.6 mg) was isolated from Fr2.20 and Fr2.21. Deuterated chloroform was dissolved and nuclear magnetic resonance identification was performed. The compound attribution is shown in Table 1. Alternatively, the samples were separated by preparative liquid phase separation (column: COSMOSIL 5C18-MS-II, 10×250 mm, 5 μm), and eluted isocratically with 100% acetonitrile-water (t R =22.4min).

[0041] Identification of compound II, colorless block crystals; optical rotation (C=1.0,CHCl3), indicating that compound II is levorotatory, HR TOFMS m / z 313.2505[M+Na] in positive ion mode + ,C 20 H 34 The calculated value of NaO is 313.2502, indicating that the actual molecular formula of the compound is C 20 H 34 O, combined with the NMR data in Table 2, the structure of the compound is determined as follows:

[0042]

[0043] Table 2. NMR data assignment of compound II

[0044]

[0045] (400MHz for 1 H NMR, 100MHz for 13 C NMR)

[0046] Preparation of compound III:

[0047] The gene vniB (cytochrome P450) from Virgaria nigra NBRC9238 was heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae, and the heterologous expression strain was fermented in large quantities and fed with compound II. After the feeding and fermentation, it was repeatedly extracted three times with acetone: ethyl acetate (1:3) and concentrated to dryness under reduced pressure. LH20 was used for crude fractionation, and the fractions containing compound IV were collected and concentrated. After preparative liquid phase separation (column: COSMOSIL 5C18-MS-II, 10×250mm, 5μm), 90% acetonitrile-water isocratic elution (t R =19.0min).

[0048] Compound IV was obtained by removing the 16-hydroxyl group through Barton-McCombie deoxygenation. The product after the reaction was passed through a normal silica gel column, eluted with n-hexane to remove tributyltin hydrogen, and then eluted with methanol to obtain a component containing compound III. After preparative liquid phase separation (column: COSMOSIL 5C18-MS-II, 10×250mm, 5μm), 75% acetonitrile-water isocratic elution, 9.0 mg of pure compound was obtained. Deuterated chloroform was dissolved and nuclear magnetic resonance identification was performed. The compound attributes are shown in Table 3.

[0049] Compound III identification: colorless oil; optically active (C=1.0, CHCl3), indicating that compound III is levorotatory, HR TOFMS m / z 289.2536[M+H-H2O] in positive ion mode + ,[M+H-H2O] + The calculated value is 289.2531, indicating that the actual molecular formula of the compound is C 20 H 34 O2, combined with the NMR data in Table 3, the structure of the compound is determined as follows:

[0050]

[0051] Table 3. NMR data attribution of compound III

[0052]

[0053] (400MHz for 1 H NMR, 100MHz for 13 C NMR

[0054] Example 5

[0055] The primary structure of a protein determines its three-dimensional structure, and the three-dimensional structure directly determines the function of the protein. Therefore, proteins with similarity in primary sequence usually have similar or identical functions. Based on this, we combined the protein sequence similarity analysis (sequence similarity networks, SSN) with NCBI blast analysis. We searched for 25 different amino acid sequences similar to VniA, as shown in the table. Through homology comparison analysis, the similarity of these sequences is between 38% and 89%.

[0056] Serial number Strain Accession number 1 Podospora aff.communis PSN243 KAK4445355.1 2 Metarhizium guizhouense ARSEF 977 KID83933.1 3 Aspergillus tanneri THC91728.1 4 Immersiella caudata KAK0633951.1 5 Metarhizium majus ARSEF 297 KID93931.1 6 Metarhizium majus ARSEF 297 KID91854.1 7 Schizothecium vesticola KAK0745701.1 8 Podospora aff.communis PSN243 KAK4451285.1 9 Poronia punctata KAI1814037.1 10 Daldinia grandis KAI0096559.1 11 Metarhizium album ARSEF 1941 XP_040681777.1 12 Daldinia decipiens XP_049098172.1 13 Schizothecium conicum KAK1834414.1 14 Daldinia childiae XP_033441764.1 15 Xylariaceae sp.AK1471 KAI3325431.1 16 Aspergillus tanneri XP_033430655.1 17 Purpureocillium lilacinum OAQ83778.1 18 Aspergillus nanangensis KAF9890543.1 19 Purpureocillium lilacinum OAQ90558.1 20 Purpureocillium lilacinum KAK4095383.1 21 Schizothecium vesticola KAK0746713.1 22 Xylogone sp.PMI_703 KAH8797665.1 23 Beauveria brongniartii RCEF 3172 OAA33865.1 24 Metarhizium album ARSEF 1941 XP_040674972.1 25 Cercophora newfieldiana KAK0649781.1

[0057] The dgrA gene (NCBI accession number KAI0096559.1) of Daldinia grandis CBS114736 strain and the xylA gene (KAI3325431.1) of Xylariaceae sp. AK1471 strain were selected and expressed in Aspergillus nidulans by plasmid construction. The results of fermentation tests were as follows: Figure 3 The results showed that the two genes also had the function of producing compound II, so genes homologous to VniA could synthesize and produce compound II.

[0058] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. (-)-vinigrol biosynthesis pathway genes, Features: The genes include vniA gene, vniB gene and vniC gene; The nucleotide sequence of the vniA gene is shown in SEQ ID NO.1 or a sequence homologous to the vniA gene and having the same function; The nucleotide sequence of the vniB gene is shown as SEQ ID NO.2 or a sequence homologous to the vniB gene and having the same function; the nucleotide sequence of the vniC gene is shown as SEQ ID NO.3 or a sequence homologous to the vniC gene and having the same function.

2. Using the (-)-vinigrol synthesis pathway gene of claim 1 to construct a (-)-vinigrol synthesis engineering bacterium, Features: The engineering bacteria is Aspergillus nidulans or Saccharomyces cerevisiae that heterologously expresses vniA gene, vniB gene and vniC gene.

3. A method for biosynthesizing (-)-vinigrol compounds, Features: expressing (-)-vinigrol biosynthesis pathway genes in Aspergillus nidulans or Saccharomyces cerevisiae, wherein the genes include vniA gene, vniB gene and vniC gene; The nucleotide sequence of the vniA gene is shown in SEQ ID NO.1 or a sequence homologous to the vniA gene and having the same function; The nucleotide sequence of the vniB gene is shown as SEQ ID NO.2 or a sequence homologous to the vniB gene and having the same function; the nucleotide sequence of the vniC gene is shown as SEQ ID NO.3 or a sequence homologous to the vniC gene and having the same function.

4. The method for biosynthesizing (-)-vinigrol compounds according to claim 3, Features: The method for expressing the (-)-vinigrol synthesis pathway gene in Aspergillus nidulans or Saccharomyces cerevisiae is to construct a recombinant expression vector expressing vniA gene, vniB gene and vniC gene, and transform Aspergillus nidulans or Saccharomyces cerevisiae to obtain the gene.

5. The method for biosynthesizing (-)-vinigrol compounds according to claim 4, Features: The recombinant vector expressing the vniA gene was constructed by ligating the vniA gene into the NotI site of the pANU vector; The recombinant vector expressing the vniC gene was constructed by ligating the vniC gene into the NotI site of the pANU vector; The recombinant vector for expressing vniB gene was constructed by ligating vniB into the BamHI site of pANR vector.

6. The method for biosynthesizing (-)-vinigrol compounds according to claim 3, Features: The vniB gene is expressed in Aspergillus nidulans or Saccharomyces cerevisiae, and compound II is fed during the fermentation process, and the fermentation product contains a (-)-vinigrol compound; the structural formula of the compound II is as follows:

7. The method for biosynthesizing (-)-vinigrol compounds according to claim 4, Features: The compound II is heterologously expressed by the gene vniA in Aspergillus nidulans or Saccharomyces cerevisiae, and the fermentation product is repeatedly extracted three times with acetone:ethyl acetate in a volume ratio of 1:3, concentrated to dryness under reduced pressure, separated by a forward silica gel column, eluted with a solution with a gradient ratio of n-hexane:ethyl acetate of 100:0, 50:1, 20:1, and 15:1, and the components containing the compound II are collected and concentrated.

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